Aspects of the Metabolism and Physiology of Gibberellins

Aspects of the Metabolism and Physiology of Gibberellins

ALAN CROZIER Department of Botany. University of Glasgow. Glasgow GI2 8QQ. Scotland

I . Introduction .

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I1. Analytical Methods . . . . . . . . A . General Observations . . . . . B . Extraction and Partitioning Techniques C . Group Purification Procedures . . . D . Separatory Techniques . . . . . E . Identification Procedures . . . . . F. Verification of Accuracy . . . . .

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44 44 46 48 51 62 79

. . . . . . . . . . . . . . Mevalonic Acid to Ent-Kaurene . . . . . . . . . . Ent-Kaurene to C A I , aldehyde . . . . . . . . . . . Pathways beyond GA,, aldehyde . . . . . . . . . . Sites of Gibberellin Biosynthesis and Compartmentation . . . .

85 85 88 92 127

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111. Gibberellin Biosynthesis

A. B. C. D.

IV . Structure-Activity Relationships .

V . Conclusions . References .

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140 141

I . INTRODUCTION The gibberellins (GAS) are a group of diterpenoid acids which function as endogenous regulators of the growth and development of higher plants . General acceptance of their hormonal role is based on the observation that

34

ALAN CROZIER

GAS are natural components of the vast majority of higher plants, and that exogenous application of pg quantities can induce a wide range of plant growth responses. They are very effective in promoting stem elongation in intact plants and the response is especially pronounced in dwarf varieties of pea (Pisum sativum), maize (Zeu mciys) and rice ( O r ~ m sativa). GAScan also overcome seed dormancy by substituting for prerequisite cold, light or dark treatments as well as promoting the growth of dormant buds of woody plants and tubers. De n o w synthesis of several hydrolysing enzymes, including a-amylase, can be induced by GA treatment of the aleurone layer of cereal grains. This finds practical application in the malting industry where GA is widely used to increase the rate of starch hydrolysis. GAS can promote stem elongation and the subsequent flowering of rosette plants grown in noninductive short day photoperiods and in a similar manner can circumvent the vernalization requirements of certain biennial species. Sex expression can be modified by GAS, particularly in the Cucurbitaceae where the production of staminate flowers is strongly enhanced. The effects of GAS on reproductive organs are not restricted to angiosperms as similar responses have been observed in some coniferous species. GA application results in prolific male strobilus production in 60-day-old seedlings of Arizona cypress (Cupressus nrizonica Greene), a species that does not usually produce strobili until it is 1G-15years of age. GAS will induce parthenocarpic fruit development in a number of plants including Lycopersicum esculentum Mill., Cucumis sativus L., Solanum melongena L. and Capsicum frutescans L. GAS are widely used in vineyards as they induce the growth of large, elongated berries in open clusters, thereby making the grapes more attractive for table use. It is also reassuring to know that Californian wines prepared from GA-treated grapes and tasted by a panel of experts scored just as highly as those made from untreated berries. A further effect of GA is to retard both leaf and fruit senescence. Treated citrus fruit attached to the tree remains green for six months or more. The senescence of detached fruit is slowed from three weeks to two months by G A application. Clearly GAS have far-reaching effects on many phases of growth and development. There have been many reviews on GAS,the most recent on the physiological role of GAS being that of Jones (1973). GA metabolism has been covered by Phinney (1979), Hedden et al. (1978) and Railton (1976). Hedden (1979) has reviewed selected aspects of GA chemistry while Graebe and Ropers (1978) have published a critical and very comprehensive review of GA chemistry, biochemistry, metabolism and physiology. Agricultural and horticultural uses of GAs have been outlined by Weaver (1972). The discovery of GAS originates from an investigation of the “foolish seedling” or “bakanae” disease of rice (Kurosawa, 1926). The disease had been observed in Japan for over 150 years and infected plants were characterized by both excessive shoot overgrowth of the seedlings and lowered seed

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

35

production by mature plants. The first suggestion that a fungus might be involved was made in 1912 after hyphae were observed in infected rice plants (Sawada, 1912). The pathogen was subsequently identified as an ascomycete Gibberella ,fujikuroi (Saw) Wr. (called Fusarium nionilifornie Sheldon in the asexual stage) and it was shown that sterile culture filtrates of the bakanae fungus induced a marked growth stimulation in rice and maize (Kurosawa, 1926). In 1935 the active ingredient was isolated in a purified but noncrystalline form and given the name gibberellin (Yabuta, 1935). crystalline GA was obtained by Yabuta and Sumiki (1938) although this later proved to be a mixture of at least three compounds all of which promoted shoot growth when applied to rice seedlings (Takahashi et a / . , 1955). Many of these early reports were, contrary to popular belief, published in English, yet despite a great interest in hormonal regulation of growth by auxins, plant physiologists in the West did not become aware of the Japanese work on GA until the early 1950s when groups at the US Department of Agriculture and ICI Akers Laboratory in the UK initiated their own investigations. The British isolated “gibberellic acid” (Borrow et a / . , 1955) and the Americans “gibberellin X” (Stodola et ul., 1955). The compounds proved to be identical and the structure of gibberellic acid, or GA3 as it is now known, was fully elucidated by Cross et a / . (1959) (see Fig. I). The quantity of GA produced as a metabolic by-product by G. fujikuroi exceeds that found in higher plants by several orders of magnitude. Even so, growth promoting activity similar to that of GA was found to be present in higher plant tissues by Radley (1956) and Phinney et al. (1957) and the first characterization of a GA from a flowering plant was reported by MacMillan and Suter (1958) who isolated GA1 from immature seed of the scarlet runner bean, Phaseolus coccineus L. With the development of both improved purification procedures and analytical techniques, subsequent progress has been rapid and 62 GAS have now been characterized (Fig. 1). Nine GAShave been found only in Gibberella fujikuroi cultures, 38 are exclusive to higher plants while 15 are ubiquitous, having been detected in extracts from the fungus and higher plant tissues. Many more potential permutations of the GA structure exist and there will undoubtedly continue to be additions to this list for some time to come. So as to avoid confusion, the trivial nomenclature GA1-GA62 has been adopted and there will be a sequential allocation of numbers GA,,-GA, to any new naturally occurring, fully identified GA (MacMillan and Takahashi, 1968). Familiarity with the various GA structures is not such a daunting task as first impressions of Fig. 1 might suggest. All the GASpossess an ent-gibberellane skeleton and can be divided into two groups by virtue of the possession of either 19 or 20 carbon atoms (Fig. 2). The C19-GAs have lost carbon-20 and all but one possess a 19- 10 y-lactone bridge, the exception being GA, which has a 19+2 linkage. The C20-GAsare characterized by the presence of carbon-20 which can exist as either a CH3, C H 2 0 H , CHO or COOH

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--OH H

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r

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COOH

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Fig. I . Structures of GA,-GA6,. F, endogenous component of Gihherellafuji~uroi;H, higher plant GA

CHZ

40

ALAN CROZIER

20

enl-gibberellane skeleton

CH3

u

COOH C - 2 0 methyl C ~ O - G A

O----CHOH

CHO

7

CH3

‘1 ‘\

H

H

,

CH3

COOH COOH

C-20aldehydic C ~ O - G A

m

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6-lactor CpO-GA

COOH

CH3

\

‘,

COOH

CH2

COOH C - 2 0 carboxylic C m G A

H CH3

COOH

C*Z

y - l o c t o n i c C19-GA

Fig. 2. Ent-gibberellane skeleton and basic structures of CI9-and C?o-GAs.

function. The 20-CH20H group forms a 19+20 d-lactone bridge and the 20-CHO function appears to exist, in solution, in equilibrium with a 19+20 Glactol ring (Harrison et al., 1968). The variations in the oxidation state at C-20 and the presence or absence of 38- and 13a-hydroxyl groups account for 20 of the GAS(Table I). The remaining GASare represented by additional

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

41

TABLE 1 GA structures bused on variations in the oxidation state at C-20 und the presence or absence of hydroxyl groups at C-3 and C-13

H ydroxylation Oxidation at C-20

None

38

13a

38, 13a

modifications to these basic configurations in the form of 2,3 and 1,lO epoxide groups, C-3 and C-12 keto groups, 8-hydroxylation at C-1, C-2, C-12 and C-15, a-hydroxylations at C-1 and C-2, C-12 and C-16, oxidation of the 18-methyl group to carbonyl and carboxyl functions and the introduction of 1,2 and 2,3 double bonds. GA-glucose conjugates have also been found in higher plants. The glucose is present in the pyranose form and the 0(3)B-~-glucosylether of G A I and GA3, the 0(2)~-D-glucosylether of GAB,GA26,GA2, and GA29the 0(11)/?-~glucosyl ether of GA35 and the 0(7)B-~-glucosylester of G A T ,GA4, GAS, GA9, GA3,, GA38and GA4, have all been characterized. The n-propyl ester of GA3 has been identified in cucumber seed extracts while O(3)P-acetyl GA, and O(3)B-acetylGA3 are the only conjugated GASto have been isolated from Gibberella fujikuroi cultures. In addition a sulphur containing derivative of GA3, called gibberethione, has been isolated from seed of Pharbitis nil (Yokota et al., 1974, 1976). The application of combined gas chromatography-mass spectrometry (GC-MS) to the analysis ofendogenous GAS(Binks et al., 1969; MacMillan, 1972) is the main reason why GAS have now been identified in more than 26 species of higher plants (see Graebe and Ropers, 1978). Nine GAShave been identified in extracts from immature seed of Calonyction gladiata and 13 in Phaseolus coccineus. Immature seed has proved to be a rich source of GAS and can contain up to 100 mg kg- fresh weight. It is therefore not surprising that the vast majority of GAs identified in higher plants have originated from seed material. The GASpresent in immature seed seemingly can become conjugated as the seed develops and seeds have also been the source of almost all the conjugated GAS that have been identified to date. It is debatable whether or not the high concentrations of GAS found in immature seed have a hormonal function. The little evidence that is available is equivocal and has been obtained from experiments utilizing either 2isopropyl -4 - (trimethylammonium chloride) - 5 - methylphenyl- 1- piperidine carboxylase methyl chloride (AMO-1618) or B-chloroethyltrimethylam-

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42

ALAN CROZIER

monium chloride (CCC). Both these compounds inhibit GA biosynthesis in Gibberellu fujikuroi cultures and cell-free systems from higher plants by preventing the conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate (Robinson and West, 1970b; Shechter and West, 1969) but when applied to seedlings inhibition of growth is often mediated by other less specific effects (see Crozier et a/., 1973; Graebe and Ropers, 1978). Baldev rt ul. (1965) have shown that treatment of cultured immature Pisum sativum seed with 5 mg AMO-1618 1- results in a 60% fall in GA levels while the rate of growth is unchanged. This implies that at least a sizable portion of the GA pool is not involved in seed development. Application of CCC to immature seed of Phurbitis nil also produces depleted GA levels without adversely affecting seed growth (Zeevaart, 1966). When mature, the CCC-treated seeds were viable and germinated giving rise to dwarf seedlings with a reduced GA content. However, no conclusions can be drawn as to whether or not these symptoms are a consequence of lowered GA levels during seed maturation as the mature seed and germinating seedlings contained residual CCC which would inhibit the rate of stem elongation. It has been suggested that GAS produced during seed development are stored in the mature seed as GA conjugates and during the early stages of germination these biologically inactive conjugates are hydrolysed to release free GASwhich enhance the rate of stem elongation (see Lang, 1970). There is however no conclusive evidence for such a role and the available data imply that it is an unlikely proposition as hydrolysis of many GA conjugates yields 2P-hydroxy GAS which exhibit relatively little biological activity (see Section IV). Quantities of GA in tissues other than seed are rarely higher than 5@100 ,ug kg- fresh weight and as a consequence there are relatively few reports of GASbeing identified in extracts from such material (Table 11). Although they have been isolated from conifers (see Pharis and Kuo, 1977), there are only two examples of GAS being characterized in lower plants other than Gibberellufujikuroi. Yamane er nl. (1979) detected GA9 methyl ester in extracts from prothalli of the fern Lygodiumjaponicum while Rademacher and Graebe (1979) and Graebe et a / . (1980) have identified GA4 and small amounts of GAg, GA13,GA14 and GAZ4in culture media of Sphaceloma manihoticola, a pathogenic fungus that is a member of the Melanconiales and causes “superelongation disease” of cassava (Munihot esculentu). In contrast to this dearth of information there are many hundreds of reports of bioassays being used to detect GA-like activity in all types of higher plant tissues and organs as well as the occasional moss, fern, alga, fungus and bacterium. The data have been used to implicate endogenous GAS in many varied aspects of plant growth and development. While the use of bioassays in such circumstances is understandable, in view of their simplicity and the lack of readily available alternative methodology, it is none the less unfortunate as it is becoming increasingly apparent that bioassays are an unreliable analytical tool

TABLE I1 Identification of higher plant GAs from tissues other than seed niaterial

Gibberellin

Species

Tissue

Water sprouts Shoot apices and flower buds Shoot apices Phyllostachys edulis Althea rosea Shoot apices Seedlings Phaseolus roccineus Bryophyllum daigremontianum Shoots Sonneratia apetala Leaves Rhizophera mucranata Leaves Pinus attenuata Pollen Shoots Pinus attenuata Citrus reticulata Nicotiana tabacum

GA20>

GA29

Picea sitchensis Cupressus arizonica Juniperus scopulorum Pseudotsuga menziesii O r p a sativa Humulus lupulus Stevia rebaudiana Spinacea oleracea Ribes nigrum

Needles Shoots Shoots Shoots Seedlings Shoots Shoots Shoots Shoot apices

Pisum sativuni

Seedlings

Reference Kawarada and Sumiki (1959) Sembdner and Schrieber (1965) Murofushi er a / . (1966) Harada and Nitsch (1967) Bowen et a / . ( 1 973) Gaskin et al. (1973) Ganguly and Sircar (1974) Ganguly and Sircar (1974) Kamienska et a / . (1976) Crozier, Morris and Bell (unpublished data) Lorenzi et a / . (1976, 1977) See Pharis and Kuo (1977) See Pharis and Kuo (1977) See Pharis and Kuo (1977) Kurogochi et a / . (1978) Watanabe er a/. (1978) Alves and Ruddat (1979) Metzger and Zeevaart (1980) Crozier, MacMillan and Schwabe (unpublished data) Sponsel and Albone (unpublished data)

44

ALAN CROZIER

with which to monitor qualitative and quantitative changes in GA levels (Reeve and Crozier, 1975, 1980; Graebe and Ropers, 1978; Letham et d., 1978). Acceptance of this view implies that much of the available information on topics such as sites of GA biosynthesis and cellular compartmentation of GASin seedling tissues and the involvement of endogenous GAS in developmental processes such as dormancy, photoperiodism, leaf expansion, and dwarfism, may be based on something other than a firm experimental foundation, and so requires critical re-investigation using more definitive methods of analysis. Clearly, this is a contentious issue and the following sections will discuss the special problems associated with the analysis of trace quantities of endogenous GASand outline various approaches that can be used to overcome them. 11. ANALYTICAL METHODS A . GENERAL OBSERVATIONS

Theoretical concepts associated with the identification of hormones in plant extracts have been proposed by Reeve and Crozier (1980) who point out that to fully understand the nature of the problems encountered in practice it is necessary to take a general view of analytical theory. It is important to realize that the distinction made between “qualitative” and “quantitative” analysis is a semantic convenience rather than a logical reality. Because it is impossible to quantify an unknown in meaningful terms, quantitative analysis is in fact inherently qualitative. The converse also applies, since the statement that “X is C A I ” implies that ALL of sample X has ALL the properties associated with the chemical concept of C A I . Reeve and Crozier ( 1980) further argue that quantitative analysis displays all the enigmas of scientific induction. The identification and quantification of a substance can never be absolute, and thus must be considered in association with a complex probability term which defines the chances of making an error when concluding that Y = x p g of compound Z . Two types of error, namely precision and accuracy, independently contribute to the complex probability term. Precision is a measure of random errors that determine run-to-run variability. Thus when given a series of estimates of the same sample it is possible to use statistical methods to calculate the standard deviation (SD) of the data and, with a minimum of assumptions, state that the probability of the precision being no worse than k2SD is 0.95. An averaging process can be applied to enhance the precision of an analysis in proportion to the square root of the number of estimates averaged. Accuracy, however, refers to the non-random or systematic error of the analysis and its error and confidence limits are inordinately more difficult to ascertain. Understanding the distinction between accuracy and precision is critical as it is essential to realize that, regardless of the number of estimates con-

45

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

tributing to the final average, there is no guarantee that accurate results will be obtained as non-random error will apply the same bias to each estimate and so be present undiminished in the averaged result. A “target” analogy such as that illustrated in Fig. 3 is a useful means of demonstrating the total independence of the terms accuracy and precision. It is evident from Fig. 3 that a rifle must not only be aimed accurately but must also be designed so as to closely group its shots, i.e. it must be precise if there is to be a high probability of hitting the “bull’s eye”. In the case of plant hormones it is a common mistake to assume that an analysis is accurate because it offers adequate precision. It is apparent from Fig. 3 that the degree of repeatability provides no such assurance of accuracy. In practice, verification of accuracy is the single most crucial, yet neglected, factor in the analysis of plant hormones. Reeve and Crozier (1980) have proposed that in a general context verification of accuracy consists of (a) defining, in terms of probability limits, the complexity of the analytical problem, and (b) relating this to the amount of pertinent information obtained during an analysis. At present, practical solutions to this problem require making a number of less than ideal assumptions. Even so figures for accuracy obtained by such methods will be more reliable than those ascertained by conventional procedures where the criteria for verification can range from the whims of technical fashion to standards of intuition that vary enormously from one investigator to another. In order to put a complex situation into perspective it is necessary to review the practical procedures employed in GA analysis before discussing them in the context of the proposals of Reeve and Crozier (1980) for verification of accuracy. At a practical level the analysis of endogenous GAScan be divided into the

Inoccurote and imprecise

lnoccurote and precise

Accurate ond imprecise

Accurate and precise

Error

Error

Error

Error

Fig. 3. Target analogy demonstrating the independence of the error terms accuracy and precision.

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ALAN CROZIER

following sequential steps : (i) extraction and partitioning, (ii) group purification procedures, (iii) separation and (iv) identification. GAS are comparatively major components in extracts from Gibberrllri ,fiijikur.ai so little purification is necessary before identification is attempted. In contrast, they are minor trace constituents in extracts from higher plants and substantial purification is essential before attempts are made at characterization. This problem is especially severe with vegetative tissues because the GA levels are several orders of magnitude lower than those encountered in developing seeds. The chances of inaccurate analysis are substantially enhanced in such circumstances as the possibility of mistaking an impurity for a G A are increased. Sample losses invariably occur during purification and this also adversely affects the accuracy of quantitative estimates. Such errors can be corrected through the use of an appropriate internal marker which is added to every sample at the extraction stage. The most suitable internal markers are isotopically labelled analogs of the particular GA under study. ['HI, [3H]and [I4C]GAs tend to behave in the same manner as their endogenous counterparts yet can be differentiated by mass spectrometry or radioassay. When the GA under investigation is not available in an appropriately labelled form the best alternative internal standard is a GA of similar structure. However, recourse to such a procedure increases the possibility of some degree of separation occurring between the standard and the endogenous GA before the ultimate analytical step, thereby degrading accuracy. B. EXTRACTION AND PARTITIONING TECHNIQUES

Over the years an array of GA extraction and partitioning procedures have been used and this must cause confusion to many budding gibberellinologists. Tissues are usually extracted with methanol or ethanol, although aqueous buffers have also been tried. In [3H]GA metabolism studies methanol removes >95% of the radioactivity in Phn.seo/u,sseedlings (Reeve and Crozier, 1978). It is, however, an open question as to whether or not endogenous GAS are removed from all cellular sites with equal efficiency. Browning and Saunders (1977) reported that extraction of isolated chloroplasts of Tviticurii ciestivum with the detergent Triton-X yielded far higher levels of GA4 and GAg than methanol extracts. Unfortunately, similar results have not been obtained when the experiment has been carried out in other laboratories (Railton and Rechov, 1979). Buffer extracts from pea seedlings contain fewer impurities and more GA-like activity than methanolic extracts (Jones, 1968). However, it does not necessarily follow that buffer is the more effective in removing GAS from plant tissues as the bioassay data could just as well reflect reduced inhibitor concentrations as increased GA levels. When methanol and buffer extracts are subjected to several purification steps prior to bioassay, the methanolic extract yields higher levels of G A-like activity (Reid and Crozier, 1970).

47

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

Macerate tissue and extract 3 times with an excess of cold methanol. Combine methanolic extracts and reduce to the aqueous phase in i~ucuo.Add at least an equivalent volume of pH 8.0,0.5 M phosphate buffer and if necessary adjust extract to pH 8.0

+

Partition at least five times against volumes of toluene Toluene

Aqueous phase Slurry with PVP and polyamide (50 mg ml-') filter

I

I PVP and polyamide

Aqueous phase Adjust to pH 2.5 and partition against 5 x + volumes of ethyl acetate

I

1 Acidic, ethyl acetate-soluble fraction

Aqueous phase Partition against 3 x f volumes of n- butanol

(Free G A S , and unknown amounts of GA conjugutes)

I

Acidic butanol-soluble fraction

I

Aqueous phase

Fig. 4. Flow diagram of extraction and partitioning techniques.

The procedures that are currently in routine use in my own laboratory are shown in Fig. 4. Tissue is macerated and extracted three times with an excess of cold methanol. The combined methanolic extracts are reduced to the aqueous phase in vaciio and the aqueous residue diluted at least twofold with pH 8.0,0.5 M phosphate buffer.This stabilizes the pH and ensures a minimum ionic strength during the ensuing partition procedures. At pH 8.0 the

48

ALAN CROZIER

aqueous phase is sufficiently basic to retain even the less polar GAS when partitioning against toluene yet not so basic as to risk isomerization. Petroleum spirit could be used instead of toluene but our experience is that the aromatic solvent has a higher solubilizing power for compounds with a large number of conjugated double bonds (i.e. pigments) and it therefore removes more impurities. Furthermore, emulsion problems are less likely to arise when toluene is used. Many investigators partition the aqueous phase against ethyl acetate or diethyl ether although the GA partition coefficient data of Durley and Pharis (1972) clearly show that this results in the removal of significant quantities of non-polar GAS. After partitioning at pH 8.0 the buffer phase can be further purified by slurrying with insoluble polyvinylpyrrolidone (PVP) (Glenn et a / . , 1972) and polyamide before acidification to pH 2.5 and extraction with 5 x 2/5 volumes of ethyl acetate. At this pH the partition coefficients are such that the bulk of the free GAS are removed by the ethyl acetate. The tetrahydroxy compound GA32is the only known free GA that will be retained by the buffer to any extent (Yamaguchi et a/., 1970). Metabolism experiments with [3H]GAs indicate that certain GA conjugates also migrate into the ethyl acetate. It is difficult to assess what proportion of the conjugated GAS this represents as little is known at present about their partitioning behaviour . It is however possible to extract conjugates from the acidified aqueous phase with n-butanol. The GA moiety of GA conjugates is best released by enzymatic hydrolysis and the efficiencies of various enzymes has been investigated by Knofel et a / . (1974). C . GROUP PURIFICATION PROCEDURES

The concentration of GAS in the acidic, ethyl acetate-soluble fraction is usually very low so a multistep analytical procedure has to be employed in order to attain a degree of purity that facilitates an accurate determination of GA content. Both the GA levels and the nature and amounts of contaminants vary greatly from one tissue to another so that the exact combination of procedures to be used is best determined by an on the spot assessment rather than the application of “cookbook style recipes”. When deciding what particular techniques to utilize some general points should be borne in mind. In the initial stages of purification, the substantial sample weights encountered dictate the use of chromatographic techniques with a high sample capacity. It is also advisable at this stage to use procedures which separate the GAS as a group from other components in the extract, otherwise unwieldy numbers of sub-fractions are quickly generated and there will be a marked decrease in the overall speed of analysis. Finally, purification is most effectively achieved if the individual techniques display widely different separatory mechanisms. Gel permeation or steric exclusion chromatography (SEC) has proved useful as a preparative, group separatory procedure (Reeve and Crozier,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

49

1976, 1978). The system consists of two 25 x 1000 mm columns connected in series, packed with Biobeads SX-4 and eluted with tetrahydrofuran (THF) at a flow rate of 2 ml min- '. This is the maximum flow rate the support can tolerate without excessive compression of the bed. The gel has an operating range of (k1500 M W and solutes elute in decreasing order of molecular size. The sample capacity is high and is readily realized because of the excellent solubilizing power of THF, 1.5 ml of which will readily dissolve up to 1.0 g of an acidic, ethyl acetate extract; recoveries, estimated with a range of [3H]GAs, are >90%. The absence of adsorption effects ensures that even with the most impure extracts all solutes will be eluted by a volume of solvent (630 ml) which corresponds to the total volume of the column (V,). This allows the system to be used repeatedly without fear of sample overlap. 13H]GA9 and [3H]GA43,which represent the extremes of the molecular weight range of the free GAS, have respective retention volumes (V,) of 550 ml and 470 ml in this system with peak widths (w) of 40 ml. Endogenous GAS in extracts can therefore be purified by collecting the 45Cb.570 ml zone. While this technique is well suited for large-scale extracts a high performance SEC procedure has recently become available which offers a very rapid speed of analysis for the purification of smaller sized samples (Crozier et a / . , 1980). It involves the use of a p-Spherogel support" with a nominal exclusion limit of < 2000 MW. p-Spherogel is a macroporous cross-linked polystyrene divinylbenzene copolymer support that has been specifically designed for high performance liquid chromatography (HPLC) (Krishen, 1977). An 8 x 300 mm column eluted with 0.5% acetic acid in T H F generates 9000 theoretical plates and has a sample capacity of > 100 mg. The exclusion or void volume (V,) is 5.5 ml and V , is 9.5 ml. The V R of [3H]GA43is 7.0 ml and that of [3H]GA9,7.6 ml. In both instances w=0.4 ml. Thus collection of the 6.8-7.8 ml zone provides a very simple means of separating the free GAS as a group from the many extraneous components typically present in plant extracts. The speed of analysis is greatly enhanced as at a flow rate of 1 ml min- samples can be analysed every 9.5 min. The salient features of the two SEC systems are summarized in Table 111. Grabner et a / .(1976) used DEAE Sephadex A25 anion exchange chromatography to separate abscisic acid (ABA), GA3, GA, and ~(3)/3-D-glucosyl ether of GA,. This procedure is readily adapted for use as a group separatory procedure for free GAS. A 25 x 150 mm column of DEAE Sephadex A25 charged in the acetate form is eluted with four void volumes (600 ml) of methanolic 0.1 M acetic acid to remove neutral and weakly acidic impurities. The GAS are then eluted with c. 90% efficiency with two void volumes of ~ acid. The technique has been used to reduce the methanolic 1 . 0 acetic weight of a Pinus attenuata shoot extract from 550 mg to 45 mg and similar

'

* Altex Scientific Inc., Berkeley, California.

50

Perfbrniuiic~choructrristics

ALAN CROZIER

TABLE 111

of' SEC using Bioheads SX-4 and p-Sphrrogd supporis" Biobeads SX-4

p-Spherogel

-.

Nominal exclusion limit Column dimensions Solvent Flow rate Sample capacity

v,,

v/.

N N,, H Speed of analysis Elution zone of G A S Analysis time "

1500 amu Two 25 x I000 mm THF 2 mI min 1 gm 350 ml 630 ml 3600 650 0.55 mm 0. I9 plates s - I 450-570 ml 320 min

-'

< 2000 amu 8 x 300 mm 0.57" acetic acid in THF I ml mm > 100 mg 5.5 mi 9.5 ml 9000 1600 0.03 mm 15.8 plates s 6.8-7.8 ml 9.5 mill

N-efficiency in theoretical plates. NeJ, efficiency in effective plates, H-plate height.

reductions in sample size have been achieved with other tissues (Crozier and Bell. unpublished data). As long ago as 1939 purification of GAS was achieved by exploiting the unusual reverse phase effects of charcoal adsorption chromatography (Yabuta and Hayashi. 1939). As currently employed the sample is dissolved in 1-2 ml of 203:, aqueous acetone and applied to the top of a 20 x 120 mm charcoal-celite ( 1 :2) column. Weakly adsorbed impurities are eluted with 100 ml of 207; aqueous acetone which is equivalent to four column volumes. The GAS are then removed with 200 ml of acetone. The sample capacity of charcoal is high and a column of the dimensions described can accommodate extracts weighing up to 500 mg (Reeve and Crozier, 1978). The method is also readily adaptable for use as a simple slurry procedure in which case large numbers of extracts can be treated in a matter of minutes. The recovery of GAS from charcoal is usually 75-85"/,. However, inexplicably high losses do occur from time to time, even with the same batch of charcoal, and as a consequence the procedures should only be used when replicate samples are readily available. Other group separatory procedures have been used for the purification of GAS. Although PVP adsorption chromatography can significantly reduce the dry weight of an extract (Glenn et [)I., 1972), very low column efficiencies and long analysis periods are associated with this technique. Furthermore, it involves the use of aqueous solvents which is undesirable because of the risk of GA rearrangements (Pryce, 1973). It is therefore safer, easier and almost as effective to make use of the PVP slurry treatment at the partitioning stage,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

51

as shown in Fig. 4. A Sephadex G-I0 column eluted with 0.1 M, pH 8.0 phosphate buffer will retain GAS by virtue of uncharacterized adsorption phenomena and can be of value as a purification tool (Crozier rf o/., 1969). However as this procedure also involves exposure of the extract to mildly alkaline conditions for several hours. with attendant risks of degradation, it should be avoided if the required degree of purity can be achieved without recourse to a hydrolytic environment. Counter-current distribution has been used as a preliminary purification procedure for GAS (Crozier rt ( I / . , 1969) but it is now somewhat outmoded and usually does little that cannot be more effectively achieved with other techniques. 1). SEPARATORY TECHNIQUES

In most instances extracts which have been subjected to a range of group purification procedures will still require further purification before successful attempts can be made at GA analysis. This is achieved through the use of analytical methods which separate the GAS to some degree. Originally paper chromatography (PC) was the method of choice (see Phinney and West, 1961) but it was superseded by thin layer chromatography (TLC) (MacMillan and Suter. 1963; Kagawa c’t ( I / . , 1963) which is still widely used especially in conjunction with bioassays. However liquid-liquid partition column chromatography systems offering a high peak capacity (Giddings, 1967) have the ability to simultaneously resolve a large number of components and as a consequence provide much better separations than TLC. Several such systems have been used with GAS although on occasions some extraordinary but unrepeatable separations have been claimed. In general, good separations have been obtained with techniques utilizing either silica gel or dextran gel supports. Adequate results can be achieved, without recourse to expensive instrumentation. with a silica gel partition column (Powell and Tautvydas, 1967), originally developed to analyse indole-3-acetic acid ( I A A ) and other indoles (Powell, 1963). The system involves partitioning a O-lOO~:( gradient of ethyl acetate in hexane against a 400,; ( v / w )0.5 M formic acid stationary phase on a Mallinckrodt CC-4 silica gel support. Separation is primarily determined by the degree of hydroxylation : G A S with no free hydroxyl groups elute at an early point in the gradient and in turn are followed by the mono-, di- and tri-hydroxy G A S as the solvent strength increases. The elution pattern of 12 GAS is presented in Table IV. Durley rt a / . (1972) have reported that the technique works well only with certain batches of silica gel as columns tend to temporarily “dry out” at an early point in the gradient. Because of this they developed an alternative procedure using a Woelm silica gel support with a 15% water stationary phase. While this technique provides better results than TLC, it does not perform as well as the Powell and Tautvydas column. This is probably due to the mixed nature of the separatory mechan-

52

ALAN CROZIER

TABLE IV Retention characteristics of GAS on a straight phase silica gel partition column (Durley et al., 1972)" Fraction number

Gibberellin

2 4 5 6 8

11 13-14 18 "Column: 13 x 200 mm Mallinckrodt CC-4 silica gel; stationary phase: 40%. 0-5 M formic acid; mobile phase: 160 min gradient, @loo% hexane in ethyl acetate; flow rate: c. 3 ml min -' ; sample: G A S as indicated; detector: 25 successive 20 ml fractions collected and G A content determined by gas chromatography.

ism which varies during the course of a gradient from silica gel adsorption to partitioning against a stationary phase that changes from water to 0.5 M formic acid. The "drying out" phenomenon experienced with the PowellTautvydas system is, in fact, due to out-gassing of the solvents and is easily overcome by degassing the hexane and ethyl acetate under reduced pressure, immediately prior to use. Re-absorption of atmospheric oxygen by the ethyl acetate can be suppressed by entraining a stream of nitrogen over the solvent reservoir (Reeve et al., 1976). When these precautions are taken the procedures of Powell and Tautvydas (1967) are reproducible and batch-tobatch variations in Mallinckrodt silica gel are not apparent. There is in fact nothing especially magical about Mallinckrodt CC-4 silica gel. Other silica gels work equally well and in certain instances their performance is far superior. In the early 1970s advances took place in liquid chromatography technology, especially the development of efficient microparticulate silica gel supports (see Majors and MacDonald, 1973). This facilitated vast improvements in the performance of the silica gel partition system, which is especially well suited for the separation of GAS in plant extracts, as a 10 x 450 mm column can accommodate multicomponent samples weighing up to 100 mg. The high sample capacity is a direct consequence of the high (40%)stationary phase loading. The relatively high miscibility of the ethyl acetate mobile phase and formic acid stationary phase does, however, present special problems that must be overcome if high column efficiency is to be maintained. This can be achieved through the use of a stationary phase trap in the solvent delivery line and a precolumn, which, along with the analytical column, is held at 30f0-05"C to ensure equilibration of the incoming mobile phase with the stationary phase (Reeve et al., 1976; Reeve and Crozier, 1978).

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

53

Reeve et a / . (1976) assessed the performance of various types of silica gel in this system by chromatographing UV absorbing phenol under various conditions and calculating column efficiencies by established procedures (see Karger, 1971). Three silica gel supports were used : (i) Mallinckrodt CC-4- irregularly shaped particles with a wide size range (approximately 60-250 pm). The support used by Powell and Tautvydas (1967). (ii) Merckogel SI 200-spherical, I 40-63 pm particles with a 200 A pore diameter. (iii) Partisil 20- irregularly shaped 20 pm particles of closely controlled size distribution with 55-60 8, pore diameters. The relationship between plate height, H , and linear solvent velocity, v, for columns packed with these gels is presented in Fig. 5. In each instance the concentration of ethyl acetate in the mobile phase was adjusted so that phenol eluted with a capacity factor, k', of 1.5. The performance of both the Mallinckrodt CC-4 and Merckogel SI 200 falls off at increased flow rates, the effect being much more marked with CC-4. Much better H values were obtained with Partisil 20 and no significant fall off was evident at higher solvent velocities. From the practical viewpoint Partisil 20 is clearly the superior support as it can generate high efficiencies without sacrificing the speed of analysis. Subsequently, 5 and 10 pm silica gel supports have become Mallinckrodt C C - 4

-2

1.0

E E

Merckogel

SI 200

0.5-

I-

o.oJ,

0

Partisil 20

1

2

I

3

1

4

1

5

v ( m m s-') Fig. 5. The relationship of plate height ( H )to linear solvent velocity (v). Column: 10 x 450 mm packed with Mallinckrodt CC-4 Silicar (triangles), Merckogel SI 200 (circles) and Partisil 20 (squares). Sample: phenol. Stationary phase: 40%, 0.5 M formic acid. Mobile phase: hexaneethylacetate, ratioadjusted togivea k'of 1.5 forphenol. Detector:absorbancemonitorat (Reeve et al., 1976.)

54

ALAN CROZIER

commercially available and they enhance performance even further although operating pressures are higher. A 10 x 450 mm column packed with Partisil 10 and eluted at a flow rate of 5 ml min- ',which corresponds to v = 1.5 ml s- ', generates up to 3800 theoretical plates for a solute with a k' of 1.2. Thus plate height and speed of analysis can be calculated at 0.12 mm and 1.1 effective plates per second. Depending upon solvent composition, a column inlet pressure of 14C200 p.s.i. is required. Recovery from the column is 95% for a wide range of compounds. These performance figures represent a considerable improvement in both efficiency and speed of analysis, when compared with classical liquid chromatography techniques used for the separation of GAS. The system is some ten times faster and twenty times more efficient than the silica gel partition column of Powell and Tautvydas (1967) from which it was derived. The transition from a silica gel partition to a high performance system necessitates the use of more elaborate instrumentation. The preparative HPLC that is used is illustrated in Fig. 6, along with an on-stream homogeneous radioactivity monitor which is employed in GA metabolism studies to detect [jH] and [14C] solutes eluting from the column. A dual pump gradient generator delivers mixtures of hexane and ethyl acetate saturated with 0.5 M formic acid to the analytical column via a pulse dampening network, a stationary phase trap and a precolumn. Samples are dissolved in the mobile phase and introduced into the analytical column via a six-port sample valve. Solvent emerging from the column is directed to a UV monitor before entering a stream splitter which subtracts a pre-set portion of the column eluant and restores the original flow rate with a make-up solvent of ethyl acetate :toluene (1 :1). After the addition of scintillation cocktail supplied from a metering pump the mixture is cooled to - 5°C and passed through a spiral glass flow cell positioned between the photomultiplier tubes of a manual scintillation counter. The output is processed by a spectrometer/ ratemeter and displayed along with the UV-absorbance trace on a dual channel recorder. The inclusion of a radioactivity monitor in the system requires a suitable compromise be made between chromatographic resolution and speed of analysis and detector sensitivity (Reeve and Crozier, 1977).This is achieved by selecting an appropriate scintillant :eluant ratio, matching the flow cell volume and geometry with the minimum chromatographic peak width and adjusting the overall flow rate to give an optimum value for flow cell transit time. When these parameters are optimized the monitor has a relative sensitivity of 3 x lo3 d.p.m. for and 1 x lo3 d.p.m. for I4C for a solute eluting with a k' of 1.7. The monitor does not contribute to the total bandspreading of the chromatograph for solutes where k' > 1.7. By manipulation of the hexane-ethyl acetate ratio a wide range of mobile phase solvent strengths can be used to provide rapid and effective separations. Figure 7 illustrates the use of a gradient designed for the analysis of samples

+ Stationary

i

Pulse dampener

Gradient generator and pumps

Splitter control unit Constant temperature circulotor (30°C)

73$

5

-

1

Make-up solvent reservoir

V 0

0

0

a

0

-

'u

Pump

c

) .

a

I

I

Solvent reservoirs

Sample volve

I I L - -- - - - -

Two-pen recorder

I I I

Hold-up coil

€3

-

Scintiliont reservoir

pump

Splitter

r

UV cell

p P

Constant temperature

Q

Collect

Flow cell in scintillation Counter

UV monitor

.--U Spectrometer / ratemeter

____

Fig. 6. Preparative HPLC with UV and radioactivity monitors. (Reeve and Crozier, 1978.)

56

ALAN CROZIER E 0

h

r

0

~

1 1-

1

60

120

Retention time ( min)

Fig. 7. Preparative HPLC of radioactive GAS and GA precursors with UV-absorbing internal markers. Column: 10 x 450 mm Partisil 20. Stationary phase: 40%. 0.5 M formic acid. Mobile phase: 2 h gradient O-lOO% ethyl acetate in hexane. Flow rate: 5 ml min-'; sample: c. 24,000 d.p.m. ["C] ent-kaurene, 50,000 d.p.m. [14C]GA3, [3H]GA5, ['4C]GA12. [14C]GA15 and [3H]GAZo;100,000 d.p.m. [3H]enr-kaurenoic acid, [3H]GA1, [3H]GA4, [3H]GAs, ['H]GA9, [3H]GA1zaldehydeand t3H]GA14, and uncalibrated amounts of gibberic acid, allogibberic acid and gibberellenic acid. Detectors : radioactivity monitor I800 c.p.m. full-scale deflection, absorbance monitor at AZs4(Reeve et a / . , 1976).

whose components span a wide range of polarities. The GAS and GA precursors separate according to the degree of hydroxylation. Compounds with no hydroxyl groups such as ent-kaurene, ent-kaurenoic acid, GA, aldehyde, GA9, GA15and GA12elute first, followed by the monohydroxylated GAS (GAL,GAI4,GAS and GA,,), the dihydroxylated compounds GAl and GA3,and finally GA, which has three hydroxyl groups. When wide-range gradients of this type are employed UV-absorbing markers such as gibberic, allogibberic and gibberellic acid can be incorporated into the sample to allow precise determination of the relative retentions of radioactive peaks. In metabolic studies, metabolism of the applied G A often involves successive hydroxylations and the products are usually chromatographically distinct from each other and from the precursor molecule. In such cases a considerable saving can be made in the analysis time because good separations are obtainable without the need for high effective k' values. This is shown in

,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

0

Retention time (rnin)

57

3'0

Fig. 8. Preparative HPLC of G A , , GA4 and GABusing a restricted solvent gradient designed for rapid analysis. Column: 10 x 450 mm Partisil 20. Stationary phase, 40%, 0.5 M formic acid. Mobile phase: 20 min gradient 80-100% ethyl acetate in hexane. Flow rate: 5 ml min-'; sample: c. 20,000 d.p m. [3H]GA,, ['HIGA, and [3H]GA8. Detector: radioactivity monitor, 600 c.p.m. full-scale deflection. (Reeve er at., 1976.)

Fig. 8, where the solvent programme has been adjusted to allow the repeated separation of GA4, GAI and GA8 at 30-min intervals. Silica gel supports with a chemically bonded stationary phase are now available (see Locke, 1973). Columns packed with this type of support are extremely stable and present fewer problems to the inexperienced chromatographer than the preparative HPLC system designed by Reeve et al. (1976). However, the stationary phase content of these supports is rarely more than 15% and is usually only 5% (Majors, 1975; Cooke and Olsen, 1979). As a consequence, the sample capacity is very limited when compared with a silica gel support carrying a 40% stationary phase loading. Reverse phase chromatography of free GAS using a chemically bonded octadecylsilane (ODS or C18) stationary phase has been reported by Jones et al. (1980). Four 6.5 x 600 mm columns connected in series, packed with Bondapak C18 (Waters Associates) and referred to as a preparative system, were eluted with a 25-min, 3&100% gradient of methanol in 1% aqueous acetic acid at a flow rate of 9.9 ml min-'. Thirty fractions were collected and the location of GA standards determined by gas chromatography (GC). The separations obtained are presented in Table V. An analytical system using a single 4 x 300 mm p-Bondapak c18 column (Waters Associates) gave similar results. The procedures were used to separate endogenous G A Sin an extract from immature seed of Pharbitis nil. Zones of biological activity from the

58

ALAN CROZIER

TABLE V Reverse phase chrornatogruphy of GAS (Jones et al., 1980)" Fraction number

Gibberellin

11 12 13 15 17 18 19 21 22 23 24 28 "Column: four 6 . 5 ~ 6 0 0mm Bondapak C,,/Porasil B, columns in series; mobile phase: 25 min gradient. 30-10070 methanol in 12, aqueous acetic acid; flow rate: 9.9 ml m i n - ' ; sample: GAS as indicated; detector: 30 successive 9.9 ml fractions collected and GA content determined by gas chromatography.

preparative column were rechromatographed on the analytical column, fractions from which were bioassayed and the active components subsequently identified as GA3, GAS,GA,,, GAI9, GA20, GA29and GA44 by GC-MS. Despite the successful identification of these GAS it would be unfortunate if other investigators were to use reverse phase chromatography procedures in the manner described by Jones et al. (1980). Although figures for column efficiency are not given, from the data presented, it would seem that the preparative columns, despite their length, generate fewer than 400 theoretical plates ( H = 6 mm), while N = 1600 and H=0.19 mm for the analytical column. A system comprising either a single Whatman Magnum 9 x 500 mm ODs-2 or Shandon 8 x 250 mm ODs-Hypersil column would be much cheaper yet would provide a higher sample capacity and far superior efficiency and peak capacity than the combined efforts of the five columns used by Jones et a l . (1980). In order to fully utilize the separatory capacity of such a system at least 150 fractions must be collected for analysis by bioassay. Jones et al. (1980) collected only 30 fractions and therefore the effective resolution of their reverse phase systems is, in practice, no better than that of a silica gel-formic acid partition column (Powell and Tautvydas, 1967) which requires neither an elaborate solvent programmer nor expensive pulse-free pumps. High column efficiencies and good G A separations have been achieved with dextran gels as a stationary phase support. The procedures of Pitel et a/.

59

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

(1971a) and Vining (1971) using Sephadex G-25, separate the double bond isomers G A , / G A 3 and G A 4 / G A 7 and some of their closely related derivatives. These columns are, however, of restricted general value as they do not provide adequate resolution of other groups of G A S(Durley rt ul., 1972). In contrast methods devised by MacMillan and Wels (1974) do separate a wide range of GAS and G A precursors. A biphasic solvent of light petroleumethyl acetate-acetic acid-methanol-water (100 :80 :5 :40 :7) was prepared and the aqueous phase used to swell the Sephadex LH-20 support and act as a stationary phase. The gel was packed into a column and eluted with the organic phase. Five thousand five hundred theoretical plates were generated on a 15 x 1450 mm column and excellent G A separations were obtained (Fig. 9). The sample capacity of this column is 100-200 mg so most plant extracts can be easily accommodated. The method has the advantage that it is relatively simple and does not require expensive, complex equipment. However, the 30-h analysis period is a major problem as far as routine analyses are concerned, as it severely limits sample throughput. The speed of analysis, calculated from GA3 in Fig. 9, is only 0.05 effective plates per second. Because of a lack of gel rigidity, it is unlikely that this situation could be improved by either increased solvent velocities or solvent programming (see Bombaugh, 1971). Despite this drawback the procedure is an attractive proposition for occasional use.

0)

C

P

?

D

LL

10

20

30

40

50

60

70 80 90 Fraction number

100

110

120

130

140

150

Fig. 9. Separation of GAs by liquid-liquid partition chromatography on a Sephadex LH-20 support. Column: 15 x 1450 mm Sephadex LH-20. Stationary phase: aqueous phase of light petroleum%thyI acetate-acetic acid-methanol-water (100:80:5:40:7) mixture. Mobile phase: organic phase of above. Flow rate: 50 ml h -l. Sample: ( I ) mi-kaurene, (2) ent-kaurenoic acid, (3) GAI2aldehyde,( 4 ) GA,,alcohol, ( 5 ) ent-7a-hydroxykaurenoic acid, GA9, (6) steviol, G A I 2 , (7) GA15, (8) GA~~aldehyde, (9) GA24, (10) GA4, (1 I ) GAT,(12) GA25. (13) GAT,GA3,. (14) GAI4,(15) GAS,( 16) mevalonic acid, (17) G A 3 6 . (1 8) C A I 6 , (19) GA2,(20) C A I ,G A l 3 , GA, ,, (21) GA3 and (22) CA,, GAZ8.Detector: 150-ml fractions collected and analysed by GC with a flame ionization detector (MacMillan and Wels, 1973).

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

61

Fig. 10. The response of (a) the Tanginbozu dwarf rice leaf sheath and (b) the cucumber hypocotyl bioassays to GA3.Controls on the left, plant on the right treated with 1 pg GA3. (c) The response of the lettuce hypocotyl bioassay to GA,. Controls on the left, seedlings on the right grown in 1 pg GA, ml-'.

Whatever method of chromatography is used to separate endogenous GAS,their subsequent detection can be a time-consuming process because of the lack of a specific label. [3H]and [14C]GAsare of course an exception as they can be readily detected with a radioactivity monitor. If a known GA is being analysed the appropriate zone of the chromatogram can be collected and subjected to additional analysis to facilitate identification and quantification. When the identity of the endogenous GAS are unknown, bioassays are commonly used to detect peaks of GA-like activity which are then subjected to physicochemical procedures such as GC-MS in order to characterize the active components. Very often, however, endogenous GASare not identified in this manner and analyses go no further than bioassay with the GA content of samples being compared on this basis.

62

ALAN CROZIER

E. IDENTIFICATION PROCEDURES

I . Bioussays und Rudioinznzunoussuys Historically bioassays have played an important role in the discovery of GAS. Many GAS were originally detected in plant extracts because of their biological activity, and without such an indicator it is unlikely that the extensive purification that must precede rigorous chemical analysis could have been achieved. Indeed it is interesting to speculate how much would currently be known about GAS if rice seedlings did not elongate so markedly when infected with G . fujikuroi. Over the years numerous bioassays have been devised. Bailiss and Hill (1971) listed 33 test systems based on processes such as coleoptile, leaf sheath, epicotyl, mesocotyl and radicle growth, bud dormancy and seed germination, a-amylase synthesis, leaf expansion and senescence and flower and cone induction. GA-induced elongation of the leaf sheath of Tanginbozu dwarf rice and hypocotyls of lettuce and cucumber seedlings is illustrated in Fig. 10. Typical dose-response curves for the barley aleurone, Tanginbozu dwarf rice microdrop, lettuce, cucumber and dwarf pea bioassays are shown in Fig. 11, while the essential features of these and other widely used GA assays are reviewed in Table VI. The relative activities of the individual GAS in some of these test systems are listed in Table VII. The data are compiled from Crozier et uI. (1970), Yokota ef a / . (1971), Fukui et u / . (1972), Yamane et a / . (1973), Reeve and Crozier (1975), Hoad et a / . (1976) and Sponsel et a / . (1977). When used to detect GAS in plant extracts, bioassays are moderately selective, especially when compared with many physicochemical detectors. However no known GA bioassay is entirely free from interaction with extract impurities. Thus, regardless of the repeatability of bioassay data, the accuracy is always open to question until such time as verification is achieved by reference to a more definitive technique. The following example involves the estimation of GA levels in Phuseolus cocciiwus seedlings and illustrates the problems that severely restrict the interpretation of bioassay data. The acidic, ethyl acetate-soluble fraction obtained from a methanolic extract of light grown Phuseolus seedlings was partially purified by Sephadex G- 10and charcoal-celite column chromatography and then divided into two. One portion was subjected to TLC and the other to liquid chromatography (LC) on a silica gel partition column (Powell and Tautvydas, 1967). When a 1/60 aliquot of each chromatographic fraction was tested in the Tanginbozu dwarf rice bioassay the LC fractions induced a greater overall response and revealed more zones of biological activity than did the TLC fractions (Fig. 12). Seemingly the higher peak capacity of LC resulted in a better separation of the GAS from one another as well as from impurities. Support for this view was obtained when a 1/120 aliquot of each chromatographicfraction was assayed. The LC fractions showed the expected

u n

I”

20

Cucumber bioassay 10

o

lo+

10-l

loo

,-.

E

E

I

f av) l r

n

c 0

W

100

_I

50

o

m3

lo-’

loo

I

E E

o

10-l

pg GA3 ml-‘

o

I O - ~ 10-1

loo

pg G A T mL-’

Fig. 1 1 . Dose-response curves of the cucumber hypocotyl, Tanginbozu dwarf rice leaf sheath. Progress No. 9 dwarf pea epicotyl. barley aleurone and lettuce hypocotyl bioassays.

TABLE VI Gibberellin bioussuys Method Tanginbozu dwarf rice leaf sheath bioassay Progress No. 9 dwarf pea epicotyl bioassay Dwarf maize leaf sheath bioassay Cucumber hypocotyl bioassay Lettuce hypocotyl bioassay Barley aleurone bioassay Rumex leaf senescence bioassay

Reference Murakami (1968) Kohler and Lang (1963) Phinney (1956) Brian, Hemming and Lowe (1964) Frankland and Wareing (1960) Nichols and Paleg (1963) Jones and Varner (1967) Whyte and Luckwill (1966)

"Test compound GA4.hTest compound GA,.

Minimum detectable level of GA,

Range of linear response to GA,

TABLE VII Relative activities ofgibberellins injive bioassay systems Gibberellin

Barley aleurone

Dwarf Pea

Lettuce hypocotyl

Dwarf rice

++++ ++++ ++++ +++ ++

+++ ++ ++++ +++ +++ ++ +++ + ++

+++ ++ +++ ++ ++ ++ ++++ + +++

+++ +++ ++++ ++ +++ +++ +++

++ +++ + + + + 0 + 0 0

+

0 0 0

+ 0 +++ ++

0 0 0 0 0

+ +++ + +++ + 0 +++ ++ ++ + -

-

Relative activities: inactive.

0 0 0

0

0

0

0

+ ++ 0 ++

0

+ +++ ++ +

0

0 0

+

0 0

+++ ++ +++ + + ++ ++ ++ +++

0 0 0

++

0

0 0

+ ++ + 0 + + +++ + ++ +

0 0 0

0

0 0

+ ++ + 0 + + ++ 0 + 0

+ ++ +++ + + + + ++ + + +++ +++ +++ 0 +++ +++ +++

0 0

+ + + ++ ++ ++++ + +

++ +++ +++ +

+

Cucumber hypocotyl

++

++ ++ +++ + + ++++ 0 +++ ++ + +

0 0

++ +

0 0 0 0 0 0 0

+++

+

0

0

0 0

+ +++

0

0 0

+++ +++ +++ + ++

0

0 0

0 0

0

0

0

0

+

+

++

+ + + +, very high; + + + , high; + +, moderate; +, low; 0, very low to

66

ALAN CROZIER

TLC

LC

r

G A S standards

+10-3/1g

26

301

(b’

t

18 14

pg

+0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1

5

10

15

Fraction number

20

I I T 1

25

Rf

Fig. 12. Tanginbozu dwarf rice bioassays of eluates from a silica gel partition column (LC) and a silica gel G thin layer chromatogram (TLC) developed with ethyl acetate-chloroformformic acid (50: 50 : 1) of a semi-purified extract from red light-grown seedlings of Phaseolus coccineus cv. Prizewinner. Eluates were tested at (a) 60- and (b) 120-fold dilutions.

reduction in biological activity, whereas the TLC fractions actually displayed enhanced activity at the lower dose (Fig. 12). Such anomalous dose-response behaviour clearly indicates that the selectivity of the bioassay is insufficient to cope with the level of interfering substances. Even if it were possible to establish that the growth promotion in Fig. 12 was exclusively due to the action of G A S ,it would still be difficult to obtain a meaningful measure of the actual amount of G A present. To express the data as pg of G A 3 equivalents is misleading because the relative activities of individual G A Svary greatly (Table VII). The threshold doses differ by several orders of magnitude; there is often no parallelism between the slopes of the response curves, and, as a further complication, the size of the dose required to saturate the response is far from uniform (Reeve and Crozier, 1974). These factors must exclude the use of a biological response as the basis for the quantification of an unknown G A . Quantitative estimates of G A levels based on biological activity may however be of more value if they can be related to a specific G A . For instance, in the case of the Phaseolus extract,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

67

illustrated in Fig. 12, it has been established by GC-MS that the material contains GAl which elutes from the LC column in fraction 14 (Bowen et al., 1973). It can therefore be argued that there are grounds for expressing the biological activity in fraction 14 as ng of GA1 by reference to the regression of the response on log-dose GA1. As this involves a log-normal distribution the estimate will be a median rather than a mean value and will have asymmetric confidence limits. Table VIII contains estimates of the GA1 content of the Pkaseolus extract based on the growth response induced by 1/60 and 1/120 aliquots of LC fraction 14 in the dwarf rice bioassay. The accuracy of estimates is dependent upon the validity of the assumption that the doseresponse curve of fraction 14 exactly mirrors that of the GA1 dose-response curve. As halving the dose size had no significant effect on the GA1 estimates it would seem that the assumption is at least partially valid and that LC fraction 14 is acceptably pure. However, interference from extraneous material need not necessarily be revealed by assaying at more than one dose level. It was therefore of interest to analyse the extract in other bioassays which offered different selectivities. When LC fraction 14 was tested in the lettuce hypocotyl and barley aleurone a-amylase bioassays, the GA1 estimates obtained were much higher than those based on the dwarf rice bioassay (Table VIII). Without further investigation it is impossible to establish which figure is the more accurate, so under the circumstances the best estimate of the GAl content of the Phuseolus extract is 4-1400 ng. It should be noted that at no stage has rigorous proof of accuracy been obtained and thus there is no guarantee that the actual GA1 content of the extract lies within even this broad range. The Pkaseolus analysis cited above is by no means a “worst case” example as the extract underwent a two-step purification and LC fractions were tested in three bioassays at various dilutions. In many published instances, purification is almost non-existent and estimates of GA content are based on TLC of crude, acidic ethyl acetate-soluble extracts and a single bioassay at TABLE VIII Estimated GA1 content of an extract from 60 light-grown Phaseolus coccineus seedlings Estimated G A , levels (ng) Bioassay Dwarf rice Lettuce hypocotyl Barley aleurone

’

Median value

Upper and lower 95% confidence limits

18“ 20b 600’ 700‘

5-60 448 13C-1400 3W1200

“1/60 aliquot assays; ljl20 aliquot assays; 1/6 aliquot assays.

68

ALAN CROZIER

one dilution. The relationship between estimates based on such data and the actual GA content of a sample is likely to range from minimal to nonexistent. Although immunological assays are extensively employed in the fields of mammalian endocrinology details of their application to GAS are limited to one report by Fuchs and Fuchs (1969). This should not be taken to indicate their general unsuitability in this role as the selectivity, limits of detection and simplicity of a well designed radioimmunoassay at least rival, and often exceed, those of GA bioassays. Fuchs and Fuchs (1969) showed that antibody raised against GA3 extensively cross-reacted with GA4, GA,, GA9 and to a lesser extent G A I 3 . While this lack of specificity is detrimental to radioimmunoassay of specific GAS it is a distinct advantage in developing a general assay to monitor overall GA levels. The limited ability of the GA3antibody to distinguish between individual GAS also suggests that it would be worthwhile investigating the possible use of affinity chromatography as a GA group separatory purification procedure. 2. Physicochemical Methods ( a ) Gas chromatography and combined gas chromatography-mass spectrometry. GC of the methyl esters of GAI-GA9 using a flame ionization detector (FID) was first reported by Ikegawa et al. (1963). Subsequently Cave11 et al. (1967) separated the methyl esters and trimethylsilyl ethers of the methyl esters of GA1-GAI5, GA18and GAI9 on 2% QF-1 and 2% SE-33 columns. The application of these procedures to the analysis of endogenous GAShas however not been a great success because the purity of most extracts is such that an FID, which is a non-specific mass detector, has to cope with high background levels and numerous extraneous peaks. As a consequence the great advantage of GC, its high peak capacity, is lost as there is no guarantee that the mass peaks being measured are in fact attributable to GAS.The analytical situation is greatly simplified when G C is used in metabolism studies as radioactive precursors and metabolites can be selectively monitored with a flow-through proportional gas radioactivity monitor (Simpson, 1968). In the light of more definitive analyses, identifications based on the retention times of [3H]GAmetabolites on 2% QF-l,2% SE-30 and 1% XE-60 columns have proved very reliable (see Durley and Pharis, 1973; Durley et al., 1974a; Railton et al., 1974a). The problems encountered with an FID can be overcome when a mass spectrometer is used as the G C detector, as all the GAS can then be distinguished from each other and from extract impurities on the basis of their mass spectra. Effluent from the GC column passes through a separator, which removes most of the carrier gas, and enters the ion source of the mass spectrometer where, a t c. Torr, it is ionized by bombardment with high energy electrons. This process is known as electron impact ionization. It

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

69

should be noted that instruments offering chemical ionization in a suitable reagent gas, such as methane or ammonia, are increasing in popularity although as yet they have not been widely used with GAS. Regardless of the method of ionization, a signal relative to the total ion current (TIC) is derived either by summing the ion current values using a data system or by intercepting a portion of the unresolved ion beam. The TIC gives an indication of the amount of material in the source and is analogous to a FID trace. However, the real analytical power of mass spectrometry lies in the fact that fragment ions can be resolved according to their mass to charge ratio (m/e) by means of a magnetic sector or quadrupole mass analyser to give spectra such as that illustrated in Fig. 13. The range, pattern and variety of fragments is so vast that each compound yields a characteristic spectrum. Identifications are based on the matching of spectra with those of compounds of known structure. If reference spectra are available they can be used for comparative purposes so it is unnecessary to have standards on hand. This is an important consideration as the great majority of the GAS are not readily available. GC-MS was first introduced to plant hormone analysis by Binks et al. (1969) who published reference electron impact spectra of the methyl esters and trimethylsilyl ether of the methyl esters of GA1-GAz4. One hundred nanograms or less of GA are required to obtain a mass spectrum, and provided adequate separation is achieved by GC, acceptable GA spectra can be obtained from relatively impure plant extracts. Primarily because of these attributes, GC-MS has rapidly become a technique of great importance to GA analysis and it is widely believed that mass spectral data are essential if a “conclusive, definitive or unequivocal” characterization is to be achieved. Identifications made in the absence of such data are invariably viewed with suspicion. GC-MS can also be used for selected ion current monitoring (SICM) and the technique is of particular value in the quantitative analysis of trace quantities of endogenous GAS.Instead of scanning the entire mass range the mass spectrometer determines the intensities at one or more selected m/e values that are prominent in the spectrum of the GA under investigation. In this role the mass spectrometer acts as a selective G C detector and, as it monitors only a few ions, detection limits can be as low as one picogram. However SICM does require that the identities of GASlikely to be present in a sample be known and that reference compounds are available to quantify the detector response and determine GC retention characteristics. Mass spectrometry reveals the relative isotope content of fragment ions provided the isotope content is sufficiently high. Although the level of 3H in [3H] labelled compounds is usually too low to measure, the [14C/’2C]and [’H/‘H] ratios can often be determined at the same time as a G A is identified from its mass spectrum (Bowen et al., 1972; Bearder et al., 1974). [14C] and [’HI

M+ 504

m 0

100

200

300

400

m /e

Fig. 13. Mass spectrum of the trimethylsilyl ether of the methyl ester ofGA,.

500

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

71

GAS can therefore be used as internal standards in quantitative analysis as they behave in a similar if not identical manner as their endogenous ["C/'H] counterparts yet can be distinguished from them by mass spectrometry. The ability of mass spectrometry to determine relative isotope content is also of value in metabolic studies as it provides a means of determining whether a GA mass spectrum is that of either a ["C/'H] endogenous component of indeterminate ancestry or a metabolite originating from a ['HI or ['"C] labelled precursor (see Sponsel and MacMillan, 1979). The flexibility and potential of GC-MS is greatly enhanced when it is coupled with an on-line computer (MacMillan, 1972). Copious details of the various modes in which such instrumentation can be operated, along with examples of the types of data obtained in assorted GA analysis, are contained in two practically orientated articles by Gaskin and MacMillan (1978) and Hedden (1978). ( h i High performance liquid chromatography. As commonly practised HPLC and G C are broadly equivalent in that they display similar efficiencies and speeds of analysis with the sample capacity of HPLC being about ten times that of GC. The major difference between the two techniques lies in the thermodynamics of the partitioning process. In liquid-solid and liquidliquid processes the differences in the free energies of distribution of the solutes d(dG') are usually far greater than for gas-solid or gas-liquid systems. Thus all other factors being equal HPLC will always give a superior separation to GC. In addition d(dG') is much more dependent upon the properties of the mobile and stationary phase in HPLC than it is in GC, thus HPLC is able to offer a much wider variety of column selectivities. HPLC has been applied to numerous diverse analytical problems (see Pryde and Gilbert, 1979). High column efficiencies and peak capacities, rapid speeds of analysis, the availability of many supports each offering markedly different separatory mechanisms, operation at ambient temperatures and ease of sample recovery all contribute to the overall effectiveness of the technique. It should however be noted that the high efficiencies ( > 40,000 theoretical plates m-') are achieved on columns with a 2-5 mm bore. The sample capacity of such columns rarely exceeds 500 pg. Thus, as far as the analysis of endogenous GASare concerned, the potential of HPLC, like that of GC-MS, can only be fully exploited when applied to extracts of relatively high purity. The application of HPLC to G A analysis is still in its infancy and the first problem confronting potential users of the technique is detection of the GAS. Although refractive index and far-UV monitors are often referred to as universal detectors they are not as useful as implied by the manufacturers' advertising literature which almost invariably fails to point out that they function in only a very restricted range of solvent conditions. Faced with this situation one answer is to use bioassays to detect G A S in HPLC eluates although this is not a particularly satisfactory solution, as it is time consuming

72

ALAN CROZIER

and much of the practicality of HPLC is lost. An alternative approach taken by Reeve and Crozier (1978) is to convert GAS to derivatives which absorb in an accessible region of the UV spectrum. GA benzyl esters (GABEs), synthesized by esterification with N ,N’ dimethylformamide dibenzylacetal, have a A,,,, of 256 nm and can be readily detected in a wide range of solvents with a standard UV monitor operating at 254 nm. The GABEs can be analysed on a silica gel adsorption column which generates up to 8000 theoretical plates (H=0.06 mm) and provides good separations of isomers because of its ability to distinguish subtle differences in the spatial relationships of the polar groupings of structurally similar molecules. An added advantage is that the selectivity of the silica gel can be substantially altered simply by changing the reagent used to modify the mobile phase. This point is illustrated in Fig. 14, which shows the separation of isomeric GABEs in hexane-dichloromethane based solvents modified with dimethylsulphoxide (DMSO) and THF. In the DMSO system the elution order is GA,,BE>GA,BE> GA,BE>GA,BE and GA,BE>GA,BE. The 13a-hydroxy GABEs (GA20BEand GA,BE) elute before their 3b-hydroxy equivalents (GA4BE and GA,BE) while the A ’ . and d 2 %isomers (GA3BE, GA7BE and GA,BE)

’

Mobile phase Hexane-dichloromethane - DMSO

Mobile phase Hexane-dichloromethane - DMSO (25 75 1)

GA.BE

0

8 12 16 20 Retention time (min )

4

Retention time (min)

Mobile Dhase Dichloromethane-THF

Mobile phase Dichlorornethane -THF

(97 31,

I

0

. 4

24

(92 81

.

8

.

12

16

Retention time (min)

20

24

I

0

.

4

8

.

12

.

.

16 20 Retention time (min)

.

24

.

Fig. 14. The influence of DMSO and T H F modifiers o n the HPLC retention characteristics of GABEs. Column : 4.6 x 500 m m Partisil 10. Mobile phase: as indicated on figure. Flow rate: 1.61111 m i n - ’ . Sample: GA,BE, GA,BE, GA,BE and GA,,BE or G A , B E and GASBE. Detector: absorbance monitor at AZS4.(Reeve and Crozier, 1978.)

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

73

are more retained than their saturated analogs ( G A I B E , G A 4 B E and GA2,BE). When T H F is substituted for DMSO there IS a complete reversal of the elution order with respect to both the location of the hydroxyl group and the degree of unsaturation. Such marked changes in column selectivity demonstrate the flexibility of silica gel adsorption HPLC and can be of value in the purification and isolation of G A S . A comprehensive discussion of silica gel adsorption HPLC of G A B E s has been published by Reeve and Crozier (1978). The procedures have been used in conjunction with an onstream HPLC radioactivity monitor and direct probe mass spectrometry to purify and identify [3H]GA metabolites from PhLisrolus c*occineusseedlings (Crozier and Reeve, 1977; Reeve and Crozier, 1978; Nash rt al., 1982) and lettuce hypocotyl sections (Nash " t al., 1978). Although they have proved useful in metabolism studies it should be noted that the c,,~,, of mono GABEs is 205 1 mol-'cm-' and the limit of detection at A254 is only c. 300 ng. This lack of sensitivity represents a serious constraint when it comes to fully utilizing the high resolving power of HPLC to analyse trace quantities of endogenous G A S . Other G A derivatives do however offer much greater potential in this regard. Heftmann r t u l . (1978) prepared p-nitrobenzyl GA esters (h,,, = 265 nm, c,, > 6000) using 0 pnitrobenzyl-N,N'-diisopropylisourea (Knapp and Krueger, 1975). Unfortunately, when the esters were chromatographed on a preparative silver nitrate impregnated silica gel column, the performance was very poor ( N = 1500, H = 3.25 mm) and peaks up to 200 ml in volume were obtained. As a consequence, the limit of detection achieved at A265 was 100 ng rather than the 10 ng that might have been expected if conventional HPLC procedures had been used. Morris and Zaerr (1978) used 18-Crown-6, according to the procedures of Durst et d.(1975), to catalyse the synthesis of pbromophenacyl G A esters (;.,,l,x=256 nm, F,,,,= 19,100).The p-biomophenacyl esters of G A 3 , G A 4 , G A , , GA,, G A 9 and G A I 3 were analysed on HPLC systems utilizing silica gel supports with a bonded C, or cyanopropyl stationary phase. The separations obtained with the reverse phase C, column are illustrated in Fig. 15. The limit of detection for nzoizo G A esters at was, as anticipated from their c,,,,, < 5 ng. Recently a marked increase in sensitivity has been obtained by using GA methoxycoumaryl esters (GACEs) for HPLC (Crozier et d., 1982). These derivatives are synthesized from 4-bromomethyl-7-methoxycoumarinin a Crown ether catalysed reaction (Fig. 16) that was originally used by Diinges (1977) to produce methoxycoumaryl esters of fatty acids. The GACEs are nm) (Fig. 17) and can be strongly fluorescent (A=;'= 320 nm, A5!?=400 detected at the low picogram level with a spectrophotofluorimeter after reverse phase HPLC. This is shown in Fig. 18 in which a log-log plot of relative response against sample size gives a line with a slope of 1 .O, indicating a linear response extending over almost four orders of magnitude. The limit

G

3

A254

c

0

5

15

10

Retention time ( rnin )

Fig. 15. Reverse phase HPLC of p-bromophenacyl GA esters. Column: 4 x 300 mm bBondapak,'CI8. Mobile phase: 15 min gradient. 5C-100; ethanol in 20 mmol pH 3.5 ammonium acetate buffer. Flow rate: 2 mi min - I . Sample: 1)-bromophenacyl esters of GA,, GA,, GAS, GAT.GA, and GAI3. Detector: absorbance monitor at (Morris and Zaerr. 1978.)

CH2Br 4 - bromornethyl-7- rnethoxycoumarin ( BMC 1

0 I1

R-C-OH

BMC

18- C r o w n - 6

Crystal K,CO, acetonitrile 60' for 2 h

R-C-O-CH2

Fig. 16. Crown ether catalysed synthesis of methoxycoumaryl esters.

Wavelength ( n m ) Fig. 17. Fluorescence spectra of GA,CE.

4

I

/i

Fluorescence

Excitation 320 nm

aJ

Emission 4 0 0 n m

'3x bockground noise 0

I

I

I

1

1 P9

l0pg

1oopg

1ng

Gibberellin A 3 Fig. 18. HPLC analysis of GA3CE. Column: 5 x 250 mm ODs-Hypersil. Sample: GA,CE, dose as indicated. Mobile phase: 45% ethanol in 20 mmol pH 3.5 ammonium acetate buffer (GA,CE k' = 2.3). Flow rate: 1 ml min -' , Detector: Perkin-Elmer 650-1OLC spectrophotoffuorimeter, excitation 320 nm, emission 400 nm, 10 nm slits.

76

ALAN CROZIER

of detection for niono esters is c. 1 pg as determined by the point at which the curve intersects the ordinate equivalent to three times the level of background noise. Good recoveries (>90:/,) and efficiencies ( N = 10,000, H=0.025 mm) were obtained when the GACEs were chromatographed on an ODSHypersil column. This HPLC system has the ability to distinguish between closely related GAS as the double bond isomers GA1/GA3,GA4/GA7 and GA5/GAzo,all separated with baseline resolution. It is also of interest to note the effect of solvents on column selectivity. When a methanol-buffer gradient was used GAI3CE and GA14CE co-chromatographed as did GA9CE and GA36CE (Fig. 19a). However the compounds are well resolved when ethanol is substituted for methanol (Fig. 19b). In general increasing the number of hydroxyl groups decreases retentions, 13a-hydroxylation to a greater extent than 3&hydroxylation which, in turn, is more effective than hydroxylation at either the la- or 2&positions. Similarly, A ' . 2 and GAS elute earlier than their saturated analogs. Methoxycoumaryl functions increase V R as the elution order is mono > bis> tris esters. The GACEs have been investigated by direct probe mass spectrometry. Electron impact and chemical ionization positive ion spectra were of no value as in all instances the dominant fragment was rn/e 191 with no other ions of significant intensity being present. However, chemical ionization negative ion spectra proved to be more diagnostic (Table IX). A strong molecular ion was GAS,GA3CE and GA,CE. M-189, arising from the obtained with the d loss of the ester moiety, was the main fragment in the spectra of the other C19-GACEs. Some C19-GAs (i.e. GA4CE and GA2,CE) have identical spectra but they can be readily distinguished on the basis of their HPLC retention characteristics (Fig. 19). Three CZO-GAswere analysed and M-189 was the strongest ion produced by GAI4CEwhile M-395 was the base peak in the spectra of both GA13CEand GAS6CE. As far as GAS are concerned HPLC and GC are mutually incompatible techniques. While G C derivatives such as GA methyl esters can be chromatographed on an HPLC column they are not readily detected with conventional on-line HPLC monitoring systems. Conversely GACEs and other derivatives that are suitable for HPLC are far from ideal candidates for GC as they lack the necessary volatility. This means that it is difficult to obtain mass spectra of GACEs by GC-MS. There are three ways around this problem. The first, and the one employed in obtaining the mass spectra presented in Table IX, is to use direct probe mass spectrometry. This practice has limitations with plant extracts as relatively large samples of high purity are required if acceptable spectra are to be obtained. The second approach would be to develop an effective transesterification process to convert fluorescent or UV-absorbing GA esters to a methyl ester that could be silylated and analysed by GC-MS. One potential problem with this procedure is that GA derivatives amenable to transesterification may well be somewhat unstable

',

GA13 GA14

5 16 GA9 GA36

G

GA25

i

L I

0

1

5

I

10

1

15

1

20

I

I

25

30

I

35

GA13

I

0

I

5

10

,

15

r

20

I

25

I

30

I

35

Retention time (min)

Fig. 19. Reverse phase HPLC of GACEs. Column : 5 x 250 mm ODS-Hypersil. Mobile phase: 30 min gradient (a) 6&100% methanol in 20 mmol, pH 3.5 ammonium acetate buffer, (b) 4&80% ethanol in 20 mmol, pH 3.5 ammonium acetate buffer. Flow rate: 1 ml min - I . Sample: methoxycoumaryl esters of G A 1 , G A 3 , C A I , G A S , GA,, G A a , GA9, GA13, GAL,, GA16, G A Z o ,G A Z 5 ,GA36, c. 9 ng mono, 4.5 ng bis and 3.0 ng tris esters. Detector: Perkin-Elmer 650-1OLC spectrophotofluorimeter, excitation 320 nm, emission 40 nm, 10 nm slits.

78

ALAN CROZIER

TABLE IX Methane chemical ionization negative ion mass spectra of gibbrrellin methoxycoumaryl esters Compound

Mol. wt.

GA,CE GA,CE

536 534

GA,CE GAJE GA,CE

520 518 518

GAsCE GAgCE GA 13CE

552 504 942

GA14CE GA 16CE GAZoCE GA36CE

724 536 520 738

347-100% (M-189) 534100% (M-), 34543% (M-189), 301-7% (M-233), 2837% (M-251) 331-100% (M-189) 329-100% (M-189) 518-100% (M-), 329-34% (M-189). 285-5% (M-233), 26715% (M-2-51), 2655% (M-253) 363-100% (M-189) 315-100% (M-189) 565-8% (M-377 [-189-1881), 5477100% (M-395 [-189-2061), 359-54% (M-583 [-189-188-206 and/or -189-206188]), 3155% (M-627), 3166% (M-628) 535-100% (M-189), 347718% (M-377 [-189-1881) 347-100% (M-189), 329-6% (M-207), 303-9% (M-233) 331-100% (M-189) 549-10% (M-189,343-100% (M-395 [-I89-206])

and some degree of breakdown could occur during HPLC. The long-term solution is the use of an HPLC directly coupled to a mass spectrometer. Two interfaces are currently being marketed although the technology is still in its infancy and performance, convenience and reliability have yet to be proven (see McFadden et al., 1977; McFadden, 1979; Karger et al., 1979; Arpino and Guiochon, 1979). When it becomes a practical proposition HPLC-MS will greatly increase the flexibility of HPLC. It will be possible to obtain spectra with smaller sized samples and, equally important, as far as GACEs and the analysis of endogenous GA is concerned, is that components eluting from HPLC columns could be analysed by SICM. The HPLC-fluorescence procedures can be used for GA analysis when the identity of individual GA(s) likely to be present in the sample is known or suspected and when reference compounds are available to determine HPLC retention characteristics and quantify the response of the fluorimeter. In view of the low picogram limits of detection of the GACEs it is evident that the amount of plant material that must be extracted can be reduced to gram quantities. This will make it possible to experiment with small tissues such as root caps and apical buds and individual plant parts that are either difficult to obtain and/or contain only small quantities of GA. While HPLC data may be compiled in these circumstances the amount of GA present will rarely be sufficient to enable a full scan mass spectrum to be obtained. This raises a question of great importance, namely, is it possible to identify a G A

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

79

solely on the basis of chromatographic retention indices? Although to date only about 20 of the 62 known GAShave been subjected to HPLC it is quite apparent that the separatory power of the technique is more than adequate to distinguish all known GAS. Contrary to popular belief this is not the problem. The real nub of contention, when dealing with trace components from natural sources is, do the chromatographic procedures employed have the capacity to separate the GA under study from all other components potentially present in the sample to which the detector will respond? Furthermore, and equally important, how can this capacity be demonstrated so that the accuracy of the analysis can be verified? F. VERIFICATION OF ACCURACY

Much confusion and dogma surround the entire process of verification of accuracy. Although it is widely accepted that only mass spectrometric evidence is acceptable this approach is not without its problems even in apparently favourable circumstances. The points of contention can be illustrated by a hypothetical conversation between a logician, (L), playing devil’s advocate, and a plant physiologist (PP) who has methylated and trimethylsilylated a purified plant extract prior to analysis by GC-MS, and on the basis of SICM at m/e 506 and a full scan mass spectrum has concluded that his sample contains 1 p g of GA1.Most of us will have some sympathy for the plant physiologist and consider that he has more than enough evidence to verify the accuracy of his analysis and that his data would certainly be published by even the most critical of journals. Nevertheless the points raised by the logician do demonstrate that the plant physiologist’s conclusions are based on very subjective criteria. The discussion runs as follows : PP “I estimate that the extract contains 1 pg of C A I.” L “You surely can’t mean 1~oooOOOO. . . . pg of GA,? There must be some uncertainty in the estimate.” PP “Of course, by analysing the sample five times I calculated that the 95% confidence limits are 0.1 pg.” L “Yes, that estimates the random error associated with the measurement process but is it the only source of uncertainty? Can you rule out the possibility that, say, 0.1% of the quantified response was due to compounds other than GAI?“ PP “No, I can’t be absolutely certain.” L “Perhaps then as much as 1% of the response was due to impurities.” PP “That is possible.” L “Then why not lo%, 50% or even loo%?’’ PP “Oh no. I don’t think that is at all likely.” L “Why not?” PP “Because the SICM data I used to quantify the GA, content were obtained by monitoring at m/e 506 which is the molecular ion of the trimethylsilyl ether of GA, methyl ester. This is a very selective procedure.”

80

ALAN CROZIER

L PP

L PP

L PP L

PP L PP L

“Do you mean to say that at m/e 506 the mass spectrometer responds only to the molecular ion of the G A , derivative? Surely fragment ions from other compounds at or near this nominal mass would also evoke a response.” “Yes but it seems improbable that an impurity giving rise to such a fragment would have the same GC retention time as the trimethylsilyl ether of G A , methyl ester.” “Perhaps, but how improbable is improbable? You must quantify that statement before accuracy can be defined.” “You forget that I obtained a full scan mass spectrum of the SICM peak that was used to determine the amount of G A L present in the sample.” “In that case you are transferring the source of the uncertainty of the estimate to the full scan mass spectrum.” “But this enables me to be far more certain of the accuracy of my analysis, as chemists tell me that mass spectra provide unique fingerprints of organic compounds.” “It now seems that verification of accuracy hangs on the word ‘unique’. This implies that mass spectra can distingukh between an infinite number of compounds.” “Of course not, that is impossible; but it is well known that the discriminating power of mass spectrometry is very high indeed and far exceeds that of other procedures.” “I agree, but just how high is very high indeed? It must be able to distinguish more compounds than the number that are present in your sample.” “No problem. It can certainly do that. The purity of the sample was more than adequate. It was extensively purified prior to GC-MS and was very clean indeed.” “The uncertainty in accuracy we were originally discussing has now manifested itself in the uncertainty associated with the purity of the extract. Thus you are no further forward as you must quantify this new uncertainty by deriving the numerical probability associated with your statement ‘The purity of the sample was more than adequate’. Until this is achieved the accuracy of your estimate will remain undefined and in doubt.”

At the start of the discussion between the plant physiologist and the logician the uncertainties associated with the analysis of GA, centred around whether SICM was sufficiently selective for the problem in hand. When the full scan mass spectrum was used as a basis for accuracy the uncertainty factor changed and became associated with the discriminatory power of a mass spectrometer relative to the complexity or purity of the sample. Reeve and Crozier (1980) have suggested a simple practical test, called a “Successive Approximation”, for detecting situations in which the selectivity of an analysis is inadequate. It relies on the fact that, as the purity of a sample is increased, estimates of GA concentration must show an improvement in accuracy, since even a totally non-selective method will provide accurate results with a perfectly pure sample. The successive approximation works in the following manner. When given a sample purporting to contain a given quantity of GA, ( E , ) the test for accuracy simply consists of purifying the sample by a factor of at least two and re-estimating the GA, content (E2).If

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

81

El is accurate, E 2 , taking into account the precision of the method, should not be significantly different. If a difference is found, El must be rejected as inaccurate and E2 retested by further purification and analysis. This process is continued for as long as is necessary to obtain an estimate that does not change on purification. At this point it is possible to conclude that on the basis of the available evidence, there are no grounds for believing the estimate is inaccurate. Reeve and Crozier (1980) have discussed several statistical methods that can be used to derive the probability of errors arising when making such an assumption. An alternative way to approach the verification of accuracy is through the uncertainty associated with sample purity. The nature of the problem is best grasped by viewing extracts under analysis as open-ended systems in which an infinite array of organic compounds are potentially present. For a variety of reasons only a limited number of these possible components are likely to occur in amounts that are significant in relation to the quantity of GA present. Reeve and Crozier (1980) used typical molecular weight distributions of plant extracts to draw probability limits on the number of compounds likely to be encountered. Information theory was then invoked to determine what type of mass spectrum, chromatogram or other analytical information was necessary to ensure that the discriminating power of the method was suficient to cope with the number of compounds likely to occur at a given probability level. Mass spectrometric data comprise the authoritative subjective standard that is widely used as a basis for the verification of accuracy, because of the enormous discriminating power of the technique. For instance, the mass spectrum of authentic GAzo trimethylsilyl ether methyl ester in Fig. 20a yields 436 binary digits (bits) of information according to the proposals of Reeve and Crozier (1980). This means that it can distinguish different compounds. However all this bewildering power is not transferred to the spectrum in Fig. 20b which has been used to identify GAzo in Steviu rebuudiunu extracts (Alves and Ruddat, 1979). Although Fig. 20b contains many of the features of the authentic GAzotrimethylsilyl ether methyl ester spectrum, the chances of a mistaken identification would be reduced if the sample had been purer and a more exact match had been obtained. In fact only 198 bits of information in Fig. 20b correlate with the spectrum of the standard in Fig. 20a. In accepting this less-than-perfect match the power of the technique has been reduced to such an extent that it can distinguish only one compound in lo6'. This represents an infinitesimal fraction of its potential discriminating power. Reeve and Crozier (1980) calculated that, at a probability level of 0.9, the number of different compounds potentially present in a typical plant extract is lo4' and thus c. 140 bits of information are required to ensure accuracy. The spectrum in Fig. 20b more than meets this standard so, provided the basis of the calculations is valid, it facilitates

( a ) Authentic GA2oTM Si M e

c L

z

( b

.

Putative GA20TM Si M e

80-

6040

20 10

0

50

100

150

250

200

300

350

400

450

m/e

Fig. 20. Electron impact mass spectra of (a) authentic trimethylsilyl ether of the methyl ester of GAzoand (b) putative trimethylsilyl ether of the methyl ester of G A z ofrom a purified extract of Stevia rebaudiana shoots (Alves and Ruddat, 1979).

METABCLISM AND PHYSIOLOGY OF GIBBERELLINS

83

the accurate identification of GAZ0in S. rebuudiunu. It is evident that 140 bits of information can be furnished by something less than a full scan mass spectrum although there are limits, and if too few ions are monitored and/or too many spurious fragments are present, mass spectrometric evidence will almost certainly fail to provide the necessary verification of accuracy. When the upper limit of the molecular weight range of components in an extract is limited by SEC the potential complexity of the sample is greatly simplified. If, for instance, 90% of the mass of a fraction collected from an SEC column is comprised of compounds with a molecular weight of less than 400, only 36 bits of information are required to guarantee accuracy with probability of 0.9. SEC systems that can achieve this type of fractionation were described in Section IIC. When they are incorporated into purification procedures it becomes possible to use less powerful analytical techniques than mass spectrometry to yield accurate results provided the principles and attendant assumptions laid down by Reeve and Crozier (1980) are followed. For instance, any chromatogram can be treated in an analogous manner to the mass spectra in Fig. 20. The potential information yield in bits is related, on a one-to-one basis, to the peak capacity of the chromatographic system.

I

Authentic GAx

Sample A

I 0

I

5

I

1

10

15

20

1

25

Retention time (min )

Fig. 21. Hypothetical analysis of GA, on a chromatogram with a peak capacity of c. 100.

84

ALAN CROZIER

Thus capillary G C has a potential of c . 300 bits, modern HPLC c. 100 hits, while classical procedures such as TLC and PC produce no more than 5 hits. Figure 21 illustrates a hypothetical chromatogram of an authentic sample of GA, in which the potential information by virtue of the peak capacity is 100 birs. All of this information is available for the verification of accuracy in the case of sample A, which produces a trace that is a perfect match with that of the GA, standard. Sample B, however, contains a large number of impurities and the correlation is far from perfect. Although GA, can be quantified on the basis of the appropriate peak area, the amount of information that can be used to verify the accuracy of the estimate is limited to only one bit as so few parts of the chromatogram match the authentic GA, trace. Clearly sample purity is an important consideration and cannot be ignored, as it is a major factor in determining whether or not sufficient evidence is accrued for verification of accuracy. When analysing impure samples the availability of a selective detector is advantageous as traces will yield more information than equivalent chromatograms obtained with a non-specific method. This is where the strength of SICM lies, why G C data obtained with a radioactivity monitor have proved reliable in identifying [3H]GA metabolites and why, in contrast, GC-FID analysis of endogenous GAS has produced many erroneous identifications. In this context the bioassays in Table VI can be looked upon as selective detectors for free GAS.Unforunately they are very labour-intensive and the problem is compounded as large numbers of fractions must be collected and analysed if the peak capacity of the chromatogram is to be maintained. In addition the response time can be anything from two to seven days and the precision is poor because of the log-linear doseresponse curve and the inherent variability of plant material. One appealing feature of all chromatographic methods is that by running a sample in a number of different solvent systems information can be accumulated. HPLC is particularly amenable to this approach because of the ease and efficiency of sample recovery and the variety of separatory mechanisms that are available. Indeed certain combinations of HPLC techniques can easily challenge the informing power of mass spectrometry. The analysis of GACEs is a case in point as in circumstances where the limited availability of sample prevents a mass spectrum being obtained, information can be readily accumulated by HPLC because of the low limits of detection of the spectrophotofluorimetric monitor. However, the interested reader is urged to consult the rules used to calculate the information yield of a combination of chromatographic techniques as in some situations the total information obtained is not additive (Reeve and Crozier, 1980). While the procedures of Reeve and Crozier (1980) provide a means of verifying accuracy it must be emphasized that they involve a number of less than perfect assumptions and it would be unwise, without detailed investigation, to pedantically adopt such criteria as universal standards for analysis.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

85

However, it does appear that even rough objective standards provide an intuitively acceptable assessment of many of the imponderables usually left to subjective judgement. 111. GIBBERELLIN BIOSYNTHESIS A. MEVALONIC ACID TO ENT-KAURENE

All GAS originate from a common pathway leading to GAlz aldehyde; thereafter, depending upon the plant material, the pathway can branch in an assortment of directions. Primarily as a result of investigations with cellfree systems from Gibberella fujikuroi (Shechter and West, 1969; Evans and Hanson, 1972), Marah macrocarpus* (Graebe et al., 1965; Upper and West, 1967; Oster and West, 1968), Circurbira maxima? (Graebe, 1969, 1972), Pisutn sativum (Anderson and Moore, 1967; Coolbaugh and Moore, 1971; Coolbaugh et a / . , 1973; Graebe, 1968) and Ricinus communis (Robinson and West, 1970a) it has been established that the early stages of GA biosynthesis follow the normal isoprenoid pathway. The activation of mevalonic acid to the pyrophosphate form is followed by conversion to dimethylallyl pyrophosphate via isopentenyl pyrophosphate. Three condensation steps then occur, each involving isopentenyl pyrophosphate, in which the conversion of dimethylallyl pyrophosphate-geranyl pyrophosphate-tfarnesyl pyrophosphate+geranylgeranyl pyrophosphate takes place. Geranylgeranyl pyrophosphate is a precursor of ent-kaurene which is synthesized via copalyl pyrophosphate. The enzymes involved in the synthesis of ent-kaurene from mevalonic acid are soluble, remaining in the high speed supernatant when tissue homogenates are ultra-centrifuged, and require ATP, Mg’ ’ and Mn’ ’as co-factors. Liquid endosperm preparations from seed of Cucurbita maxima at the appropriate stage of development can convert mevalonic acid to ent-kaurene with an efficiency of c . 40% (Graebe, 1969). However, in other systems, especially seedling material, the ent-kaurene yield is much lower, as mevalonic acid is preferentially converted into products such as squalene via farnesyl pyrophosphate ; phytoene and casbene via geranylgeranyl pyrophosphate ; and ( + )-stachene, ( )-sandarocopimaradiene and trachylobane via copalyl pyrophosphate (Graebe, 1968 ; Robinson and West, 1970b; Hedden and Phinney, 1979). Certain synthetic growth retardants can inhibit the conversion of geranylgeranyl pyrophosphate to ent-kaurene via copalyl pyrophosphate in cell-free systems. The first step in the sequence is affected by N,N,N-trimethyl-1methyl (2’,6’,6’-trirnethylcyclohe~-2’-en1’-yl prop-2-enylammonium iodide (Hedden et al., 1977)), AMO-1618 and its isomer Carvadan, phosphon-D,

+

*Originally referred to as Echinocystis macrocarpa. toriginally referred to as Cucurbita pepo.

86

ALAN CROZIER

phosphon-S, 4-53, 4-58 and 4-64, inhibitors of sterol biosynthesis such as S K F 3301A and SKF 525A and high doses of CCC. The conversion of copalyl pyrophosphate to ent-kaurene is more resistant and is sensitive only to 4-53, 4-58, 4-64 and the steroid inhibitors (Fall and West, 1971; West, 1973; Frost and West, 1977). In the late 1960s and early 1970s some of these retardants, in particular AMO-1618 and CCC, were widely used in physiological experiments and it was often assumed, without appropriate experimentation, that their inhibitory effects on growth were a direct consequence of reduced ent-kaurene synthesis and the resultant depletion of endogenous GA levels. Anyone questioning the simplicity of these views was likely to become embroiled in a colourful debate (see Lang, 1970). There was, none the less, much data indicating that the mode of action of the retardants was considerably more complex in vivo than in vitro. Their inhibitory effects on plant growth are not universal (Cathey and Stuart, 1961) and when dwarfism is induced it can rarely be completely counteracted by exogenous GA treatment (see Lockhart, 1962; Crozier et al., 1973). In certain circumstances low doses of CCC and AMO-1618 can actually enhance growth and/or increase levels of endogenous GA-like activity (Mishra and Pradham, 1968; Van Bragt, 1969; Wunsche, 1969; Reid and Crozier, 1970, 1972; Hdevy and Shilo, 1970). To compound the situation still further, Douglas and Paleg (1974) have shown that phosphon-D, AMO-1618 and CCC inhibit growth and sterol biosynthesis in Nicotiana tabacum and the retardation of growth can be prevented by application of either sterol or GA. Unless the role of GA is to activate sterol biosynthesis, these data prove that the retardants have more than one potential site of action in higher plants. Indeed it is now generally accepted that they are not the elegant physiological tool once envisaged and that their effects on plant growth are unlikely to be exclusively due to an inhibition of ent-kaurene synthetase. Claims to the contrary are now expected to be accompanied by unequivocal evidence rather than assumptions. Seedlings of the d 5 single gene recessive mutant of Zea mays are characterized by shortened stems and leaves and, unlike normal seedlings, they contain little or no GA-like activity (Phinney, 1961). The growth rate of d5 mutants is enhanced by treatment with GAS and some GA precursors such as entkaurene, and it has been suggested that dwarfism is a consequence of a block in the GA biosynthesis pathway prior to ent-kaurene formation (Katsumi et al., 1964). Hedden and Phinney (1979) have investigated this possibility by using a cell-free system to study the production of ent-kaurene by shoots of etiolated normal and d 5 seedlings. In preparations from both tissues, most of the radioactivity from a [14C]mevalonic acid precursor became associated with phytoene and squalene. Although only relatively minor components overall, the main diterpene hydrocarbons produced were ent-kaurene (entkaur-16-ene) and its isomer, which is not on the pathway leading to GAS,

87

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

ent-isokaurene (ent-kaur-15-ene). Normal seedlings synthesized higher levels of ent-kaurene while ent-isokaurene was the major diterpene in the d5 incubations. Similar ent-kaurenelent-isokaurene ratios were obtained when [' 4C]geranylgeranyl pyrophosphate and [3H]copalyl pyrophosphate were used as substrates. This point is demonstrated in Table X which also shows that the total incorporation of radioactivity into diterpenes was higher in cell-free preparations from normal seedlings than it was from d 5 . TABLE X Incorporation of radioactivity into ent-kaurene and ent-isokaurenefrom [2-'4C]mevalonic acid ( M V A ) , ['4C]geranylgeranylpyrophosphate ( G G P P ) and [3H]copalylpyrophosphate ( C P P ) incubated in cell-free extracts from etiolated shoots of normal and ds seedlings of Zea mays. Data expressed as c.p.m. g-' fresh weight. (After Hedden and Phinney, 1979) -~~~~~ ~

Substrate

Tissue

~~

c.p.m. g

-' fresh weight

-

ent-kaurene ent-isokaurene MVA

GGPP

Normal ds Normal

CPP

Normal

d5 d5

673 16 103 10 766 153

84

127 26 37 81 608

ent-kaurene

ratio ent-isokaurene 8.0 0.: 4.0 0.3 9.5 0.3

The data of Hedden and Phinney (1979) indicate that the normal allele of the d 5 gene controls the conversion of copalyl pyrophosphate to ent-kaurene since mutation results in a marked reduction in ent-kaurene biosynthesis. Apparently the mutated gene codes for an altered enzyme which catalyses the production of ent-isokaurene at the expense of ent-kaurene. Hedden and Phinney (1979) have suggested a possible mechanism for the enzymic formation of ent-kaurene and ent-isokaurene from copalyl pyrophosphate. It involves ent-kaurene being synthesized from copalyl pyrophosphate by the loss of a proton from the C-17 carbofiinm ion (I) while loss of a proton from C-15 gives rise to ent-isokaurene (Fig. 22). Thus any alteration of the enzyme, such as a shift in the position of the proton-accepting group bringing it closer to C-15 than to C-17, would result in increased production of entisokaurene in preference to ent-kaurene as seemingly occurs in the d5 mutant of Zea mays.

88

ALAN CROZIER

Copalylpyrophosphate

J

@ \

enl- kaurene

enf - isokaurene

Fig. 22. Proposed scheme for the synthesis of rnt-kaurene and ent-isokaurene from copalyl pyrophosphate in maize seedlings (Hedden and Phinney, 1979).

B.

ENT-KAURENE TO C A I 2ALDEHYDE

Further conversion of ent-kaurene involves sequential oxidation at C-19 to produce em-kaurenol, enr-kaurenal, em-kaurenoic acid and em-7a-hydroxykaurenoic acid (Fig. 23). All the steps have been shown to occur in cell-free us et al., systems from liquid endosperm of both Marah ~ ~ ~ a c r o c a r p(Graebe 1965; Lew and West, 1971) and Cucurbita maxirna (Graebe and Hedden, 1974) and immature seed of Pisurii sativurii (Ropers et al., 1978). Murphy and Briggs (1975) have demonstrated the sequence from ent-kaurenol in cell-free preparations from embryos and young leaves of Hordeum distichon. In Gibberella jujikuroi the conversion of ent-kaurenoic acid to ent-7ahydroxykaurenoic acid has been established (West, 1973; Bearder et al., 1975a). The enzymes involved in this section of the pathway in Marah are particulate and located in the 105,000 x g pellet (Dennis and West, 1967). Activity is dependent upon the presence of O2 and NADPH2. The oxidation of ertt-kaurene to eut-kaurenol, and that of ent-kaurenal to ent-kaurenoic acid, is inhibited by carbon monoxide and in both instances the inhibition is

18-

1 9 ~

ent - kaurane skeleton

L

ent- kaurene

enf- kaurenol

ent - kaurenal

ent- kaurenoic acid

enf - 7a - hydroxykaurenoic acid

Fig. 23. Ent-kaurane skeleton and the conversion of ent-kaurene to ~nt-7a-hydroxykaurenoicacid

90

ALAN CROZIER

counteracted by light which exerts maximal effect at 450 nm. This suggests that the conversions are catalysed by mixed function oxidases and implies an involvement of cytochrome P450(Murphy and West, 1969). The growth retardant ancymidol (a-cyclopropyl-a-[p-methoxyphenyl]-5-pyrimidine methyl alcohol) which induces a GA-reversible inhibition of root and shoot growth (Leopold, 197l), blocks the oxidation of ent-kaurene, ent-kaurenol, ent-kaurenal but not ent-kaurenoic acid in cell-free preparations of Marah macrocarpus, and it has been suggested that it interacts with cytochrome P450 in the oxidase-catalysed steps between ent-kaurene to ent-kaurenoic acid (Coolbaugh et al., 1978). In cell-free systems from Cucurbita maxima and Marah macrocarpus, ent7a-hydroxykaurenoic acid undergoes either oxidative B-ring contraction to produce the GA precursor, GA1,aldehyde, or ent-6a-hydroxylation to form ent-6a,7a-dihydroxykaurenoicacid (Graebe et al., 1972, 1974~;West, 1973; Graebe and Hedden, 1974). To date only GAlzaldehydeformation has been reported in preparations from immature Pisum sativum seed (Ropers et al., 1978). In Gibberella fujikuroi, ent-7a-hydroxykaurenoic acid gives rise to GAlzaldehyde (Hanson et al., 1972) and in addition is seemingly the intermediate in the synthesis of both ent-6a,7a-dihydroxykaurenoicacid and 7pdihydroxykaurenolide from ent-kaurenoic acid (West, 1973). The ent-6a,7adihydroxykaurenoic acid does not accumulate to any extent, being converted to fujenal (Cross et al., 1970)while 7,LLhydroxykaurenolideacts as a precursor of 7/?,18-dihydroxykaurenolide(Cross et al., 1968a). All these steps are illustrated in Fig. 24. The conversion of ent-7a-hydroxykaurenoic acid to G Al ,aldehyde requires contraction of ring B from a six to a five carbon structure with the extrusion of C-7. Evans et al. (1970) proposed that ring contraction was initiated by abstraction of the ent-6a-hydrogen as feeding experiments with Gibberella fujikuroi using stereospecifically labelled [3H]mevalonic acids showed that the ent-6a-hydrogen was lost in the conversion of ent-7ahydroxykaurenoic acid to GA3 while hydrogen at the ent-6j?-position was retained. Experiments by Graebe et at. (1975) indicated a similar process may occur in Cucurbita maxima preparations as metabolism of ent-7a-hydroxy ['4C,6-3Hz]kaurenoic acid produced GA1,aldehyde and ent-6a,7a-dihydroxykaurenoic acid with half the 3H/14Cratio of the substrate. Timecourse studies on the synthesis of these two metabolites in preparations from the 200,000 x g microsomal pellet of Cucurbita endosperm revealed that both compounds were formed simultaneously at equivalent rates. LineweaverBurk and Hill plots of the rates of synthesis were linear and the Hill plot had a slope of almost 1.0 indicating first order kinetics. Co-factor, pH and temperature requirements for both reactions were similar implying that both metabolites were being formed from the same high energy intermediate whose rate of synthesis determined the overall rate of production (Graebe

//

/

@-gQ \

oc-d \ H ',""

OH COOH

ent - 6a,7a - di hydroxy kau renoic acid

Fujenal

@- m COOH

COOH CHO

enl- 7a - hydroxykaurenoic acid

GA12 aldehyde

7p- hydroxykaurenolide

7&18 -dihydroxykaurenolide

Fig. 24. Metabolism of ent-7a-hydroxykaurenoic acid.

'R ent -70- hydroxykaurenoic acid

ent-6a,7a- dihydraxykaurenoic acld

t

QQ-(&Q R'

R

(0

c+

H/ '3 \

& c' / \

H O GA 12aldehyde

H

Fig. 25. Proposed mechanism for the conversion of ent-7a-hydroxykaurenoic acid to GA12aldehyde and enr-6a,7a-dihydroxykaurenoicacid. R = COOH (Evans et al., 1970; Hedden et al., 1978).

92

ALAN CROZIER

and Hedden, 1974). Graebe et d.(unpublished data quoted by Hedden et d., 1978) fed ent-7a-hydro~y[6a-~H,I 7-3Hz]kaurenoicacid containing 62 atoms :{ [’HI to the Cucurbitu cell-free system. The resultant GA1zaldehyde and enr-6a,7a-dihydroxykaurenoicacid had the same specific radioactivity as the substrate but contained only zero and 4 atoms :{ [’HI respectively thereby proving that they had both lost the ent-6a-hydrogen atom. The simplest mechanism commensurate with the experimental data obtained with Gibberellu and Circitr.bitcr was originally proposed by Evans et a / . (1970) and is illustrated in Fig. 25. Abstraction of the ent-6a-hydride from errt-7a-hydroxykaurenoic acid produces a putative carbonium ion intermediate which can either be hydroxylated at the ent-6a-position to give ent-6a,7a-dihydroxykaurenoic acid, or alternatively undergo B-ring contraction to form GA12aldehydevia migration of the 7,8 bond to the 6,8 position and the loss of a proton from the hydroxyl function of the extruded C-7. C. PATHWAYS BEYOND GA1 ,ALDEHYDE

I . Gibberella fujikuroi GA biosynthesis pathways have been thoroughly investigated in Gibherellu ,fi~jikur*oi. The GAS are metabolic bi-products and do not appear to be involved in mycelial growth in any way. Early studies by Cross et a / . (1968b) were suggestive of a branch at an early point in the pathway as although [ 17-’4C]GA1,aldehyde and [ I 7-14C]GA14 were effectively converted to [17-14C]GA3,[17-14C]GA12produced three unidentified acids and only relatively small amounts of GA3. Details of developments from a chronological point of view can be gauged from reviews by Cross (1968), Hanson (1971), MacMillan and Pryce (1973), MacMillan (1974), Graebe and Ropers (1978) and Hedden et N / . (1978) while an outline of the current status of knowledge is presented in Fig. 26. Much of the information in Fig. 26 was obtained from experiments using mutant strains of Gibbere//nfujikuroi.Originally genetic studies were virtually impossible because of an inability to consistently obtain the sexual stage of the fungus in the laboratory. However, Spector (1964) succeeded in routinely inducing perithecial production by growing Gibberella strains of opposite mating types on a Cirrus stem medium. Asci were removed from mature perithecia and ascospores from individual asci separated with a micromanipulator and transferred individually to potato-dextrose agar for culturing. With this technique Spector and Phinney (1966, 1968) provided direct evidence of genetic control of GA production in the fungus and demonstrated the presence of two non-allelic genes blocking different points Fig. 26. G A biosynthesis pathways beyond GA,2aldehyde in Gibberella fujikuroi. Thick arrows represent steps connecting major metabolites.

GA40

94

ALAN CROZIER

on the synthesis pathway. The first gene blocked an early stage in the metabolic sequence and controlled all GA production. The second blocked a later step as the production of only GA1 and GA3 was adversely affected. Further developments occurred because Phinney (unpublished data) was able to modify the barley aleurone a-amylase bioassay (Jones and Varner, 1967) to provide a simple non-labour intensive procedure to monitor the presence or absence of GA in Gibberellafujikuroi cultures. This facilitated a rapid screening of the GA production capacity of many thousands of strains of Gibberelln from both natural sources and mutants arising from UVirradiation of a wild type parent strain GF-la. The pathways illustrated in Fig. 26 were compiled from data obtained with the GA synthesizing strains ACC 917, GF-la and M-119 and two mutants, B1-41a and R9. R9 arose spontaneously from a wild type strain isolated from a paddyfield in Japan and is blocked for 13a-hydroxylation, producing neither GA, nor GA3 (Bearder et al., 1973a). B1-41a is a UV-induced mutant blocked between ent-kaurenal and ent-kaurenoic acid with a leakage of <3:4 for the conversion of [2-'4C]mevalonic acid to [I4C]GA3 (Bearder et al., 1974). This mutant has proved especially useful, as the absence of endogenous GASenables metabolism studies to be carried out with unlabelled precursors. The conversion of GA,,aldehyde to either G A 1 2 via C-7 oxidation or GA,, aldehyde via 3P-hydroxylation represents the major branch in the GA biosynthesis pathway (Bearder et a/., 1973b, 1975a; Evans and Hanson, 1975). GAI4aldehydeis probably the immediate precursor of GAI4as both compounds are converted via GA4 and GA? to GA3,which is the major GA in G A-producing strains of Gibberella. In addition to this main pathway, trace quantities of G A I 3 ,GA36and GA4, are synthesized from both GA14 aldehyde and GA14,while GA4 is the substrate for small amounts of GA1, GA2, GAI6 and GA4? (Bearder et al., 1975a; McInnes et ul., 1977). GAl, GA3, GA16 and GA36 are not further metabolized, nor is GA3,, which although not detected as a metabolite in these studies is an endogenous component of Cibberellu (MacMillan and Wels, 1974; Bearder et ul., 1975a). Some seemingly contradictory data have been obtained with G A I 4 feeding. Evans and Hanson (1975) have reported that it is essentially unchanged after a 24-h incubation while Cross et al. (1968a) observed a 4.7% incorporation into GA,. Both investigations used the wild type strain ACC-917. Studies by Hedden et al. (1974) with B1-41a showed that although GA14 and GA14aldehyde produced the same spectrum of metabolites, all the aldehyde was converted over a five-day period while 40% of the acid remained unmetabolized. This could be a consequence of poorer penetration of GA14 to the enzymic site in the fungal hyphae but the possibility that GA14 may not be on the direct pathway between GA,,aldehyde and GA4 cannot be discounted. The alternative pathway, from GAI2aldehyde via GA12, gives rise to

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

95

predominantly non-hydroxylated GAS (Fig. 26). [17-14C]GAI fed to strain ACC-917 yielded labelled GA9 (23%), GA15 (973, GA24 (25%) and GA25 (18”,J within six hours (Evans and Hanson, 1975). Similar results were obtained with the B1-41a mutant except that trace amounts of GA3, G A I 3and GA14 were also formed (Bearder et a / . , 1975a). Presumably this could be due to low level conversions of G A I Zto G A I 4 .GA9 was synthesized only from GA12,GA24 was converted to GA2s,which along with G A l S ,was not further metabolized. GA9 is not converted to GA4, instead it is slowly metabolized to small amounts ofthe hydroxylated G A S ,G A l o ,GA, 1 , GA20, GA4o and 16,17dihydro, 16,17dihydroxyGA9 (11) as well as the A1.’o,19,2 lactone (111) and possibly d ’GA9 (IV) as illustrated in Fig. 26 (Cross et a / . , 1964, 1968b; Bearder et a / . , 1976a). The and A ’ * ’ ’ compounds are potential precursors of GA, although this has not been experimentally tested. It is evident from the GA biosynthesis pathways illustrated in Fig. 26 that only C19-GAs undergo 13a-hydroxylation and that 3b-hydroxylation occurs only at the CIO-GA stage. Enzyme-substrate specificity, however, is low as some structural analogs of endogenous intermediates are metabolized to produce an array of natural GAS and GA analogs (see MacMillan, 1974; Hedden et al., 1978). While such studies provide information of intrinsic value they also result in the production of sizable quantities of many GAS that would otherwise be very difficult, if not impossible, to obtain. The GAdeficient mutant B1-41a is especially useful in this regard as the metabolites are not contaminated with endogenous GAS. This strain has been used to synthesize 15&hydroxy derivatives of GA1, GA3, GA4, GA7, GA9 (i.e. GA45), GA12. GA13 and GA15 from ent-l5a-hydroxykaurenoicacid (Bearder and Kybird, unpublished data). Metabolism of steviol (ent-13hydroxykaurenoic acid) by B1-41a produces ent-7a,l3-dihydroxykaurenoic acid, ent-6a,7a, 13-trihydroxykaurenoic acid, 7jl,13-dihydroxykaurenolide, 13-hydroxyfujenal, G A I , G A I 7 , G A l s , GAI9, GA20 and GA53 (Bearder et al., 1975b). In the absence of a GA deficient mutant, a similar spectrum of metabolites can be obtained, undiluted by endogenous GAS, by feeding steviol to normal Gibberella strains and inhibiting ent-kaurene synthetase with either AMO-1618, CCC or N,N,N-trimethyl-l-methyl-3-(3’,3’,5’trimethylcyclohexyl)-2-propenylammonium iodide (Murofushi et a/., 1979). There is a further facet to the interest in steviol since it occurs naturally in high concentrations as a glucoside in the leaves of Stevia rebaudiuna which is a member of the Compositae, native to Paraguay. Unlike Gibberellafujikuroi, many higher plants contain 13a-monohydroxylated C2,,-GAs and it has been hypothesized that steviol may be a key precursor in the synthesis of these compounds (Ruddat et al., 1963, 1965). It is envisaged that the pathway would run steviol+ ent-7a, 13-dih ydroxykaurenoic acid -,G A ,aldehyde+ GA53.Steviol is active in GA bioassays (Katsumi et a/., 1964) and as noted ‘ 3

96

ALAN CROZIER

above it is converted to 13a-hydroxy CZO-GAsby Gibberella fujikuroi. Similar conversions have not, however, been demonstrated in higher plant tissues and Stevia rebaudiana is the only plant in which steviol has been detected although the literature offers no indications of serious attempts being made to locate it in other species. The steviol hypothesis is interesting conjecture but no more than that as critical evidence is clearly lacking and the possibility that 13a-hydroxylation takes place after, rather than before, B-ring-contraction is equally feasible. The metabolic profile can be modified to some degree by varying the pH of the medium in which Gibberella is cultured. For instance, GAI4accumulates in 20-h feeds of ent-kaurenoic acid, ent-7a-hydroxykaurenoic acid, GA12 aldehyde and GA14aldehydeto B1-41a at pH 7.0, yet at pH 3.5 its appearance is transient, presumably because further conversion is no longer a rate limiting step (Bearder et al., 1975a). When the GA-producing strain ACC-917 is grown on synthetic medium the ratio of the GA3/GAl content is >20 and [14C]GA1is not converted to [14C]GA3.However when the fungus is grown in a glucose-soybean meal medium the GA3/GA1 ratio is < 1.5 and there is a 0.6% conversion of [1,2,3H2]GA1to [3H]GA3(Pitel et a/., 1971b; McInnes et al., 1977). Under the circumstances it seems plausible that some of the seemingly contradictory results that have been obtained in G A metabolism studies (see Hedden et al., 1978) could be due to non-uniform culturing techniques compounded by the use of different strains of Gibberellafujikuroi. 2. Cucurbita maxima When MnC12 is included in the incubating medium, the 20,000 x g supernatant from liquid endosperm of Cucurbita maxima seed converts mevalonic acid through to GA12aldehyde and GA12(Graebe et al., 1972). Oxidation of GA12aldehyde to GA12 is catalysed by both microsomal and soluble enzymes although further conversion is exclusively associated with the 200,OOOxg supernatant and requires NADPHz and Fei+ or F e + + + cofactors. The action of the soluble enzymes is inhibited by M n + + which explains why metabolism does not proceed beyond GA12in the presence of MnC12 (Graebe and Hedden, 1974). The G A biosynthesis pathways that operate in the Cucurbita maxima cellfree preparations are illustrated in Fig. 27. GA12aldehyde is primarily converted to GA,, which is metabolized to produce GA4, GA13,GA, 1 5 , GA24, GA25, GA36, GA37and GA43. Re-incubation of these compounds indicates that the main pathway from GA12 runs GA12-+GA24+GA36+GA13+ GA43 (Graebe et al., 1974a,b,c). The terminal product, GA43, is an endogenous constituent of Cucurbita maxima endosperm (Beeley et al., 1975) implying that the pathway operates in vivo as well as in vitro. GA24 feeds yield some GA25, which, on re-incubation, is converted to GA13, showing that an alternative route exists from GA24 to GA13, proceeding via GAzs

’ COOH

I

HO

H

o

COOH W C

H

z

GA43

Fig. 27. G A biosynthesis pathways beyond GA,,aldehyde in cell-free preparations from liquid endosperm of Cucurbita maxima seed. Thick arrows represent the preferred pathway based on rates of conversion and levels of metabolites.

98

ALAN CROZIER

rather than (3,436. However, this is probably a minor pathway as GA25 is converted to GA13far less efficiently than GA36.The formation of GA24 from GAlz represents two oxidation steps at C-20, and the S-lactone GA15 would seem to be the logical intermediate. In short-term incubations with [ 14C]GAI2 , a radioactive component with the G C retention characteristics of GA15accumulates and then disappears as GA43starts to build up. However, GA15is not the intermediate, as when it is added to cell-free preparations the only metabolite is GA37.Graebe et al. (1974~)postulate that the true intermediate between GA12 and GA24 may be the C-20 alcohol (V), which they suggest Iactonizes to form GAl 5 . The only CI9-GAmetabolite to be detected is GA4 which is produced in 5-10% yields from GA12aldehyde (Graebe et al., 1974b,c). It has recently been determined that GA36 is the immediate C20-GAprecursor of GA4 (Graebe et al., 1980). Although GA9 is also metabolized to GA4 the significance of the conversion is unclear because GA9 has never been detected as a metabolite in Cucurbita cell-free preparations (Graebe et al., 1974b,c). The conversion may therefore reflect a lack of specificity of the 3j?-hydroxylase involved in the production O f GA36 from GA24, although the possibility that GA9 is a natural substrate that goes undetected because it is rapidly metabolized to GA4,cannot be ruled out. When GA12aldehydeis incubated at the usual concentration of 0.1 pg 100 pl-I the main product is GA43. However when fed at 36 pg 100 plthere is a low yield of GA43rand the major metabolite is GA14aldehyde which is not detected at the lower precursor dose. GA14aldehyde feeds produce GA14, and both compounds are incorporated into GA37as well as into GA36, GA13 and GA43. On the basis of this evidence Graebe and Hedden (1974) have concluded that GA,,aldehyde is not a natural intermediate in the synthesis of GA43and that its production is induced by high concentrations of GA12aldehyde. This may be a somewhat simplified view of the. situation as it is unclear from the data provided whether there is indeed a genuine decrease in the amount of GA43 synthesized at the higher substrate concentration, or merely a reduction in relative terms compared to the increased levels of GA14aldehyde and unmetabolized GAl zaldehyde. If it is the latter, GA14aldehyde may well be a natural metabolite which under normal circumstances does not accumulate, as its rate of synthesis from GA12aldehyde does not exceed its rate of metabolism to GA14. When the substrate concentration is increased conversion to GA, becomes the rate limiting step in the pathway and as a consequence GA14aldehyde builds up and is readily detected. The data produced in biosynthetic studies can be less than straightforward even when closed in vitro systems are used. It is important to appreciate that the accumulation of metabolites merely indicates rate limiting steps in the pathway and that pool size does not necessarily reflect the importance of a compound as an intermediate in the biosynthetic sequence. It is quite poss-

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

99

ible for key intermediates in a pathway to be overlooked because they are rapidly metabolized and do not accumulate to any extent. Incubation of steviol in Cucurbita maxima preparations yields ent7a, 13-dihydroxykaurenoic acid and ent-6a,7a, 13-trihydroxykaurenoic acid (Graebe, 1974~).Surprisingly, in view of the data obtained by Bearder et a / . (1975b) with Gibberella fujikuroi there is no evidence of B-ring contraction to form 13a-hydroxy GA12aldehyde, GA53and other 13a-hydroxy CZO-GAs. Clearly there is a marked difference in the substrate specificity of GAl2 aldehyde synthetase in the two systems. 3 . Pisum sativum ( u ) Seeds. The 200,000 x g supernatant fraction from immature pea seed cv. Schnabel converts GAI2aldehydeto unknown metabolites and GA12alcohol, while the 2000 x g supernatant yields several products, two of which have been identified as the 13a-hydroxy GAS,GA44 and GAS3.Exactly how these GASare biosynthetically related has not yet been reported although the most likely sequences would seem to be GAlzaldehyde+GA12+GA53+ GA44 or alternatively GAIzaldehyde+ GA, 3aldehyde+ GAS +GA4, with GA,, alcohol being formed independently from GAl ,aldehyde in both instances. The activity of the enzymes involved in the production of these GAS is dependent upon the presence of Fe' i.and ATP in the incubating medium (Ropers et al., 1978). Although the pathways have not been fully elucidated in Pisum sativum it is evident that they are quite distinct from those operating in Cucurbira maxima and that 13a-hydroxylation occurs at a much earlier point than it does in Gibberellafujikuroi. The data provide no solace for proponents of the steviol hypothesis as 13a-hydroxylation takes place after rather than before B-ring contraction. The metabolism observed in vitro is probably closely related to that occurring in vivo as GA44 is an endogenous constituent of immature seed of Pisum sativum cv. Progress No. 9. The seeds contain relatively large amounts of GA20 and GA 29 and smaller quantities of GA9, GA17,GA3*,* GA44 and GA51 (Frydman et al., 1974; Sponsel and MacMillan, 1977). Frydman et at. (1974) used GC-SICM to quantitatively analyse GA9, GA1,, GAZ0and GA29 during seed maturation. They found that as the seed matures there is a sequential increase and subsequent decline in the levels of the individual GAS. The GA9 maxima occurs 20 days after anthesis, followed by the GA17and GAzopeaks two days later and GAZ9 at day 27 (Fig. 28). Against this background of changing endogenous GA levels, which presumably reflects changes in enzyme activity associated with *This identification has been withdrawn while further investigations are carried out as the mass spectrum upon which the characterization was based may have been that of the isomeric compound 2B-OH GAd4 (Sponsel era/., 1979).

100

ALAN CROZIER

0 15r

Days after anthesis

Fig. 28. Estimated endogenous levels of G A 9 , CAI,, G A Z Oand G A Z 9during maturation of seed of Pisum sativum cv. Progress No. 9 (Frydman et a / . , 1974).

seed development, GA metabolism studies have produced some intriguing data. The results of Frydman and MacMillan (1975), Sponsel and MacMillan (1977, 1978), Ropers et al. (1978) and Durley et al. (1979) have been used to compile the pathways illustrated in Fig. 29. At an early stage of seed development [15,17-3H]GA9undergoes 13a-hydroxylation and gives rise to [3H]GA20.However, the yields are < 10% and Sponsel and MacMillan (1977) point out that GA9 is unlikely to be the natural precursor of GAzoas at the time of the feed neither GA9 nor GAZ0are endogenous components of the immature seed. The conversion may well be a consequence of the lack of substrate specificity of the 13a-hydroxylase involved in the metabolism of GA12aldehydeto GA53.At a later stage of seed maturation, 13a-hydroxylation is suppressed and [15,17-3H]GA9acts as the precursor of [3H]GA51. This is believed to be an endogenous pathway as both substrate and product are now native constituents of the seed. [3H]12a-hydroxyGA9and a [3H]12a-

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

101

hydroxyGA, conjugate are also produced from [15,17-3H]GA9 at both stages of seed development. However, these are thought to be artifacts as neither compound has been isolated from Pisum sativum (Sponsel and MacMillan, 1977). If the biosynthesis of these compounds is due to low enzyme-substrate specificity, it seems possible that Pisum may naturally produce endogenous 12a-hydroxy GASwhich to date have not been detected. Although the CZo-GAprecursors of GAzohave not so far been determined, the conversion of GA44, or alternatively its hypothetical C-20 alcohol equivalent, to GA20via G A I 9is one option in view of the detection of GA44in Pisum seed extracts and its in vitro production from GA12aldehyde. GA19, which has recently been shown to be an endogenous Pisum GA (Ingram and Browning, 1979), may act as the precursor of G A L 7as well as GAzo(Fig. 29). The predominance of 13a-hydroxylation during the early stages of seed development, the high levels of endogenous GAZo,and the low GA, content imply that GAzo is on the main GA biosynthesis pathway and that GA9 originates via a minor route. No obvious intermediates between GA12aldehyde and GA9 have either been isolated from Pisum sativum or detected in metabolic studies. Metabolism of [ 14,15,17-3H]GA20during the later stages of seed development results in a 5&90% yield of [3H]GA29.Highest conversions are found when the time of the feed and the extraction of the tissue coincide with the respective maxima of native GAZ0and GA29. This suggests that the 2phydroxylation step is a normal endogenous process. Small quantities of conjugates of [3H]GAzo and [3H]GA29 also originate from [14,15,173H]GA20although neither conjugate has been found to occur naturally in Pisum. [3H]GA29fed to seed 27-28 days after anthesis is not metabolized to any extent over an eight day period. [3H]GA29produced in situ by feeding [14,15,17-3H]GAzois also metabolized at a very slow rate despite the fact that the endogenous GA29 pool rapidly declines (Sponsel and MacMillan, 1977). The identification of [3H]GA metabolites from Pisum sativum seed by Sponsel and MacMillan (1977) was based on GC-radioactivity retention times. Although mass spectra were obtained from mass peaks co-chromatographing with the radioactivity, this did not distinguish between ['HI endogenous and [3H] metabolite GA because the specific activity of the [3H]GAs was much too low for [3H] fragments to show up in the spectra. To overcome this problem Sponsel and MacMillan (1978) used precursor GAS labelled with [3H] and a high level of ['HI. The [3H] served as an indicator of metabolic processes while deuterated and non-deuterated species in the molecular ion cluster of mass spectra were used to estimate the ['HI metabolite and ['HI endogenous GA content. As would be anticipated from the results of previous experiments, differential rates of metabolism of exogenous and endogenous GA29 were observed

-OH

GA,,

aldehyde

CH2

CH3':

-OH+

GA44

-OH

+[Hypothetical]

COOH

0 -OH

G A 2 9 conjugate+

1

129-OH GAS

Compound B (tentative)

120-OH G A g conjugate

m,P -unsaturated ketone

Fig. 29. GA metabolism pathways in developing seed of Pisum sativum. Thick arrows represent established conversions and thin arrows indicate hypothetical steps.

103

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

TABLE XI Levels of endogenous and exogenous GAZ9 in developing Pisum sativum seed following ] (Ajtey Sponsel and MacMillan, 1978) the application of’ [ Z U - ~ H [2a-3H]GA29. Age of seed (days after anthesis)

Metabolism period (days)

21 30 33 36 40

0 3 6 9 13

Recoverable GAz9(pg seed -’) Endogenous Exogenous

7.7 3.6 1.9

1.5 1.2

5.1 4.5 4.1

3.6 2.8

2, recovery of exogenous GA29 100 79

12 63

49

when [2a-2H][2a-3H]GA29was fed to Pisum sativum seed 27 days after anthesis (Sponsel and MacMillan, 1978). At the start of the experiment each seed contained 5.7 p g of the deuterated substrate and 2.8 p g remained after 13 days, indicating a 49% recovery of the exogenous GA29. Over the same period the endogenous GA29 pool fell from 7.7 pg to 1.2 pg seed-’ which is 160,; of the original level (Table XI). The metabolism 0 f G A 2 9 resulted in a loss of label as, except for a minor accumulation of a putative GAZ9conjugate, no metabolites were detected. Presumably this was due to oxidation at C-2 and Sponsel and MacMillan (1978) have proposed that the data are consistent with the conversion of [2u-’H] [ ~ U - ~ H I to G the A ~a,B-unsaturated ~ ketone shown in Fig. 29 which is an endogenous component of both seeds and seedlings of Pisum sativurn. The data of Durley et a / . (1979) support this view, and indicate that [3H]GA29,formed from [2,3-3H]GA20,is converted to the [3H]ketonevia [3H]compound B which has been tentatively assigned the structure illustrated in Fig. 29. The [3H]ketone was the major labelled product in mature seed although moderate amounts of [3H] compound B were also present. Both compounds seem to be metabolized only slowly and Durley et ul. (1979) suggest that they serve as biologically inactive catabolite sinks for GAS produced during the development of Pisum seed. In other species this role is thought to be played by GA conjugates which are only minor constituents in mature pea seed. The [3H]a,/3-unsaturated ketone was not identified by Sponsel and MacMillan (1977) in their experiments with [ 14,15,17-3H]GA20 and [14,15,17-3H]GA29 because the relevant radioactive G C peak was attributed to the partially derivatized ether, mono trimethylsilyl ether G A 2 9 methyl ester. Sponsel and MacMillan (1978) have postulated that the slow rate of conversion of exogenous [ ~ u - ~[H~ ]u - ~ H I G compared A ~ ~ , to that of the endogenous species, is due to the cleavage of the 2a-H bond being subject to a primary isotope effect. In such circumstances, breakage of a C-2H bond

104

ALAN CROZIER

would be expected to be about eight times slower and that of a C-3H bond up to sixteen times slower than breakage of a C-’H bond. In support of this proposal Sponsel and MacMillan (1978) contend that endogenous GA29 and [lj?,3a-2H][lj?,3a-3H]GA29, produced in situ from [lj?,3~-~H][lp,3a-~H] CiAzo,are metabolized at equivalent rates by maturing Pisum seed. However the experimental data that are produced are not convincing. [1/3,3~-~H] [ ~ ~ ? , ~ U - ~ was H ] injected G A ~ ~into 23-day-old seeds and samples for analysis were harvested immediately and at two to four day intervals for a period of 13 days. The levels of recoverable endogenous and exogenous GAzo and Ga29 that were obtained are presented in Table XII. Estimates of the endogenous GA content show that the GAzo pool was depleted by day 9. Endogenous G A 2 9 was not detected in 23-day-old seed at day 0 although by day 9 it was present at a level of 10.3 bg seed-’. At the termination of the experiment four days later this had fallen to 7.7 pg seed- The estimated level of recoverable exogenous GAzo at day 0 was 5.4 pug seed-’. Thirteen days later this had all been metabolized and each seed contained 2.6 pg of GAZ9that had been formed by 2p-hydroxylation of the exogenous deuterated GAZO.This represents a 48% recovery of the applied label which is almost identical to the 49% obtained with exogenous [ ~ u - ~[H~ ]U - ~ H I G A ~ ~ . ]G metabolized A~~ at an equivalThe contention that [ lP,3a-’H] [ I / ~ , ~ U - ~ H is ent rate to endogenous GA29 while [2a-’H] [ ~ U - ~ H ] G is Ametabolized ~~ more slowly is, therefore, not based on differences in the rates of metabolism of the applied labels but on differential rates of disappearance of the endogenous GA29 pool in the two experiments. The reasons for this can be seen in Tables XI and XI1 and Fig. 28. When [2a-2H][2a-3H]GA29was applied to seed 27 days after anthesis, the endogenous GA29 pool was at its highest measured value containing 7.7 p g seed- and during the course of the 13 day

’.

’

TABLE XI1 Levels of exogenous and endogenous GAZoand GA29 in developing Pisum sativum seed following the application of [ I @ ,3a-’W [ I P , ~ U - ~ H I G(After A ~ ~Sponsel . and MacMillan, 1978)

Age of seed (days after anthesis)

23 25 28 32 36

Metabolism period (days)

0 2 5

9 13

Recoverable GA (pg seed-’) Endogenous Exogenous

% recovery of

exogenous GAZOGA19 GAzo GAZ9 GA,,andGA,, 2.4 3.2 2.3 -

-

1.3 5.3 10.3 7.7

5.4 2.9 0.9 -

-

1.6 2.4 3.2 2.6

100 83 61 59 48

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

! I

105

metabolism period it fell to 1.2 pg seed-’. When [ 1/3,3a-2H][l/3,3a-3H]GA20 was used as a substrate for [l/3,3a-2H][l/3,3a-3H]GA29the endogenous GA29 level peaked 32 days after anthesis, four days later than anticipated, and the experiment was terminated when the seeds were 36 days old. Hence, metabolism of the applied label was monitored for only four days beyond the endogenous GA29 maximum, whereas in the experiment with [2/3-’H][2/3 3H]GA29it was followed for 13 days. In view of this lack of synchrony and variance in metabolism periods it is difficult to draw any firm conclusions as [I/3,3a-3H]GA29 to the relative rates of metabolism of exogenous [ l/3,3~-~H] and [2/3-’H] [2/3-3H]GA29with respect to that of endogenous GAZ9.The dynamics of GA metabolism in developing Pisum seeds are inherently complex and experimental data are exceedingly difficult to interpret because (i) the kinetics of the system are only partially characterized, (ii) the label is applied as a pulse and not at a steady dose rate and (iii) the changing endogenous GA pool sizes clearly indicate that the system is not operating under steady state conditions. There is therefore a lack of persuasive evidence to support the suggestion that a primary isotope effect at C-2 is responsible for the slow rate of conversion of [2a-2H][2a-3H]GA29.It is, in fact, difficult to envisage how this model could account for the similarly slow metabolism of [ 14,15,17-3H]GA29.Other explanations that warrant consideration include the slow transport of exogenous GA29 to the metabolic site, although this is unlikely to be a limiting factor when labelled GA29 is produced in situ from exogenous GA20.However, differential rates of metabolism could occur if there were two pools of endogenous GA29, one arising from GAzoand the other from a different precursor. In this context it would be interesting to learn how GA51 and its precursor GA9 are metabolized by Pisum sarivum seed during the later stages of maturation. Although neither GA would be present in the seed in detectable quantities this does not necessarily eliminate them as potential intermediates in the synthesis of GA29 as endogenous pool sizes need not reflect rates of turnover. More informed discussion on this topic will only be possible when GA feeds are made throughout seed maturation instead of being restricted to the developmental stage at which the endogenous pool is maximal. ( b ) Seedlings. GA metabolism has also been studied in etiolated seedlings of Meteor dwarf pea. [3H] metabolites were identified on the basis of their GC retention characteristics using three different stationary phases, although in some instances mass spectrometric evidence was also obtained. The results are summarized in Table XIII. The fates of [3H]GA9 and [3H]GA20are similar to those observed in developing seed, while [3H]GA3accumulates after the application of 13H]GA5.The metabolism of [3H]GA14is of special interest as it involves the conversion of a Cz0-GAprecursor to two C19-GAs, GAI and its metabolite GAB.On the basis of metabolite pool sizes in a time course study, and the structural relationships of the GAS involved, Durley

106

ALAN CROZIER

TABLE XI11 Metabolism oj [ 3 H ] G A sby dark-grown Pisum sativum seedlings Precursor [17-3H]GA9

Metabolities 12a-hydroxyGA9,GAlo",GA20GA29, GA51

[2,3-3H]GA20 [l-3H]GA5 [17-3HlGAi4 [1,2-3H]GA,

GAZ9 GA3 GA,, ' 3 . 4 8 , GAi,, GA23, (3.438 GAS

Reference Railton et 01. (1974a,b) Railton et ul. (1974a,c) Durley et al. (1973) Durley et cd. (1974a,b) Durley et a / . (1974b)

This compound may be an artifact resulting from the sample being in prolonged contact with active sites on silica gel particles during either T L C or Woelm silica gel partition colum chromatography as described by Durley et a / . (1972) and discussed in Section 1I.D. Also see Grove (1961) and Hanson (1966).

rt 01. (1974a, 1974b) proposed a GAI4+GAl 8+GA38 +GA23 + G A +GAB

pathway with G A 2 8 being formed as a side branch from G A 2 3 (Fig. 30).The evidence for such a pathway is circumstantial as the metabolism of the potential CZO-GAintermediates in the sequence leading to GAl has not been investigated. It is impossible to assess the degree to which the conversions listed in Table XI11 and illustrated in Fig. 30 reflect native GA biosynthetic pathways operating in Pisum sativunz seedlings, because the low GA levels have so far precluded a detailed investigation of endogenous GAS. This is a major stumbling block and is reflected in the slow progress made in attempts to elucidate the regulatory role of GAS in stem elongation of pea seedlings. Much of the interest in this subject originates from the elegant studies of Lockhart (1956,1959) with the dwarf and tall Pisum cultivars Progress No. 9 and Alaska. These investigations showed that GA3 treatment promoted the growth rate of light-grown tall seedlings to that of etiolated plants. There was no observable effect when GA3 was applied to tall seedlings grown in darkness although when dwarf plants were used, GA3 promoted the growth of both light- and dark-grown material and the final heights were similar. Lockhart (1959) reasoned that light inhibited stem growth either by (i) inhibiting GA biosynthesis, (ii) enhancing the destruction of endogenous GA or (iii) decreasing the sensitivity of the seedling to GA. These mechanisms could also account for the reduced rate of stem elongation in dwarf varieties. Subsequent attempts to relate endogenous GA-like activity to rates of growth of pea seedlings have been based almost exclusively on bioassay data, and there are several seemingly contradictory reports in the literature. Jones and Lang (1968) estimated the GA content of light- and dark-grown Alaska and Progress No. 9 pea seedlings using agar diffusion and solvent extraction procedures. GAl-like and GA5-like compounds were detected

H CH3

‘’\

O coon

a CH2

-OH H CH3

CHZ

COOH GA8

Fig. 30. Proposed metabolism of [17-’H]GAI4by light-grown seedlings of Pisum sarivum cv. Meteor (Durley et al., 1974a,b).

108

ALAN CROZIER

when tissues were extracted, but only the GA -like component diffused from excised shoot apices into agar blocks. There were no significant differences in either diffusible or extractable GA-like activity obtained from light- and dark-grown dwarf and tall plants. Kende and Lang (1964) had previously obtained similar results to Jones and Lang (1968), but in addition demonstrated that while light- and dark-grown Pisum scrtivurn seedlings responded equally well to the CAI-like compound, the growth response of light-grown plants to the GA,-like substance was considerably less than that of darkgrown tissues. GA1 and GAS promoted growth in the same way as the endogenous GA,-like and CA,-like components. These observations support Lockhart’s third hypothesis which suggested that the inhibitory effects of light on stem growth are due to changes in the sensitivity of the seedling to GAS rather than to qualitative or quantitative changes in the GAS themselves. The physiological relevance of these experiments has, however, been questioned by Frydman and MacMillan (1973) who, after identifying GAzo and GA29 in immature pea seed suggested that these GAS and not GAS and GAl were responsible for the two GA-like compounds present in seedling extracts. Although Sponsel and Kirkwood (unpublished data quoted by Hedden et a / . , 1978) have since characterized GAzo and GA29 in Pisz.int seedlings it should be noted that GA29 induces only a relatively small response in GA bioassays (Reeve and Crozier, 1975; see Table VII). Thus, unless G A 2 9 is present in very high amounts, it is unlikely to account for the CAI-like zone of biological activity in Pisum seedling extracts. Kohler (1970) investigated GA levels in a tall variety of Pisunt (Schnabel) and a dwarf cultivar (Kleine Rheinlanderin) and estimated that light-grown plants contained more GA-like activity than dark-grown seedlings, and dwarf varieties more than tall. Thus, there was an inverse correlation between GA content and the rate of stem elongation. The projected GA levels are quite different from those determined by Kende and Lang (1964) and Jones and Lang (1968) and are not in full agreement with earlier data obtained by Kohler (1965). It is difficult to assess whether this is due to the use of different varieties of pea or merely a consequence of the use of different analytical procedures. Kende (1967) estimated that light- and dark-grown Progress No. 9 dwarf pea seedlings metabolize [l ,2-’H]GAI at a similar rate although [1-’H]GA5 is metabolized faster by dark-grown plants (Musgrave and Kende, 1970). Railton (1974a) reported that etiolated seedlings of dwarf peas cv. Meteor convert [l 7-’H]GA9 into [3H]GAzo-like and [3H]12a-hydroxyGA9-like compounds, more readily than light-grown plants, although metabolism into a polar metabolite is unaffected by light. The conversion of [2,3-3H]GA20 into a [’H]GAz9-like component appeared to be reduced in light-grown tissues. The loss of the applied label was not determined in any of these investigations ; instead the size of metabolite pools was monitored. In practice

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

109

this is of limited value as the various products are being further metabolized so that their rate of biosynthesis cannot be ascertained from pool sizes. The relative rates of metabolism in the light- and dark-grown seedlings are more readily assessed by estimating the amount of unmetabolized precursor GA remaining at set intervals after the application of a standard dose. When this is done, there is no significant difference in the rate of metabolism of [2,33H]GA9 applied to light- and dark-grown Progress No. 9 and Alaska pea seedlings (Dunbar and Crozier, unpublished data). There is, however, strong evidence to suggest that the rate of GA biosynthesis varies in these tissues. i n vitro studies by Ecklund and Moore (1974) have shown that the 78,000 x g supernatant from the shoot tips of light-grown Alaska pea seedlings has a greater capacity for synthesizing rnr-kaurene from mevalonic acid than similar preparations from Progress No. 9 seedlings. In addition, cell-free preparations from light-grown shoot tips of both varieties exhibited higher rnt-kaurene synthetase activity than equivalent extracts from their etiolated counterparts. Railton and Reid (l974a) detected six zones of GA-like activity in lightgrown Alaska pea seedlings although the total number of endogenous GAS is likely to be much higher than this figure. Other than GA2,, and GA29 their identity is at present a matter of conjecture, and the manner in which the levels of individual GAS vary in light- and dark-grown dwarf and tall seedlings, is a topic well into the realms of unproductive speculation. Investigations into endogenous GA levels in Pisum seedlings by Kende and Lang (1964), Jones and Lang (1968) and Kohler (1970) utilized what were, at the time, “state of the art” analytical procedures. However it is now evident that the experimental system is so complex and the shortcomings of bioassays, used in conjunction with chromatographic techniques of low peak capacity, so severe, that any similarity between estimates of GA-like activity and the true GA status of the samples will be extremely fortuitous. There have been considerable advances in the analytical and separatory sciences in recent years and from the discussion in Section 11, it is clear that procedures are becoming available which, if correctly used, can provide accurate analysis of trace components in highly impure samples. It will be of interest to see what progress is made when these methods are applied to the quantitative analysis of endogenous GAS from Pisum sativum seedlings. Despite the shortcomings of the estimates of GA-like activity in Pisum seedlings it is evident that slow growing tissues do contain GAS. Thus reduced growth rates are unlikely to be a direct consequence of a blockage at an early point in the GA biosynthesis pathway as it is in the d 5 mutant of Zea mays. It is therefore doubtful that comprehensive information on the levels of individual GAS in Pisum seedlings will, on its own, provide a clear view of the role played by GAS in regulating the rate of stem elongation. The dynamics of the systems will have to be carefully investigated and attempts

110

ALAN CROZIER

made to determine the rate of turnover of endogenous GAS as it is this, rather than pool sizes, which may determine the quantity of GA that is available for growth. Rates of turnover are notoriously difficult to estimate (see Brown and Wetter, 1972) and at the very least it is necessary to establish rates of synthesis, investigate the compartmentation of site of synthesis and catabolism and determine the rates of transport between them, before even rudimentary conclusions can be drawn. At the present time, the information available on G A biosynthesis pathways in seedling material in general is very limited indeed and even less is known of the kinetics involved. Railton (1974b) has reported that the rate of turnover of GA20in dwarf pea seedlings is enhanced by cytokinin treatment. Unfortunately, the data presented are insufficient to even start to meet the criteria listed above, and the conclusion must therefore be seriously questioned until a much more thorough study is undertaken. Other pieces of fragmentary evidence do, however, suggest that further patient experimentation will provide insights into the complexities of GA metabolism at the cellular and sub-cellular level. For instance, studies on the early stages of G A biosynthesis have shown that etioplasts isolated from dark-grown Pisum shoot tips, but not mitochondria, can convert [ 1-3H]copalyl pyrophosphate to [3H]ent-kaurene. In contrast [ 1-14C], geranylgeranyl pyrophosphate does not serve as an effective substrate for ent-kaurene production in the in vitro etioloplast preparations (Simcox et al., 1975). It is not known if this is due to the conversion of geranylgeranyl pyrophosphate acting as a regulatory point in the GA biosynthesis pathway, or a consequence of the instability of copalyl pyrophosphate synthetase. The biosynthesis of [ 14C]ent-kaurene from [ 14C]geranylgeranyl pyrophosphate has however been observed in sonicated chloroplast preparations from Pisum seedlings (Moore and Coolbaugh, 1976). Direct proof that mevalonic acid is converted to ent-kaurene is lacking, as isolation of the organelle in aqueous media results in the loss of mevalonic acid-kinase activity, which is required for activation of the initial steps in the terpenoid pathway. Chloroplasts isolated in a non-aqueous medium retain mevalonic acid-kinase activity (Buggy et a]., 1974), implying that they do have the capacity to synthesize ent-kaurene from mevalonic acid. Indirect evidence to support this view was obtained when Moore and Coolbaugh (1976) showed that the in vitro production of ent-kaurene from [2-'4C]mevalonic acid by 100,000x g supernatant preparations from Alaska pea shoot tips was enhanced by the addition of chloroplast enzymes. The synthesis of GAS from ent-kaurene has not been demonstrated in chloroplast preparations from peas or any other species for that matter. However, chloroplasts from Hordeum distichon incorporate ent-kaurenol into ent-kaurenal, and ent-kaurenoic acid into ent-7a-hydroxykaurenoic acid (Murphy and Briggs, 1975), while Hordeum vulgare chloroplasts have been shown to convert ['4C]ent-kaurenoic acid into an unidentified GA-like sub-

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

111

stance (Stoddart, 1969). Chloroplast-rich preparations from Pisum sutivum seedlings contain at least five GA-like substances (Railton and Reid, 1974a) and can also metabolize [3H]GA substrates (Railton and Reid, 1974b; Railton, 1977). The only report in which [3H]GA metabolites from chloroplasts have been convincingly identified involves the formation of 16,17 dihydro,l6,17dihydroxy GA9 from [17-3H]GA9,with GAlo,which may be an artifact for the reasons outlined in Table XIII, acting as an intermediary (Railton, 1977). On balance it seems that Pisum chloroplasts may well support the entire GA biosynthetic sequence. In quantitative terms it is difficult to gauge their contribution to the overall GA complement of the plant and a considerable amount of critical investigation will be necessary if detailed information on the involvement of chloroplasts in GA biosynthesis is to be forthcoming. 4. Phaseolus coccineus and Phaseolus vulgaris ( a ) Seeds. Large numbers of GAs have been found in seeds of the broad bean Phaseolus vulgaris and the scarlet runner bean Phuseolus coccineus. GAl,GA4,GA5,GA6, G A S , G A l 7 GA20, , GA29r GA37,GA38 and GA44r as well as GASglucosyl ether and the glucosyl esters of GA1,GA,, GA37and GA,,, have all been shown to be present in extracts from developing or mature seed of Phaseolus iiulgaris (West and Phinney, 1959; Durley er a/., 1971 ; Hiraga et a/., 1972, 1974a,b; Yamane et ul., 1977). The spectrum in Phaseolus coccineus seed is very similar as G A , , GA3,GA,, GAS,GA6,GAs, GA17, GA19, GA20,GA28, GA3,, GA38and GA,, have all been identified along with the glucosyl ether of GAS and undetermined conjugates of GA17, GAZoand GA28 (Jones, 1964; Sembdner et a/., 1968; Schreiber et al., 1970; Durley et al., 1971; Gaskin and MacMillan, 1975; Sponsel and Albone, unpublished data). Studies by Durley et al. (1971) with Phaseolus coccineus seeds of increasing size, indicate that the GA levels vary as the seeds mature (Fig. 31). The pattern is similar, although not identical, to that observed in Pisum sativum seed with the main GA present in the later stages of development being GASrather than GA29. Consideration of the structures of endogenous C19- and C 2 0 - G Ain ~ Phaseolus coccineus and Phaseolus vulgaris, with regard to hydroxylation at the 3P- and 13a-positions (see Table XIV) suggests that two biosynthetic pathways may be operating as the result of a branch at an early point in the sequence. One branch would involve 3P-hydroxylation and lead to GA37 and GA,, while the alternative route would result in 13ahydroxylation of a C20-GAand give rise to G A , 7 , GA19 and GA,, as well as GAS,GA6 and GA20.Convergence of the pathways would facilitate the production of 3P,13a-dihydroxy GAS, such as G A , , GA3, GAS,GA28 and GA38.More precise details of the operation of such pathways are not available as there are no reports on the metabolism of potential C20-GAinter-

3fi-hydroxy, 13a-hydroxy und 3fi.13a-dihydroxy

Species Phaseolus vulgtrris

TABLE XIV

c,9-and CzO-und C 2 0 - G Afrom ~ seed of Phaseolus vulgaris and Phaseolus coccineus. (After Sponsel et al., 1979)

__-

3fi-OH

~~

I3a-OH

~

~~~

3fi.13a-diOH

3B-OH

13a-OH

3/?,13a-diOH

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

4-

0 -

-

-

c

.

-

-

--

113

GA5/20

Seed length (rnm)

Fig. 31. Estimated changes in the endogenous levels of GA,, GASIZO, GA6 and GABduring maturation of seed of Phuseo/us coccineus (Durley ei d., 1971).

mediates in seed of either Phaseolus vulgaris or Phuseolus coccineus. However [3H] labelled endogenous C19-GAs have been fed to developing seed of Phaseolus vulgaris c. 10 days and 18 days after anthesis (Yamane et al., 1975, 1977). The conversions that were observed are summarized in Fig. 32. [1,2-3H]GA1applied to 10-day-old seed yielded [3H]GA8, [1,2-3H]GA4 was metabolized to [3H]GA8via [3H]GA1,while [2,3-3H]GAzowas converted to [3H]GAz9as well as [3H]GA1and [3H]GA8.[l-3H]GA5 did not yield any identifiable metabolites. Evidence of the existence of similar pathways was obtained with feeds to 18-day-old Phaseolus vulgaris seed. However, the kinetics of the system were different primarily because of a marked enhancement of the activity of glycosylating enzymes with [3H]GA8glucosyl ether being the major metabolite obtained from [l ,2-3H]GA1, [l ,2-3H]GA4, [1-3H]GA8and [2,3-3H]GA20.In addition, as shown in Fig. 32, [’H]GA1 glucosyl ester, [3H]GA4glucosyl ester and putative glucosyl ethers of both [3H]GA1and [3H]GA20were also formed. [1-3H]GA5 applied to the older

GA4glucosyl ester

GAI glucosyl ester

0

3 p - hydroxylation pathway

+-b

GAB glucosyl ether

HO CH3

COOH

CHZ

GA4

130- hydroxy lat ion pathway

-b+

G A ~ oglucosyl ester (tentative) GA20

GAS glucosyl ester (tentative ) GAS

t t

13a- hydroxylation pathway

GA29

Fig. 32. G A metabolism pathways in developing seed of Phaseolus vulgaris (Yamane et a!., 1975, 1977).

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

115

seeds yielded a putative glucosyl ether of t3H]GAS,['HIGAS glucosyl ether and [3H]GAB.['H]GA6 is the expected intermediate in the conversion of [1-3H]GA5to ['HIGAB. However, it was not detected although GAGis a naturally occurring constituent of the seed and it has been shown to be converted to GAB by Phaseolus coccineus (Sembdner et a/., 1968). The origins of GASare unknown and there were no indications of it being synthesized from either [1,2-3H]GA1or [2,3-3H]GA2~. The data suggest that the main GA biosynthesis pathway in Phuseolus vulgaris seed leads to GABand that its 3-deoxy analog, GA29ris on a minor route while in Pisum sativum the major pathway appears to proceed via GA29. The subsequent fate of the main 2P-hydroxy GAS in Phaseolus vulgaris and Pisum sativum is also different. In Pisum, catabolism of GA29 gives rise to an @-unsaturated ketone and compound B, with only small quantities of a putative conjugate of GA29 being detected (see Fig. 29). Glycoxylation is the dominant mechanism in Phuseolus vulgaris with GAB being readily converted to GAB glucosyl ether. However, the 3P-hydroxy derivative of the a,Punsaturated ketone has been detected in Phaseolus vulgaris seed (Sponsel et a/., 1979) implying that a portion of the GABpool may undergo catabolism by a route analogous to that operating in Pisum sativum. The 2P-hydroxylation of GAl to form GABhas been studied using a cellfree preparation from the cotyledons of germinating Phaseolus vulgaris seed (Nadeau and Rappaport, 1972; Patterson and Kappaport, 1974; Patterson et al., 1975). Enzyme activity located in the 95,000 x g supernatant, requires F e + + or F e + + + ,NADPH and O2 co-factors and is insensitive to CO. The conversion of [1,2-'H]GA1 to ['HIGAS involves the loss of [3H] from the 2-position which results in the stoichiometric formation of [3H]H20. The cell-free system exhibits high stereospecificity as it does not 28-hydroxylate ~ ~16-keto ) GA1. either pseudo GAl [ 3 ~ - h y d r o x y G A or ( b ) Seedlings. GC-MS has been used to identify GAl, GA4, GA5 and GAZ0in extracts of Phaseolus coccineus seedlings although bioassays indicate the presence of many more as yet unidentified GAS (Crozier et ai., 1971; Bowen et a/., 1973). The metabolism of a number of [3H]GAs has been investigated using seven-day-old light-grown seedlings (Nash and Crozier, 1975; Nash, 1976; Nash et al., 1982). [3H]GAs injected into the apical bud were retained, with little metabolism, in the apical region, whereas injection into the hypocotyl, 5 mm below the cotyledonary node, resulted in redistribution of label throughout the seedling and a more extensive conversion of the applied [3H]GA to other products. These observations are illustrated in Table XV with typical data obtained using [l ,2-3H]GA1. Basal injection of [3H]GAs was also associated with a more substantial accumulation of metabolites. This method of application was therefore used when investigating GA metabolism pathways in Phaseolus coccineus seedlings. Although bioassays indicate that light-grown Phaseolus coccineus seed-

116

ALAN CROZIER

TABLE XV Distrihutioir qf radioartivity in Phaseolus coccineus srrdlings 24 h nfter injecting [I,23H]GA1 inro either the upicnl bud (A) or the hypocotyl (B) (Nash and Crozier, 1975) distribution of [3H]" Tissue Apical bud Stem Cotyledons H ypocotyl Roots

A

B

95.8 0.5 2.5 0.3 0.9

12.1 55.4 11.7

18.2 2.6

"Overall recovery of applied label 66.37, (A). 24.6';b (B) ~

lings contain only nanogram quantities of endogenous GAS (Fig. 12, Table VIII), preliminary feeds with [3H]GAsrevealed that there were no qualitative changes in the [3H]metabolite profile when the substrate levels were increased from c. 50 ng to 10 pg seedling- '. The investigation into the effects of precursor dose was combined with an evaluation of the metabolism periods required to produce an accumulation of optimal levels of conversion products. Experiments using purification and analytical procedures outlined in Section I1 were then carried out to identify the various [3H]GA metabolites. After extraction of the plant tissues, acidic ethyl acetate-soluble and acidic n-butanol-soluble fractions were obtained. Ethyl acetate extracts were purified by SEC (Reeve and Crozier, 1976) which provided a convenient means of separating low molecular weight (LMW) free GAS from high molecular weight (HMW) GA conjugates. The putative conjugates in the HMW ethyl acetate fraction and the butanol extract were hydrolysed with cellulase to release free GAS. The extracts were then further purified on charcoal-celite before being subjected to preparative HPLC (Reeve et al., 1976). [3H]GA metabolite peaks were benzyl esterified and re-examined by preparative HPLC prior to being analysed by silica gel adsorption HPLC (Crozier and Reeve, 1977; Reeve and Crozier, 1978). Identification of the [3H]GAs was based on HPLC retention indices obtained with an on-line radioactivity monitor (Reeve and Crozier, 1977). When sufficient sample was available, characterizations were confirmed with mass spectra obtained by direct probe mass spectrometry. In view of the low levels of native GAS in Phaseolus coccineus seedlings, it is valid to conclude that the GA benzyl ester mass spectra were derived from metabolite GAS and not endogenous species. [1,2-3H]GA1, [1,2-3H]GA4 and [1-3H]GA8 feeds indicated a GA,+ GA1-+GA8 pathway with all three GAS yielding HMW conjugates. [2,3-3H]GAzo gave rise to a [3H]GAz0 conjugate and trace amounts of

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

1 I7

[3H]GA3.Although the seedlings metabolized [1-3H]GA5,no metabolites 3GA9was converted to [3H]GA5and [2,3-3H]GA9 were detected. [1-3H]d2~ to [3H]GA20and [3H]GA3.However, as neither precursor has been isolated from Phuseolus coccineus, it is unlikely that native GAS and GA20are synthesized by these routes. Their appearance as metabolites is probably the result of the low substrate specificity of the enzymes involved in the 13ahydroxylation of endogenous CZO-GAs.The results are summarized in Fig. 33. It is interesting to note that [1-’H]GA8 was metabolized far more rapidly than any of the other [3H]GAs,with only 7.9% of the applied radioactivity being recovered after a 2 h metabolism period. Much of the radioactivity became associated with a volatile component, presumably [3H]Hz0,which was lost via transpiration and during preliminary partitioning of extracts. The only [1-3H]GA8 metabolite to be detected was a butanolsoluble conjugate with properties similar to those of GAS glucosyl ether. This compound was present at low levels indicating that it was either a minor catabolite or that it was being converted to other products. The detection of potential catabolites of [1-3H]GA8 and the [3H]GA8conjugate may well have been obscured by rapid rates of turnover, as well as low specific activities resulting from partial or complete expulsion of [3H] from the 1 position on the ent-gibberellane skeleton. In an effort to obtain information on CZO-GAprecursors of GA4, GAS and GA20in Phuseolus coccineus seedlings, the metabolism of GA1zaldehyde and GA14 was investigated. The seedlings rapidly metabolized [17-3H]GA14. After 8 h, only 52% of the applied radioactivity remained, distributed between [17-3H]GA14(35.5%) and a [3H]GA14conjugate (16.5%). No free [3H]GAmetabolites were detected in the LMW ethyl acetate extract, perhaps because of an absence of rate limiting steps in the ensuing metabolic sequence or conversions associated with a loss of [3H] from C-17. O n occasions, preparative HPLC traces indicated the presence of very small amounts of LMW [3H]GAmetabolites but their appearance was transitory and attempts to induce their accumulation with GA3and GA4cold traps were unsuccessful. In contrast to [17-3H]GA14, [17-3H]GA12aldehyde gave rise to several metabolites. This is illustrated in Fig. 34 which shows preparative HPLC traces of the LMW and hydrolysed HMW ethyl acetate extracts and of the hydrolysed butanol fraction. Most of the metabolite peaks yielded two to three products on benzyl esterification. Despite their number, none of the metabolites had HPLC retention characteristics corresponding to those of either G A l , GA4,GA5or GAzo.Because the applied label became associated with so many products, none of the metabolites was present in sufficient quantity to permit identification by mass spectrometry. The experiments therefore provide no positive information on the endogenous C20-GAprecursors of C1,-GAS in Phaseolus coccineus seedlings. Crozier and Reid (1971, 1972) investigated the involvement of roots in

GA4 conjugate

A2v3 GA,

GA, conjugate

GA5

Fig. 33. G A metabolism pathways in light-grown Phaseolus roccineus seedlings (Nash, 1976; Nash et al., 1982).

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

119

Retention time (min 1

Fig. 34. Preparative HPLC traces of LMW and hydrolysed H M W ethyl acetate extracts and the hydrolysed butanol-soluble fraction obtained from light-grown Phaseolus coccineus seedlings eight hours after applying [l 7-’H]GAI2aldehyde. Column, stationary phase, mobile phase and flow rate: as in Fig. 7. Detector: radioactivity monitor 1800 c.p.m. full-scale deflection (Nash, 1976; Nash et al., 1982).

GA biosynthesis in light-grown Phaseolus coccineus seedlings. Extracts from

control plants and seedlings from which root apices had been removed, were chromatographed on a Powell and Tautvydas (1967) silica gel partition column. Twenty-five fractions were collected and analysed with the Tanginbozu dwarf rice (Murakami, 1968) and barley aleurone bioassays (Jones and Varner, 1967). Several zones of GA-like activity were detected with major

120

ALAN CROZIER

peaks being located in fractions 7-9 and 12-15. A comparison of the biological activity in fractions 12-15 with the data of Crozier et al. (1971) suggested that GAl was present. On similar grounds, it was thought likely that fractions 7-9 contained GA19.However, GAS and GAZ0also elute in this zone and the identification of these GAS in extracts from Phaseolus coccineus seedlings by Bowen et al. (1973) implied that they, rather than GA19,were responsible for the GA-like activity in fractions 7-9. Removal of the root apices from seedlings resulted in the disappearance of the GA,-like activity, which was the major peak in control plants, and a concomitant increase in activity in the GAS and/or GAZ0zone. The data therefore suggest that shoot-synthesized GAS and/or GAZ0may be transported to the root apices where conversion to GA1 occurs. Subsequent studies have, however, failed to produce any evidence to support this hypothesis. [1-3H]GASand [2,3-3H]GAz0applied to the shoot apex of Phaseolus coccineus seedlings are not transported to the roots in substantial quantities ; neither precursor appears to be conkerted to GA1 and there are no marked differences in the [1-3H]GASand [2,3-3H]GAzometabolite profile obtained from intact seedlings, excised shoots and excised roots (Nash and Crozier, 1975; Nash, 1976; Nash et al., 1982). Railton (1979) has also reported that the transport of [2,3-3H]GAZ0is seemingly not compatible with the proposals of Crozier and Reid (1971, 1972). Further detailed experimentation will therefore be necessary if the significance of the changes in GA-like activity in Phaseolus coccineus seedlings following root surgery is to be determined. The effects of light on the GA metabolism and growth of Phaseolus coccineus seedlings have been investigated by Bown et a / . (1975) and the results are somewhat different to the data obtained with Pisum sativum discussed in Section III.C.3b. Light inhibits the rate of stem elongation in Phaseolus coccineus seedlings and GA promotes growth in light but not in darkness. This indicates that the availability of GA is a limiting factor only in the growth of light-grown plants. However, the effect of light is not solely to reduce the amount of GA available for growth as its inhibitory effects on stem elongation are not completely counteracted by application of exogenous GA (Fig. 35). The rate of metabolism of applied [3H]GAsis much higher in light-grown plants. In addition, light lowers the estimated level of endogenous GA-like activity c. 20-fold, although the means by which this reduction occurs is by no means clear as pool sizes merely represent the balance between rates of GA formation and utilization. There is a lack of information on rates of GA synthesis so it would be premature to assume that the increased capacity for GA metabolism is the major causal factor in the reduction of GA levels in light-grown plants. This is an important consideration as ultimately the amount of GA available for growth is more likely to be related to the rate of GA synthesis than be regulated by GA pool sizes. While the relevance of the metabolism of [3H]GAs to the growth process is un-

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

121

'"1

--g 120h

c

g

w

100-

80-

60-

40)

0

,

01

10

70

100

1

700

Gibberellin A, ( p g )

Fig. 35. Effect ofexogenous GA4 on stem growth of light- and dark-grown Phaseolus coccineus seedlings. Data expressed as mean stem length i standard error (Bown et al., 1975).

determined, the possibility of secondary control of GA availability through the operation of a competitive utilization pathway cannot be excluded. For instance, although there is a reduced capacity to metabolize GAS in darkgrown Phaseolus coccineus seedlings a greater proportion may be involved in growth than in light-grown plants in which metabolism could be primarily associated with de-activation or diversionary pathways. 5 . Conversion of CIO-GAs to CI9-GAs The studies by Graebe et al. (1974a,b,c, 1980) with Cucurbita maxima cell-

free preparations, indicate that the oxidative sequence at C-20 prior to the formation of C19-GAs,follows the general scheme outlined in Fig. 36, with the C-20 aldehyde acting as the immediate precursor of both y-lactonic CI9-GAsand C-20 carboxylic CIo-GAs. The main point of uncertainty in the pathway is the identity of the intermediate between the C-20 methyl and C-20 aldehydic CIo-GAs. d-lactonic CIo-GAs do not appear to fulfil this role as GA15undergoes 3P-hydroxylation to form GA37which is not further metabolized. Although critical evidence is lacking, it may be that the hypothetical C-20 alcohol shown in Fig. 36 is the intermediate in the conversion. On extraction, C-20 alcohol CIo-GAs would almost certainly lactonize to

122

R

ALAN CROZIER

Q-1-

CH3

RJ-J]-RG}-R CH,OH H

CH \H 3 COOH C - 2 0 methyl Cz0-GA

CH3 \COOH C-20olcohol CZaGA [ hypothetical I

H

CH \COOH C-20aldehydic C2cGA

CH3 7 - lactonic C,,-GA

‘a} Jj

0 ---- CH, R

CH3 8-lactonic C G -,A

\H CH3 COOH C - 2 0 carboxylic C2G ;P

Fig. 36 Oxidation sequence at C-20 prior to the conversion of C,,-GAs to CI9-GAs by Cucurbita maxima cell free preparations. R-H or OH

produce S-lactonic C20-GAswhich would explain why they have never been isolated as endogenous constituents. This suggestion also implies that 6lactonic GAS such as GA15,GA37rGA38 and GA44 are artifacts, although the possibility that they are also natural products of GA metabolism cannot be ruled out. Gibberella fujikuroi has been widely used to investigate the conversion of Czo-GASto C19-GAs although the results are less straightforward than those obtained with Cucurbita maxima. Fungal cultures convert GA12 to GA9 and GA14to C A I but the point in the pathway at which the C-20 group is expelled has not been demonstrated (Fig. 26). The Czo-GASoriginating from GA12 and GAI4, represent successive stages in the oxidation of the C-20 methyl group and it was originally thought that a C-20 carboxyl function might be removed by oxidative decarboxylation. This now seems unlikely as neither GA13nor GAZ5are converted to CI9-GAsby Gibberella jujikuroi (Cross e t a / . , 1968b; Bearder e f al., 1975a). Dockerill et al. (1977) observed that GAl 3aldehyde is incorporated into GA4,7;(0.9%)and GA3 (12.9%). However, Bearder and Phinney (1979) have shown that the B1-41a mutant converts GA13aldehydeanhydride to GA13 aldehyde (80%) and GAI3(2073, while GA13 alcohol anhydride is metabolized to GA13alcohol(98%) and trace quantities of GA13and GA,,aldehyde. As no C19-GA metabolites were detected it was concluded that GA13 aldehyde, GAl 3alcohol and their anhydrides (Fig. 37) do not act as precursors of CI9-GAs in Gibberella. Although Hanson and Hawker (1972) have reported the conversion of GA13anhydride (Fig. 37) to GA3 by Gibberellu cultures the data are equivocal as only 0.015% of the applied label was incorporated into GA,. Evidence obtained by Bearder et al. (1976b) con-

123

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

GA12 alcohol

HO CH3 COOH

CH20H

CHZ

GA13alcohol anhydride

GA13 alcohol

COOH

GA13 aldehyde anhydride

GA13aldehyde

0----co

GA,3 anhydride

Fig. 37. Structures of GAI,alcohol, GA,,alcohol. GA,,alcohol anhydride, GA,,aldehyde, GA,,aldehyde anhydride and G A , ,anhydride.

vincingly demonstrates that the h-lactone G A , 5 is not an intermediate in the conversion of G A 1 2to GA9, and by analogy, the involvement of GA3, in the biosynthesis of GA4 from GA14 is similarly excluded. However, the data do not rule out the possible involvement of the corresponding C-20 alcohol CIO-GAs in the production of C19-GAs. Unexpectedly, in view of the synthesis of GA, from GA36by Cucurbitu maxima, Gibberella fujikuroi does not convert the C-20 aldehydes, GA24 and GA36rto CI9-GAs (Bearder et ul., 1975a). This could, however, be due to poor substrate penetration of the fungal hyphae. Cross and Norton (1966) speculated that C-20 was lost by decarboxylation of a B,y-unsaturated acid. This theory was disproved when Hanson and

124

ALAN CROZIER

peracid oxidation

Fig. 38. A Baeyer-Villiger oxidation.

0

\ /

0

\ /

CH3

C

0

CH3

C I

'80

@-D

Fig. 39. A biological Baeyer-Villiger reaction : the metabolism of pregn-4-ene-3,20-dione by CIadesporrum resinae (Nakano et a / . , 1968).

White (1969) demonstrated that all the hydrogen atoms at the C-1, C-5 and C-9 positions were retained in the transformation of C2,-GAs to C ,-GAS by Gibberella jiijikuroi. It was postulated by Hanson and White (1969) that the C-20 group might be lost at the aldehyde stage by a Baeyer-Villiger (1 899) type oxidation. Baeyer-Villiger oxidations are unusual in that they involve the insertion of an oxygen atom a to a carbonyl function to form an ester (Fig. 38). Traditionally, the reaction is associated with the action of a peracid. Although this is difficult to envisage in a biological system, the existence of analogous enzymic processes is a distinct possibility as BaeyerVilliger type oxidations are quite common in the microbial degradation of steroid ketones (Fig. 39) (Smith, 1974; see Bearder and Sponsel. 1977). In

TABLE XVI Metabolites produced from [19-"0]GA12 alcohol, containing 55 atom % ["O], by Gibberella fujikuroi mutant Bl-4Ia (Bearder et al., 1976bj Metabolite

"801%

125

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

the case of the C-20 aldehydic C20-GAstructure (VI) illustrated in Fig. 40, a Baeyer-Villiger type oxidation would produce an intermediate (VII) which could undergo acyl-oxygen fission, hydrolysis and lactonization to form a CIg-GA (VIII)

a 1 a] H

\/

O

/O

H-C?

' 0

Baeyer - Villiger

R

Alkyl- oxygen

____t

oxidation

R

CH3 "0

2,

"OH

(E)

F Lfission yI-oxygen

R

H

CH3

(mn)

Half label retained

7-

} J ? H' $ 80R

H

H+

H3

k H +

] J - J R

H CH3

(X) All label retained

Fig. 40. Proposed mechanisms for the conversion of C2,-GAs to CI9-GAs by Cihberelln fujikuroi. R = H or OH (after Bearder and Sponsel, 1977).

Bearder et al. (1976b) obtained an insight into the mechanism involved in the loss of the C-20 group during the conversion of C 2 0 - G Ato ~ C19-GAsby Gibberellafujikuroi. [19-'80]GA12alcohol (Fig. 37) containing 55 atom % of ['*O] was fed to cultures of the GA deficient mutant B1-41a and the metabolites analysed after periods of two and five days. In the virtual absence of native GASany fall in the ["O] content of the metabolites, as determined by mass spectrometry, is due to loss of label rather than dilution by endogenous ['60]GAs. The results presented in Table XVI show that both oxygen atoms are incorporated into the y-lactone in the C-19 carboxyl group of C20-GA~ ring ofC19-GAs.Bearder et al. (1976b) concluded that GA,,alcohol and its metabolites are not covalently bound through the C-19 carboxyl group to enzymes catalysing the formation of C19-GAs, although this view is debatable since knowledge of the enzymes involved and their surrounding

126

ALAN CROZIER

microenvironment is lacking. However, the data do show that the conversion of CZ0-GAs to C19-GAs involves an intermediate with an electrophilic centre at C-10 which undergoes nucleophilic attack by the C-19 carboxyl group to form the 1'-lactone. Bearder and Sponsel (1977) point out that this rules out the lactonization mechanism of VII discussed above, as this would involve loss of half the ['*O]label (Fig. 40). They propose an alternative mechanism in which the intermediate ester (VII) is eliminated by alkyl-oxygen fission rather than acyl-oxygen fission. The carbonium ion (IX) could then cyclize by attack of the C-19 carboxyl group to generate the 7-lactone C19-GA (X) which would contain both oxygen atoms from the C-19 carboxyl group (Fig. 40). A pragmatic touch, in view of the uncertainty concerning the immediate CzO-GAprecursor of CI9-GAsin Gibberella, is that the model is flexible as in addition to the C-20 aldehyde (VI), the carbonium ion (IX) could be generated from Czo-GAs with either a C-20 carboxyl or alcohol group. Dockerill and Hanson (1978) have reported that the C-20 carbon of CZO-GAsformed from ['4C]ent-kaurene is released as ['4C]COz during the production of C I9-GAs by Gibberella fujikuroi. Dilution analysis failed to find any radioactivity associated with either formaldehyde or formic acid. Dockerill and Hanson (1978) postulated that the entire sequence from a putative peracid intermediate (XI) to the formation of a y-lactone CI9-GA (XII) may occur via a single concerted process (Fig. 41). This proposal, along with that of Bearder and Sponsel(l977) illustrated in Fig. 40, is highly speculative. The data presented by Dockerill and Hanson (1978) should not be regarded as irrefutable proof of the immediacy of C 0 2 to the expulsion of the C-20 carbon from CZo-GAs.It is possible, for instance, that the ['4C]C02 arose from small ['4C]labelled pools of either formaldehyde or formic acid which were not detected because of the inherent lack of sensitivity of dilution analysis. At present the only firm conclusion that can be made is that the conversion of CZO-GAsto C19-GAs by Gibberella fujikuroi must in effect follow the general scheme in Fig. 42. Far too little is known to permit meaningful speculations about the oxidative level at C-20 prior to its expulsion. The fact that Czo-GAs representing all stages of C-20 oxidation are produced by the fungus is of little value since ifa Baeyer-Villiger type mechanism is operating, and this is by no means certain, the insertion of oxygen between C-10 and C-20 could occur at any stage from the alcohol to acid. Alternatively, the entire sequence from a C-20 methyl Czo-GA onwards could take place without the intermediates involved leaving the enzyme complex. It seems likely that progress will be enhanced by the development of a cell-free system from Gibberella fujikuroi which can efficiently convert CZo-GAsto C19-GAS. In view of the in vitro metabolism of GA36 to GA4 by Cucurbita maxima (Graebe et al., 1980), it will be of interest to learn if the inability of Gibberella

127

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

(XII) Fig. 41. Concerted decarboxylation and lactonization of a hypothetical C,&A intermediate. R = H or O H (Dockerill and Hanson, 1978).

peracid

Fig. 42. General schematic for C,,-GA formation in Gibberellafujikuroi.

fujikuroi to carry out similar conversions in vivo is due to either (i) a major difference in the mechanism of C19-GA biosynthesis or (ii) a limited availability of substrate at the enzymic site in the fungal hyphae. Exact details of the conversion mechanism will, however, probably remain a mystery until such time as a purified, characterized enzyme is isolated that can synthesize a CI9-GAfrom a CZO-GAsubstrate. D. SITES OF GIBBERELLIN BIOSYNTHESIS AND COMPARTMENTATION

The sequential changes in endogenous G A levels associated with maturation of Pisum sativum seed occur in material grown in vitro as well as in vivo (Sponsel and MacMillan, 1977). It is also known that the endogenous GA pools in detached cultured Pisum fruits are depleted by AMO-1618 treatment (Baldev et a / . , 1965). Thus, if Pisum sativum is typical, it appears that GASare synthesized in the developing seeds rather than being imported from the parent plant. The incorporation of [2-14C] mevalonic acid into GAS by liquid endosperm preparations from Marah macrocarpus (West, 1973) and Cucurbita maxima (Graebe et al., 1972) provides direct evidence of GA biosynthesis in seeds as does the conversion of ['4C]ent-kaurene to1 GA aldehyde by cell-free preparations from Pisum sativum seed (Ropers et a/., 1978). Coolbaugh and Moore (1972) have shown that ent-kaurene synthetase is principally located in the cotyledons of immature Pisum seed and is not found in either the embryo or the seed coat.

128

ALAN CROZIER

The embryo of germinating barley grain may be a site of GA biosynthesis although the evidence is circumstantial and based on reports that (i) the production of GA-like activity in excised embryos is inhibited by CCC and phosphon D, and (ii) exogenous GA can substitute for the embryo in the induction of a-amylase synthesis in the aleurone layer of germinating seed (Paleg, 1960; Yomo, 1960; Yomo and Iinuma, 1966; Radley, 1967). Despite careful experimentation, Murphy and Briggs (1973) were unable to obtain any metabolic conversion of ['4C]ent-kaurene by cell-free preparations from germinating barley seeds. They did, however, show that the endogenous entkaurene content of the grain falls during the first twelve hours of germination. While this implies that in vivo metabolism of ent-kaurene takes place, it does not necessarily mean that GASare among the products. There is a paucity of critical evidence on sites of GA biosynthesis in seedling systems. Most, ifnot all, of the available evidence is physiological rather than biochemical in nature and is based on the detection of GA-like activity in xylem sap (Carr et al., 1964; Phillips and Jones, 1964) and the subsequent experiments of Jones and Phillips (1966, 1967) which indicated that the young leaves of the apical bud and the root apices of Helianthus annus seedlings act as sites of GA biosynthesis. Jones and Phillips (1966,1967) showed that when excised apical buds from Helianthus seedlings were incubated on agar for twenty hours, more GA, as determined by PC and bioassay, diffused into the agar than was extracted from the tissue. As no reduction in the extractable GASwas observed during the diffusion period, it was concluded that the apical buds had synthesized GAS in amounts corresponding to the level of diffusible GA-like activity. Similar results were obtained with root apices, although stem internodes and sub-apical root sections appeared not to act as sites of GA biosynthesis. Other publications followed with GA-like activity being detected in phloem exudate (Hoad and Bowen, 1968) and in the xylem sap of a range of species (Reid and Carr, 1967; Skene, 1967; Jones and Lacey, 1968; Luckwill and Whyte, 1968; Reid and Burrows, 1968) and the levels were shown to be affected by CCC (Reid and Carr, 1967), excision of various organs (Sitton et al., 1967; Crozier and Reid, 1971, 1972) and flooding of the root system (Reid et al., 1969; Reid and Crozier, 1971). In strictly biochemical terms, these reports demonstrated very little regarding sites of GA production and as a consequence, the widely accepted view that GAS are synthesized in root and shoot apices is, in essence, solely based on the results of Jones and Phillips (1966, 1967). This is unfortunate as these data provide only indirect physiological evidence, and with the passage of time they have begun to look somewhat dated and less convincing. This is because the supposition that root and shoot apices synthesize GA is dependent upon the PC-bioassay data of Jones and Phillips (1966,1967) providing a relatively accurate quantitative measure of the GA content of the tissues and agar diffusates. In view of the

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

129

shortcomings of bioassay-based estimates discussed in Section I1 .E.1, it is difficult to accept such data at face value especially as comparisons were made between GA-like activity in highly impure plant extracts and diffusates containing considerably less inhibitory material. There is therefore, despite much interest in the subject, an absence of compelling evidence on potential sites of GA biosynthesis in seedlings. Although in the 1960s there were no viable alternatives to the analysis of GASin shoot and root apices and diffusates by bioassay, this situation is now changing. If the main endogenous GAS in Heliunthus unnus seedlings could be identified, it should be feasible to employ the highly sensitive GC-SICM or HPLC procedures reviewed in Section II.E.2. to monitor diffusible and extractable GA levels. The Helianthus system appears to be particularly suitable for a detailed re-investigation and it is perhaps surprising that it has not already taken place, as Phillips (1971) reported that addition of mevalonic acid to the agar on which excised tissues are incubated, increased the levels of GA-like activity diffusing from shoot apices to such an extent, that microgram quantities of GA3 equivalents were produced by only fifty apical buds. This holds out the prospect of a considerable easing of the analytical situation. What is more, if the increased GA levels are due to enhanced biosynthesis, it may be possible to demonstrate an incorporation of radioactive mevalonic acid into GAS and this would certainly constitute unequivocal evidence of shoot apices acting as a site of GA biosynthesis. Evidence for the compartmentation of GAS in chloroplasts has already been outlined in Section III.C.3b. Other cellular components that might be involved in GA biosynthesis and metabolism include etioplasts, proplastids and vacuoles. Both vacuole and proplastid fractions from Hordeum vulgare convert [I ,2-’H]GA1 to [3H]GA8 and [3H]GA8 glucosyl ether (Rappaport and Adams, 1978). It is also known that proplastid extracts from Marah macrocarpus endosperm catalyse the synthesis of ent-kaurene from geranylgeranyl pyrophosphate and copalyl pyrophosphate although similar preparations from etiolated Pisum sutivum shoot tips and Ricinus communis endosperm convert only copalyl pyrophosphate to ent-kaurene. There is little or no enf-kaurene synthetase activity in the mitochondria1 fraction from these tissues (Simcox et a / . , 1975). Chloroplasts are impermeable to mevalonic acid so the precursor must be produced in situ (Rogers et ul., 1966). In contrast mevalonic acid can be transported across etioplast membranes although the capacity is lost following illumination (Cockburn and Wellburn, 1974; Wellburn and Hampp, 1976). There is currently no biochemical evidence to implicate etioplasts in the later stages of GA biosynthesis although bioassay data has been used to establish a link between GA-like activity and phytochrome in etioplasts of Hordeum vulgare and Triticum aestivum. The subject has attracted much attention and there is a general belief that compartmentation of endogenous

130

ALAN CROZIER

growth regulators coupled with environmentally-mediated release mechanisms could offer a ready solution to many problems of plant development including the interchangeability of red light and exogenous GA in the control of leaf unrolling in etiolated cereals (see Loveys and Wareing, 1971b; Stoddart, 1976). Reid et al. (1968) were the first to show a substantial transient rise in endogenous GA-like activity 15 min after treating etiolated barley leaf segments with red light (660 nm). This was thought to be the result of an enhanced rate of GA biosynthesis as no increase in activity occurred when the leaf sections were pretreated with either AMO-1618 or CCC. Evans and Smith (1976a) obtained similar data with authenticated barley etioplast preparations and also showed that far-red light (730 nm) reverses the red lightinduced increase in GA-like activity (Fig. 43). Thus, the effects of light on G A-like substances are mediated via phytochrome which appears to be located in the etioplast envelope (Evans and Smith, 1976b). It is also known that the GA-like activity that accumulates after red light treatment becomes associated with the incubating medium and is not retained by the etioplasts to any extent and that sonication of non-irradiated etioplast preparations results in a four-fold increase in extractable GA (Table XVII) (Evans and Smith, 1976a). Evans and Smith (1976a) propose that red light induces the photoconversion of phytochrome from the P, ,to the P,, form so facilitating changes in membrane permeability which enable GAS in the etioplast to be

Fig. 43. The effect of red and far-red light on the level of GA-like activity extracted from intact Hordeum vulgare etioplasts. GA-like activity is measured by a modification of the barley aleurone bioassay of Jones and Varner (1967). LSD-least significant difference when P=0.05 (Evans and Smith, 1976a).

131

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

TABLE XVll GA-like uctivity extracted f r o m intuct Hordeum vulgare rtioplust prrpurutions und rtioplasr prrpurutions ultrusonicutrd prior to rxtruction. ( A f t e r Evuns und Smith, 1 9 7 6 ~ ) “

Etioplasts

Dark

5 min red light

T

T

T

P

S

4.0 17.5

10.8 17.9

22.0 24.9

3.0

24, I

5 min red light+5 min dark

_________~~ ~

Intact Sonicdted

~

-

GA-like activity determined by a modified barley aleurone bioassay (Jones and Varner, 1967) and data expressed as pmol maltcse rnin mg I protein. Extracts made from either the total suspension (T) or the pellet (P) and supernatant (S) obtained by centrifugation o f T for 1 min at 6000 x g.

-’

released into the surrounding medium. They further suggest that the depletion in GA levels resulting from this efflux leads to increased production of active GASpossibly by release of feed back control of late steps in the biosynthetic pathway. Similar investigations have been carried out with wheat, in which a 5 rnin exposure of etiolated leaf segments to red light also induces a large increase in endogenous GA-like activity which peaks after about 15 rnin and thereafter rapidly declines (Beevers et a/., I970 ; Loveys and Wareing, 1971a). The response has been associated with etioplasts and it has been proposed that the controlling influence of phytochrome is exerted through changes in the permeability of the etioplast envelope to GAS, the release of GAS from a “bound” form and an enhancement of de novo GA biosynthesis (Cooke and Saunders, 1975a,b; Cooke et al., 1975). Cooke and Kendrick (1976) further suggest that the etioplast envelope may be a site of GA metabolism and one effect of red light could be to induce the release and subsequent metabolism of “bound” GAS. However, when the experimental data are closely scrutinized, these proposals appear to be distinctly speculative. Estimates of GA-like activity in the wheat etioplast preparations are based on lettuce hypocotyl bioassay data. In most instances the bioassay responses are far too small to command confidence and the claimed variations in GA levels must therefore be of questionable significance (Graebe and Ropers, 1978). What the data from Triticum aestivum and Hordeum vulgare do demonstrate is that the red-far-red reversible changes in GA-like activity are controlled by phytochrome which seems to be located in the etioplast membrane. As Hedden et al. (1978) noted, it is unclear whether the increases in GA-like activity in etioplast preparations are due to an enhanced rate of synthesis, increased metabolism of specific precursor GAS, release of “bound” GAS from membranes, increased membrane permeability or a

132

ALAN CROZIER

combination of these and other factors. Progress in identifying the mechanisms involved is unlikely to be spectacular because the technical problems encountered require investigators to have not only expertise in GA analysis and metabolism but, also, a theoretical and practical knowledge of phytochrome and experience of procedures for the isolation of organelles from plant tissues. IV. STRUCTURE-ACTIVITY RELATIONSHIPS The relative activities of individual GAS in the barley aleurone, dwarf pea, lettuce hypocotyl, Tanginbozu dwarf rice and cucumber bioassays are presented in Table VII. The data are compiled from Crozier et a / . (1970), Yokota et a / . (1971), Fukui et a / . (1972), Yamane et a / . (1973), Reeve and Crozier (1975), Hoad et al. (1976) and Sponsel et a / . (1977). Most of these reports, as well as those of Brian et a / . (1964, 1967) contain data on other bioassays but the information is less comprehensive, at least as far as natural GASare concerned. There is, unfortunately, little or no data available on the biological activity of most of the more recently discovered GAS. It can be seen from Table VII that each GA exhibits a range of biological activities and that each bioassay responds to a characteristic spectrum of GAS. High activities are shown in most bioassays by GA1, GA3, GA, and GA32; GAS, GA6, GA36and GA3, all induce a good response but of a lower order. Other GAS, in particular GA9, GAlo, GAZ3,GA24 and GA3s show a tendency for species specificity, being highly active in some bioassays yet inducing poor response in others. Consistently low activity is exhibited by GAS, GA1 GAi2,GAi3rGA14,GAi7,GA21,GA25,GA27,GA2srGA29,GA33,GA34

and GA40; while GA26,GA40, GA43rGA46 and G A 5 1 are inactive in most test systems. Some GA glucosyl esters and ethers induce a response in certain bioassays. However, it is thought that they are probably inactive per se and that their activity is a consequence of the release of the aglycone following hydrolysis by plant enzymes and micro-organisms (Yokota er a / . , 1971; Sernbdner et a/., 1976). The individual bioassays vary in their specificity. The barley aleurone bioassay, for instance, responds only to a limited number of GAS while, in contrast, the dwarf rice bioassay responds in some degree to all the GAS that have so far been tested with the exception of GA21rGA25, GA26, GA43rGA46 and GA51. It has been hypothesized that there are two main factors playing an important role in GA structure-activity relationships (Brian et al., 1967; Crozier et a/., 1970). The “goodness of fit” theory contends that the activity of a GA arises from the degree to which it fits a hypothetical receptor molecule or site. Thus any alteration in the “ideal” GA structure will lead to a lowering of biological activity. Variations in the shape of the receptor molecule from one plant species to another will account for the different re-

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

133

sponse spectra of the assay systems. It is further proposed that the response of a bioassay can be affected by the ability of the plant tissues to metabolize the applied GA. In such circumstances the response induced by a GA can be influenced by the relative ease with which it is converted to the “active” structure. The main problem appears to be assessment of the degree to which GA interconversions participate in a particular test system. The two extremes are represented by the high specificity of the barley aleurone bioassay and the ubiquitous response of the Tanginbozu dwarf rice. Perhaps in the former instance, “goodness of fit” is the major selectivity-deciding factor, while in the latter, a capacity for extensive GA metabolism would seem more likely than a lack of specificity at the receptor site. The information obtained from a comparison of GA structures and biological activities is limited because of the fragmentary nature of the bioassay data. Slight variations in the bioassay techniques can radically affect the sensitivity of the response. A further complication is that GAS are rarely tested over a full concentration range from sub-threshold to saturation levels. It is therefore only strictly valid to compare the activities of GAS in a particular bioassay when all the compounds have been tested at the same time. An additional reason for exercising caution, is the purity of the GA standards. An inactive GA need only contain 0.1%of a highly active contaminant GA to erroneously exhibit low to moderate activity. Thus, it is not unexpected that certain GAS have shown widely varying relative activities when tested by different investigators. Table VII was drawn up by ranking GAS in five categories ranging from very high biological activity to inactive. Even this general comparison is very subjective and, where appropriate, details should be checked by reference to the original bioassay data, most of which are either presented by Hoad et al. (1976) and Sponsel et al. (1977) or have been compiled by Reeve and Crozier (1975). The greatest degree of structural diversity among the 62 characterized GAS, is mediated by the relative state of oxidation at C-20 (Fig. 2) and the presence or absence of 3p- and 13a-hydroxyl groups (Table I). Thus a comparison of the biological activity of the appropriate GA pairs, makes it possible to assess the effect of each substituent as well as interactions between groups of substituents. Table XVIII provides the basis for such a comparison. With the exception of 2a- and 2,O-hydroxy GASthere are usually insufficient analogs to allow firm conclusions to be drawn on the effects of other groups on biological activity. Table XIX shows the activities of 2a- and 2p-hydroxy GAS and their deoxy analogs. An estimate of the relative effects of the different GA configurations on biological activity, is summarized in Table XX. Comparison of the activities of the appropriate GA combinations listed in Table XVIII, shows that in the barley aleurone bioassay, a 3P-hydroxy-glactone structure is more or less mandatory if a GA is t c exhibit high activity.

134

ALAN CROZIER

TABLE XVlII Rdativr biological activities of C ,9- and Czo-GAswith and without hvdroxyl groups at the C-3 and C-13 positions" ~~~~

Bioassay Barley aleurone

Dwarf rice

Dwarf pea

Lettuce

Cucumber

___ GA configuration None

20-CH3 6-lactone 20-CHO y-lactone 20-COOH 20-CH 3 ii-lactone 20-CHO y-lactone 20-COOH 20-CH3 ii-lactone 20-CHO y-lactone 20-COOH 20-CH3 d-lactone 20-CHO y-lactone 20-COOH 20-CH3 b-lactone 20-CHO y-lactone 20-COOH

0 0 0

+

0

~~

Hydroxylation 30OH

0

++ ++ +++ +

13a-OH

0

-

+++ + +++ +++ + ++ +++ ++ +++

0

0

0

0

0

0

0

-

++ 0 +++ 0

+ ++ +++ +++

+

++ ++ +++

++ + ++ +

0

++f

+++ +++

0

++ ++++

0

0

+ + ++

+

+

0

++

0

-

0

+ +++ +++ ++ +

+ ++ +++

38,13a-diOH

+++ +++ +

+

0

+ + +++

-

0

0

0

0 0 0

0

+ +++

Relative activities: + + + +, very high; + + +, high; + + , moderate; inactive. See Table I to identify individual GA configurations.

+ ++

0

0

+, low; 0, very low to

Neither group by itself contributes significantly to activity. Among other 3P-hydroxy GAS, only those possessing either a 8-lactone function or a C-20 aldehyde group, which may be in equilibrium with a d-lactol ring (Fig. 2) (Harrison et al., 1968),exhibit appreciable activity. It is possible that these GASare active because the six-membered h-lactone and h-lactol functions can partially substitute for a five-membered y-lactone ring at the receptor site. The activity of a GA is also considerably enhanced by the additional presence of a 13a-hydroxylgroup, That this less rigorous requirement

135

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

TABLE XIX Relnrive biological activities of 2a- and 2b-hydroxy G A Sm d their 2 - d e o x ~analogs

Bioassay Substitution Gibberellin

Barley aleurone

+

2-deoxy 2aQH 2fl-OH

-

2-deox y 2a-OH 2fl-OH

-

Dwarf pea

++

-

0 0

+++

+++

Lettuce hypocotyl

+++ +

Dwarf rice

++ +

Cucumber hypocotyl

+++ ++

0

0

0

+++ ++

0

++ +

++ +

0

++ + +

0

2-deoxy 28-OH

0 0

0 0

0 0

0 0

0

2-deox y 2j-OH

-

0 0

0

0

+++ + ++

+++ + +++ + +++ +

2-deoxy 2fl-OH 2-deoxy 28-OH 2-deoxy 2b-OH Relative activities: inactive.

+

++++ +++ + + ++ ++ + 0 + + + 0

+

0

+++

0

+

+

0 0

++

0

+++

0 0

0

+ + + +, very high; + + + , high; + + . moderate; +, low; 0, very low to

can be mimicked by a 16a-hydroxyl group, would appear to support the suggestion that “goodness of fit” is the dominant mechanism controlling biological activity in this bioassay. There is virtually no interaction between C-3 and C-I3 hydroxylation and the nature of the C-20 group in the Tanginbozu dwarf rice bioassay. Activity appears to be related to the degree of oxidation of the C-20 function irrespective of hydroxylation at C-3 and C-13. The activity of C20-GAs is much higher than in the barley aleurone bioassay. The y-lactonic CI9-GAs and C-20 aldehydic C2&iAs show broadly similar activities. Although &lactonic and C-20 methyl C 2 0 - G Aare ~ also active, the response is of a lower order in most instances. The C-20carboxyl CZo-GAsexhibit only low activity. Ifa GA metabolism pathway such as that illustrated in Fig. 36 were operating in Tanginbozu rice seedlings, it would be possible to visualize the activity of

136

ALAN CROZIER

TABLE XX Relative efSeect o f G A structural groups on hiologicd activity” Bioassay

Barley aleurone

Structural configurations Activating

Deactivating

y-lactone*3P-OH* 13a-OH (v. high) y-lactone*3/l-OH (high) y-lactone*d2, (moderate) 6-lactone*3/l-OH (low) 20-CHO*3p-OH (low)

la-OH (moderate) 2/1-OH (moderate)

18-COOH (high) 20-COOH (high) 2p-OH (high) 2a-OH (moderate) 13a-OH (low)

Dwarf pea

y-lactone*3P-OH (high) y-lactone*’d 2 , (high) y-lactone (low) 3P-OH*13a-OH (high)

Lettuce hypocotyl

y-lactone*d (high) y-lactone (high) h-lactone (low)

Dwarf rice

Almost all configurations except those listed in next column

la-OH (high 2P-OH (high) 3-keto (high) 18-COOH (high) 20-COOH (high) 2a-OH (moderate)

Cucumber hypocotyl

y-lactone (high) 6-lactone (high) 20-CHO (high)

la-OH (high) 12a-OH (high) 13a-OH (high) 2a-OH (moderate)

18-COOH (high) 2P-OH (high) la-OH (moderate) 2a-OH (moderate) 12a-OH (moderate)

“An asterisk * between groupings refers to the combined presence of the groups and in practice often bears no relation to the sum of the individual effects.

both C-20 methyl and aldehydic C2,,-GAs being determined by both the efficiency with which they are converted to CI9-GAs and their ability to substitute for the optimized y-lactone structure at the receptor site. In the case of &lactonic GAS, which may be artifacts that are not metabolized to C19-GAs, activity would be determined by “goodness of fit”. The isomeric 20,4y-lactone of GA4 (Fig. 44) is known to induct: a similar response to GA4

137

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

HO

.pJa CH3

COOH

20,4y-lactone isomer of GA4

CHZ

0

CH3

COOH

CH2

1 9 . 2 ~ - l a c t o n eA'.'o isomer of GA3

Fig. 44. Structures of the 20,4y-lactone isomer of G A L and the 19,2y-lactone d'.''isomei of GA,.

in the dwarf rice bioassay (Sponsel et al., 1977), while the 19,2y-lactone A's "isomer of GA, (Fig. 44) is only marginally less active than GA3 (Hoad eta!., 1976).This implies that a 19,lOy-lactone bridge per se is not an absolute requirement for high activity and that other structures of similar configuration can serve as effective substitutes. Oxidation of the C-20 aldehyde to form a C-20 carboxylic acid, would be a side branch in the synthesis of C19-GAs(Fig. 36). It may be that C-20 carboxyl C20-GA~are relatively inactive because they do not match the receptor site and further oxidation of the carboxylate ion does not occur. Other GAS showing low activity in the dwarf rice bioassay are also higher oxidation products. That they may likewise represent metabolic "dead ends" could be an explanation for their negligible activity. For instance, oxidation of GAZ0at the C-18 locus to give GAzl would be analogous to the CZo-GAs mentioned above. However, one cannot discount the possibility that the greater polarity of the tricarboxylic GAS adversely affects their ability to penetrate plant tissues. Leaf sheath elongation in rice is stimulated by a much wider range of GAS than a-amylase production in the aleurone cells of the same species (Ogawa, 1967). This may indicate that the GA receptor sites in the two tissues have different configurations. Alternatively, the receptor molecules may be similar but the leaf sheath responds to more GAS because it is capable of carrying out more extensive GA conversions than the aleurone layer. Evidence of the possible involvement of GA metabolism in leaf sheath elongation comes from studies on two dwarf varieties of rice, Tanginbozu and Waito C (Murakami, 1970a). GAS with a 38-hydroxyl group show similar relative activities when applied to the two varieties. However, the response of Waito C to 3-deoxy GAS is significantly lower than that of Tanginbozu. This suggests that the possession of a 38-hydroxyl group is obligatory if a GA is to fit the receptor site and that Waito C, unlike Tanginbozu, is unable to 38hydroxylate 3-deoxy GAS. Pseudo GAL (3a-hydroxy GA20) is much less active than GA1 in both Tanginbozu and Waito C (Murakami, 1970b). It therefore seems that a 3a-hydroxyl group cannot substitute for a 38-hydroxyl

138

ALAN CROZIER

function at the receptor site. The low activity of pseudo GA, compared to that of GA20.further suggests that the 3a-hydroxyl function is reasonably stable as far as dehydroxylation and conversion of pseudo GA, to GAIOis concerned. On purely theoretical grounds, it is improbable that further hydroxylation of pseudo GA, at the C-3 locus would occur. 2P-hydroxy GAS are relatively inactive in the dwarf rice and other bioassays. The deactivation is fairly stereospecific since 2a-hydroxylation is not as effective in reducing biological activity (Table XIX) (Sponsel et ul., 1977). GA, and GA4 both undergo 2,&hydroxylation in rice seedlings to yield GAB and GA34 respectively (Durley and Pharis, 1973; Railton et a/., 1973). 2f2-hydroxylation of GAS is frequently observed in plant tissues including those used for bioassays. GA, is metabolized to GA8 by barley aleurone layers (Nadeau and Rappaport, 1972; Nadeau et ul., 1972), lettuce hypocotyls (Silk rt ul., 1977), and cucumber hypocotyls (Rudich et a/., 1976) as well as pea seedlings (Durley et ul., 1974b) which also convert GAzo to GA29 (Railton et ul., 1974a,c). The fact that the 2P-hydroxy y-lactonic C19-GAs are much less active than their immediate precursors, indicates that they do not fit the receptor site and that the 2P-hydroxylation step may be a means whereby plant tissues deactivate GAS.In order to study further the biological effects of C-2 hydroxylation MacMillan and co-workers (unpublished data quoted by Hedden, 1979) investigated the biological activity of GA derivatives in which 2P-hydroxylation was blocked. In the d 5 maize mutant bioassay (Phinney, 1956),2P-methyl GA4 was found to be more active than GA4, although 2P-methoxy GA9 exhibited reduced activity equivalent to that of its 2P-hydroxy analog, GAS The response to 2,2 dimethyl GA4 was quite dramatic as it was 100 times more active than GA3with the growth rate being enhanced for a prolonged period of time. Further results are awaited with interest. C19-GAs,GA3 and G A 7 ,are the most active GAS in the dwarf The pea bioassay. The data in Table XVIII show that high activity is confined to 3fl-hydroxy and 3b,13a-dihydroxy GAS which, surprisingly, have not so far been identified as native constituents of Pisum sutivum tissues. Within these two groups, the best response is elicited by y-lactonic C ,9-GAs.C-20 methyl, 6-lactonic and C-20 aldehydic C20-GAsalso exhibit activity but of a lower order. Once again the C-20 carboxylic acids are inactive. These broad trends can be interpreted in terms of either a “goodness of fit” hypothesis or a GA interconversion mechanism. The conversion of [17-3H]GA,4 to CAI by dwarf pea seedlings (Durley et ul., 1974a,b) does not necessarily lend support for the latter proposal because GA14 is inactive in the dwarf pea bioassay at the 1 pg seedling- level, although Brian et ul. (1967) report that higher doses do elicit a response. A small portion of [1,2-3H]GA,fed to dwarf pea epicotyls becomes noncovalently bound to two protein fractions with estimated molecular weights

,.

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

139

of c. 60,000 and 500,000 daltons. The binding is specific in so far as GAS and GAsglucosyl ether, which are metabolites of G A , , are not associated with either protein fraction (Stoddart rt ul., 1974). In view of the high biological activity of G A 1 in the dwarf pea bioassay, it is tempting to speculate that the GA ,-protein complexes could contain the primary GA receptor. Alternatively, enzymes responsible for the metabolism of C A I may be present and the observed binding could represent enzyme-substrate complexes. At present, critical evidence is lacking and on the basis of the available data it is impossible to assess whether or not the GA-protein complexes have any physiological relevance. The lettuce hypocotyl bioassay appears to be fairly specific. It can be seen from Table XVIII that activity is almost completely restricted to the ylactonic C19-GAs,although in some instances the h-lactonic C20-GAsact as effective substitutes. Unlike the barley aleurone bioassay, there does not appear to be any significant interaction between the lactone requirement and C-3 and C-13 hydroxylation. However, data obtained by Nash rt u / . (1978) with excised lettuce hypocotyls, imply that the response to GA9 may be dependent upon its conversion by 13a-hydroxylation to GA20. Although some of the h-lactone GAS are moderately active, their C-20 aldehyde equivalents show only low activity. This perhaps suggests that applied C z O GASare not efficiently converted to active CI9-GAs,and that the activity of the S-lactone compounds results from the six-membered ring mimicking the y-lactone at the receptor site. Brian et a / . (1967) tested over one hundred GAS and GA derivatives and observed that the presence of a 13a-hydroxyl group resulted in a very marked reduction in activity in the cucumber hypocotyl bioassay. This finding contrasts with other assay systems where the effect of 13a-hydroxylation is less clear cut. At first sight, the obvious explanation for the unusual specificity of the cucumber seedlings is that the main requirement for activity is the presence of a lactonic bridge (the y-lactone displays similar activities to both the S-lactone and the C-20 aldehyde which, as mentioned earlier, may be in equilibrium with a 6-lactol structure) and for steric reasons a 13a-hydroxyl group results in a marked mismatching of the GA and the receptor molecule. With perhaps more surety it can be said that in the cucumber bioassay, 13a-hydroxy GAS per se possess low activity, and any mechanism to convert them to their more active 13-deoxy counterparts is apparently lacking. Further support for this view can be obtained from the data of Yaniane et ul. (1973).GA30 is the 12a-hydroxy analog of GA, and the presence of the 12ahydroxyl group is sufficient to strongly deactivate the molecule in the cucumber bioassay. Similarly, GA3 induces no response, whilst deoxy GAS (A2, 3GA9)is quite active. Brian et ul. (1967) showed that the 3-keto derivatives of GA4 and GA7 are active in the cucumber hypocotyl test but not in the dwarf pea or lettuce hypocotyl bioassays. This illustrates the cucumber

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system’s lack of dependence on the nature of the C-3 substituent. The 3-keto derivatives of GA1 and GA3, on the other hand, are inactive, once again demonstrating the powerful deactivating influence of the 13a-hydroxyl group. In keeping with this reasoning, it is likely that the inactivity of GA33 in the cucumber bioassay, results from the presence of the 12a-hydroxyl group rather than the keto group at the C-3 position. However, as Yamane et a / . (1973) point out, in this instance the possible influence of the 1/3hydroxyl group cannot be discounted. Thus far it has been shown that the activities of GAS in cucumber hypocotyl elongation can be predicted on the grounds that either a 19,lOor 19,20 lactone bridge is essential for activity. This activity is in turn severely reduced by the presence of either a 12a- or 13ahydroxyl group. An exception to this rule, which defies a ready explanation, is the high activity of GA32since the presence of both 12a- and 13a-hydroxyl groups should in theory render this GA completely inactive. It seems too simple an explanation to suggest that the 15P-hydroxyl group reactivates the molecule. Despite the fact that large numbers of GASand GA derivatives have been tested in the various bioassay systems, only the broadest of trends in structure-activity relationships can be observed. This is perhaps to be expected in view of compounding factors such as penetration of the applied GA to the site of action, effects of the solvent in which the GA is dissolved and the relative stability of the exogenous GA in plant tissues. Although relatively little detailed information is available on the cellular and subcellular localization of GAS, it does seem unavoidable that application of exogenous GA will destroy this compartmentation to some extent. It is therefore possible that at least a part of the bioassay response results from an abnormal chemical modification of the applied GA that takes place before a normal state of compartmentation can be re-established. For instance, the biological activity of certain GA glucosides may be due to the release of free GASby the action of non-specific glucosidases which usually would not come into contact with endogenous GA glucosides. Despite these complications, if the general trends in bioassay specificity are borne in mind, it is possible to make reasonably accurate predictions about the potential activity of untested compounds of known structure.

V. CONCLUSIONS It is evident, if judged only by the proliferation in the number of GAS isolated from natural sources, that there has been significant progress in GA biochemistry in recent years. Techniques such as SICM and HPLC have the potential to routinely analyse sub-nanogram quantities of endogenous GAS and methods have been proposed that will enable the accuracy of such estimates to be assessed. The use of these procedures should permit substantially

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better measurements of GA pool sizes than have previously been possible. In vivo and in vitro metabolism studies have enabled details of G A biosynthesis pathways to be unravelled to varying degrees in Gibberella jujikuroi and several higher plants. This is however only the beginning, as a comprehensive study of G A biosynthesis must embrace the kinetics of the system under study and to this end methods will have to be devised to ascertain rates of turnover of individual G A pools. Studies on the sub-cellular compartmentation of GAs are still in their infancy although preliminary experimentation indicates that G A Sare located in both etioplasts and chloroplasts. The ultimate aims and aspirations of many gibberellinologists are focused on the means whereby G A S affect plant growth and development. Despite the progress that has been made in elucidating G A biosynthesis pathways and the generalized correlations that can be made between the structure, metabolism and biological activity of GAS, an understanding of the mechanisms through which G A Sinfluence growth is as far away as ever. At present nothing is known about either sites of G A action or the nature of the G A growth interface. The underlying problem appears to be that growth is an ill-defined multifarious phenomenon and the sheer vagueness of our concept of the process limits attempts to relate precise knowledge of G A levels, metabolism, catabolism and compartmentation to events such as stem elongation and leaf unrolling. This situation is unlikely to change until growth can be reduced to a number of simple, well defined sub-phenomena such as mechanical properties of the cell wall, microfibril orientation and the axial rate component of cell surface expansion. Without this information advances in G A biochemistry are likely to remain an enigma as far as the control of plant growth and development is concerned.

REFERENCES Alves, L. M. and Ruddat, M. (1979). Plant and Cell Physiol. 20, 123-130. Anderson, J. D. and Moore, T. C. (1967). Plant Physiol. 42, 1527-1534. Arpino, P. J. and Guiochon, G. (1979). Analyt. Chem. 51,682-701. Baeyer, A. and Villiger, V. (1899). Ber. Dtsch. Cheni. Ges. 32, 3625. Bailiss, K . W. and Hill, T. A. (1971). Bot. Rev. 37,437479. Baldev, B., Lang, A. and Agatep, A. 0.(1965). Science 147, 155-157. Bearder, J. R. and Phinney, B. 0.(1979). Abstracts of Tenth International Corlference on Plant Growth Substances. Madison, WI., p p . 42. Bearder, J . R. and Sponsel, V. M. (1977). Biochem. Soc. Trans. 5,569-582. Bearder, J . R., MacMillan, J. and Phinney, B. 0.(1973a). Phyrochern. 12,2655-2659. Bearder, J. R., MacMillan, J. and Phinney, B. 0.(1973b). Phyrocheni. 12,2173-2179. Bearder, J . R., MacMillan, J . , Wels, C . M., Chaffey, M. B. and Phinney, B. 0.(1974). Phytochern. 13,911-917. Bearder, J. R., MacMillan, J. and Phinney, B. 0. (1975a). J . Chem. Soc. Perkin 1, 72 1-726. Bearder, J . R., MacMillan, J., Wels, C. M. and Phinney, B. 0.(1975b). Phytochem. 14, 1741-1748.

142

ALAN CROZIER

Bearder, J . R.. Frydman, V. M.. Gaskin, P., Hatton, I. K., Harvey, W. E., MacMillan, J . and Phinney, B.O. (1976a). J . Cficm. Soc. Perkin 1, 178-183. Bearder, J. R . , MacMillan. J . and Phinney, B. 0.(l976b). J. Cheni. Soc. Cheni. Cornm., pp. 834835. Beeley, L. J., Gaskin, P. and MacMillan, J. (1975). Phytochem. 14, 779-783. Beevers, L., Loveys, B.. Pearson. J . A . and Wareing, P. F. (1970). Plurita 90,286-294. Binks, R., MacMillan, J . and Pryce, R . J. (1969). Phytochem. 8, 271-284. Bombaugh, K. J. (1971). In “Modern Practice of Liquid Chromatography” (J. J. Kirkland, Ed.), pp. 237-285. Wiley-Interscience, New York. Borrow, A., Brian, P. W., Chester, V. E. Curtis, P. J., Hemming, H. G., Henehan, C., Jefferies, E. G., Lloyd, P. B., Nixon, I . S., Norris, G. L. F. and Radley, M . (1955). J . Sci. Food Agric. 6, 34&348. Bowen. D. H., MacMillan, J. and Graebe, J. E. (1972). Phytocheni. 11,2253-2257. Bowen, D. H., Crozier, A,, MacMillan. J. and Reid, D. M. (1973). Phytochern. 12, 2935-2941. Bown, A. W . , Reeve, D. R. and Crozier, A. (1975). Planta 126,83-91. Brian, P. W . , Hemming, H. G. and Lowe, D. (1964). Ann. Bot. 28, 369-389. Brian, P . W., Grove, J. F. and Mulholland, T. P. C. (1967). Phytochem. 6, 1475-1499. Brown, S. A . and Wetter, L. R. (1972). In “Progress in Phytochemistry” (L. Reinhold and Y . Liwschitz, Eds), Vol. 3, pp. 1 4 5 . Interscience Publishers Inc., New York. Browning, G . and Saunders, P. F. (1977). Nature 265,375-377. Buggy, M . J., Britton, G . and Goodwin, T. W. (1974). Phytochern. 13, 125-129. Carr, D. J. and Reid, D. M. (1968). In “Biochemistry and Physiology of Plant Growth Regulators” (F. Wightman and G. Setterfield, Eds), pp. 1169-1 185. Runge Press, Ottawa. Carr, D. J., Reid, D. M. and Skene, K. G. M. (1964). Planra 69, 382-392. Cathey, H. M. and Stuart, N. W. (1961). Bot. Gaz. 123,51-57. Cavell, B. D., MacMillan, J., Pryce, R . J . and Sheppard, A. C. (1967). Phyrochem. 6, 867-874. Cockburn, B. J. and Wellburn, A. R . (1974). J. exp. Bot. 25,3&49. Cooke, N. H. C. and Olsen, K. (1979). American Laboratory 11 (8), 45-60. Cooke, R. J. and Kendrick, R . E. (1976). Planta 131,303-307. Cooke, R. J. and Saunders, P. F. (1975a). Planta 123, 299-302. Cooke, R. J. and Saunders, P. F. (1975b). PIanra 126, 151-160. Cooke, R. J., Saunders, P. F. and Kendrick, R. E. (1975). Planta 124,319-328. Coolbaugh, R. C. and Moore, T. C. (1971). Phytochem. 10,2395-2400. Coolbaugh, R. C., Moore, T. C., Barlow, S. A. and Eckland, P. R. (1973). Phytochem. 12, 1613-1618. Coolbaugh, R. C., Hirano, S. S. and West, C. A. (1978). Plant Physiol. 62,571-576. Cross, B. E. (1968). In “Progress in Phytochemistry” (L. Reinhold and Y . Liwschitz, Eds), Vol. 1, pp. 195-222. Interscience Publishers Inc., New York. Cross, B. E. and Norton, K. (1966). Tetrahedron Letters, pp. 6003-6007. Cross, B. E., Grove, J. F., MacMillan, J., Moffatt, J. S., Mulholland, T. P. C., Seaton, J. C. and Sheppard, N. (1959). Proc. Chem. Soc., pp. 302-303. Cross, B. E., Galt, R. H. B. and Hanson, J. R. (1964). J . Chem. SOC.,pp. 295-300. Cross, B. E., Galt, R. H. B. and Norton, K. (1968a). Tetrahedron 24,231-237. Cross, B. E., Norton, K. and Stewart, J. C. (1968b). J . Chem. SOC.Cornm., pp. 10541063. Cross, B. E., Stewart, J. C. and Stoddart, J. L. (1970). Phytochem. 9, 1065-1071. Crozier, A. and Reeve, D. R. (1977) In “Plant Growth Regulation” (P. E. Pilet, Ed.), pp. 67-76. Springer-Verlag, Heidelberg.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

143

Crozier, A. and Reid, D. M. (1971). Canad. J . Bot. 48, 967-975. Crozier, A . and Reid, D. M. (1972). In “Plant Growth Substances 1970” (D. J. Carr, Ed.), pp. 414419. Springer-Verlag, Heidelberg. Crozier, A,, Aoki, H. and Pharis, R. P. (1969). J . exp. Bot. 20, 786795. Crozier, A., Kuo. C. C., Durley, R. C. and Pharis, R. P. (1970). Canad. J . Bot. 48, 867-877. Crozier, A., Bowen, D. H., MacMillan, J., Reid, D. M. and Most, B. H. (1971). Plaiira 97, 142-154. Crozier, A., Reid, D. M. and Reeve, D. R . (1973). J . exp. Bot. 24,923-934. Crozier, A., Zaerr, J . B. and Morris, R. 0. (1980). J . Chromatogr. 198,57-63. Crozier, A., Zaerr, J. B. and Morris, R. 0. (1982). J . Chromutogr. (in press). Dennis, D. T. and West, C. A. (1967). J . B i d . Chem. 242, 3293-3300. Dockerill, B. and Hanson, J . R. (1978). Phyrochem. 17,701-704. Dockerill, B., Evans, B. and Hanson, J. R . (1977). J . Chem. Soc. Chem. Comm., pp. 919-921. Douglas, T. J. and Paleg, L. G . (1974). Plant Physiol. 54, 238-245. Diinges, W. (1977). Analyt. Chem. 49,442445. Durley, R. C. and Pharis, R. P. (1972). Plunta 109, 357-361. Durley, R. C., MacMillan, J. and Pryce, R. J. (1971). Pliytochem. 10, 1891-1908. Durley, R. C., Crozier, A., Pharis, R. P. and McLaughlin, G . E. (1972). Phytochem. 11, 3029-3033. Durley, R. C., Railton, I . D. and Pharis, R. P . (1973). Phytochem. 12, 1609-1612. Durley, R. C., Railton. I. D. and Pharis, R . P. (1974a). In “Plant Growth Substances 1973”, pp. 285-293. Hirokawa Publishing Co., Tokyo. Durley, R. C., Railton, I. D. and Pharis, R . P. (1974b). Phytochem. 13,547-551. Durley, R . C., Sassa, T. and Pharis, R. P. (1979). Plant Physiol. 64,214-219. Durst, D., Milano, M., Kitka, E. J., Connelly, S. A. and Grushka, E. (1975). Analyr. Chem. 47, 1797-1801. Ecklund, P. R. and Moore, T. C. (1974). Plant Physiol. 53, 5-10. Evans, A . and Smith, H. (1976a). Proc. Nut. Acad. Sci. U S A 73, 138-142. Evans, A. and Smith, H. (1976b). Nature 259,323-325. Evans, R. and Hanson, J. R. (1972). J . Chem. Soc. Perkin I , 2382-2385. Evans, R. and Hanson, J. R. (1975). J . Chem. Soc. Perkin I , 633-666. Evans, R., Hanson, J. R. and White, A. F. (1970).J . Chem. Soc. Comm., pp. 2601-2603. Fall, R. R. and West, C. A. (1971). J . Biol.Chem. 246,6913-6928. Frankland, B. and Wareing, P. F. (1960). Nature 185,255-256. Frost, R. G. and West, C. A. (1977). Plant Physiol. 59, 22-29. Frydrnan, V. M. and MacMillan, J. (1975). Pluntu 125, 181-195. Frydman, V. M., Gaskin, P. and MacMillan, J. (1974). Plunta 118, 123-132. Fuchs, S. and Fuchs, Y . (1969). Biochim. Biophys. Acta 192, 528-530. Fukui, H., Ishii, H. Koshimizu, K., Katsumi, M., Ogawa, Y. and Mitsui, T. (1972). Agric. Biol. Chem. 36, 1003-1012. Ganguly, S. N. and Sircar, S . M. (1974). Phytochem. 13,1911-1913. Gaskin, P. and MacMillan, J. (1975). Phytochem. 14, 1575-1578. Gaskin, P. and MacMillan, J. (1978). In “Isolation of Plant Growth Substances” (J. R. Hillman, Ed.), pp. 79-95. Society for Experimental Biology Seminar Series No. 4. Cambridge University Press, Cambridge. Gaskin, P., MacMillan, J. and Zeevaart, J. A. D. (1973). PIunta 111, 347-352. Giddings, J. C. (1967). Anulyt. Chern. 39, 1027-1028. Glenn, J. L., Kuo, C. C., Durley, R. C. andPharis, R. P. (1972). Phytochem. 11,345-351. Grabner, R., Schneider, G. and Sembdner, G. (1976). J . Chromatogr. 121, 110-115.

144

ALAN CROZIER

Graebe, J . E. (1968). Phytochem. 7, 2003-2020. Graebe, J. E. (1969). Planta 85, 171-174. Graebe, J. E. (1972). In “Plant Growth Substances 1970” (D. J. Carr, Ed.), pp. 151-157. Springer-Verlag, Heidelberg. Graebe, J. E. and Hedden, P. (1974). In “Biochemistry and Chemistry of Plant Growth Regulators” (K. Schreiber, H . R. Schiitte and G. Sembdner, Eds), pp. 1-16. h a d , Sci. German Democratic Republic Inst. Plant Biochem., Halle (Saale). Graebe, J . E. and Ropers, H. J . (1978). In “Phytohormones and Related Compounds: A Comprehensive Treatise” (D. S. Letham, P. B. Goodwin and T. J. V. Higgins, Eds), Vol. I , pp. 107-203. Elsevier/North-Holland, Biomedical Press, Amsterdam. Graebe, J . E., Dennis, D. T., Upper, C. D. and West, C. A. (1965). J . Biol. Cheni. 240, 1847-1854. Graebe, J. E., Bowen, D. H. and MacMillan, J . (1972). PIanta 102,261-271. Graebe, J. E., Hedden, P., Gaskin, P. and MacMillan, J. (1974a). Phytochem. 13, 1433-1 440. Graebe, J. E., Hedden, P., Gaskin, P. and MacMillan, J. (1974b). Planta 120,307-309. Graebe, J. E., Hedden, P. and MacMillan, J. (1974~).In “Plant Growth Substances 1973”, pp. 26&266. Hirokawa Publishing Co., Tokyo. Graebe, J. E., Hedden, P. and MacMillan, J. (1975). J . Chem. SOC.Chem. Comm., pp. 161-162. Graebe, J. E., Hedden, P. and Rademacher, W. (1980). In “Gibberellins- Chemistry, Physiology and Use” (J. R. Lenton, Ed.). British Plant Growth Regulator Group, Monograph No. 5 , pp. 3147. Grove, J. F. (1961). J . Chem. SOC.,pp. 3345-3347. Halevy, A. H. and Shilo, R. (1970). Physiol. Plant 23 82&827. Hanson, J. R. (1966). Tetrahedron 22, 701-703. Hanson, J. R. (1971). Fortschr. Chem. Org. Naturst. 29, 395416. Hanson, J. R. and Hawker, J. (1972). Tetrahedron Letters, pp. 42994302. Hanson, J . R. and White, A. F. (1969). J . Chem. SOC.Comm., pp. 981-985. Hanson, J. R., Hawker, J . and White, A. F. (1972). J . Chem. SOC.Perkin. 1 , 1892-1895. Harada, A. and Nitsch, J. P. (1967). Phytochem. 6, 1695-1703. Harrison, D. M., MacMillan, J . and Galt, R. H. B. (1968). Tetrahedron Letters 27, 3 137-3 139. Hedden, P. (1978). In “Proceedings of the Plant Growth Regulator Working Group” (M. Abdel-Rahman, Ed.), pp. 3344. Plant Growth Regulator Working Group, Syracuse, New York. Hedden, P. (1979). In “Plant Growth Substances” (B. Mandava, Ed.), pp. 19-56. ACS Symposium Series 111, Amer. Chem. SOC.,Washington, D.C. Hedden, P. and Phinney, B. 0. (1979). Phytochem. 18, 1475-1479. Hedden, P., MacMillan, J. andPhinney, B. 0.(1974). J . Chem. SOC.Perkin 1,587-592. Hedden, P., Phinney, B. O., MacMillan, J. and Sponsel, V. M. (1977). Phytochem. 16, 1913-191 7. Hedden, P., MacMillan, J . and Phinney, B. 0. (1978). Ann. Rev. Plant Physiol. 29, 149- 192. Heftmann, E., Saunders, G. A. and Haddon, W. F. (1978). J. Chromatogr. 156,71-77. Hiraga, K., Yokota, T., Murofushi, N. and Takahashi, N. (1972). Agric. Biol. Chem. 36,345-347. Hiraga, K., Kawabe, S., Yokota, T., Murofushi, N., and Takahashi, N. (1974a). Agric. Biol. Chem. 38,2521-2527. Hiraga, K., Yokota, T., Murofushi, N. and Takahashi, N. (1974b). Agric. Biol. Chem. 38,251 1-2520.

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

145

Hoad, G . V. and Bowen, M. R. (1968). Planta 82,22-32. Hoad, G. V., Pharis, R. P., Railton, I. D. and Durley, R. C. (I 976). Planfa 130,113-120. Ikegawa, N., Kagawa, T. and Sumiki, Y. (1963). Proc. Japan. Acad. 39,507-512. Ingram, T. J. and Browning, G. (1979). Planta 146,423432. Jones, D. F. (1964). Nature 202, 1309-1310. Jones, M . G., Metzger, J. D. and Zeevaart, J. A. D. (1980). Plant Physiol. 65,218-221. Jones, 0 .P. and Lacey, H. J. (1968). J . Expr. Bot. 19, 52653 1. Jones, R. L. (1968). Planta 81,97-105. Jones, R. L. (1973). Ann. Rev. Plant Physiol. 24,571-598. Jones, R. L. and Lang, A. (1968). Plum Physiol. 43,629-634. Jones, R. L. and Phillips, I . D . J. (1966). Plant Physiol. 41, 1381-1386. Jones, R. L. and Phillips, I . D. J. (1967). Planta 72, 53-59. Jones, R . L. and Varner, J. E. (1967). Planta 72, 155-161. Kagawa, T., Fukinbara, T. and Sumiki, Y. (1963). Agric. Biol. Chem. 27,598-599. Kamienska, A,, Durley, R. C. and Pharis, R. P. (1976). Phytochem. 15,421424. Karger, B. L. (1971). In “Modern Practice of Liquid Chromatography” (J. J. Kirkland, Ed.), pp. 3-53. Wiley-Interscience, New York. Karger, B. L., Kirby, D. P., Vouros, P., Foltz, R. L. and Hidy, B. (1979). Analyt. Chem. 51, 2324-2328. Katsumi, M., Phinney, B. O., Jefferies, P. R. and Hendrick, C. A. (1964). Science 144, 849-850. Kawarada, A. and Sumiki, Y. (1959). Bull. Agric. Chem. SOC.Jap. 23,343-344. Kende, H. (1967). Plant Physiol. 42, 1612-1618. Kende, H. and Lang, A. (1964). Plant Physiol. 39,435440. Knapp, D. R. and Krueger, S. (1975). Analyt. Letters 8,603-610. Knofel, H. D., Miiller, P. and Sembdner, G. (1974). In “Biochemistry and Chemistry of Plant Growth Regulators” (K. Schreiber, H. R. Schiitte and G. Sembdner, Eds), pp. 121-124. Acad. Sci. German Democratic Republic Institute Plant Biochem., Halle (Saale). Kohler, D. (1965). Planta 67,4&54. Kohler, D . (1970). Z . P’anzenphysiol. 62,42&435. Kohler, D. and Lang, A. (1963). Plant Physiol. 38, 555-560. Krishen, A. (1977). J . Chromatogr. Sci. 15,434439. Kurogochi, S., Murofushi, N., Ota, Y. and Takahashi, N . (1978). Agric. Biol. Chem. 42, 207-208. Kurogochi, S., Murofushi, N., Ota, Y. and Takahashi, N. (1979). Planta 146,185-191. Kurosawa, E. (1926). Trans. Hist. Soc. Formosa. 16, 213-227. Lang, A. (1970). Ann. Rev. Plant Physiol. 21, 537-570. Leopold, A. C . (1971). Plant Physiol. 48,537-540. Letham. D. S., Higgins, T. S. V., Goodwin, P. B. and Jacobsen, J. V. (1978). In “Phytohormones and Related Compounds- A Comprehensive Treatise” (D. S. Letham, P. B. Goodwin and T. J. V. Higgins, Eds), Vol. 1, pp. 1-27. Elsevierporth Holland, Biomedical Press, Amsterdam. Lew, F. T. and West, C. A. (1971). Phytochem. 10,2065-2076. Locke, D. C . (1973). J . Chromatogr. Sci. 11, 12C128. Lockhart, J. A. (1956). Proc. Nat. Acad. Sci. USA 42,841-848. Lockhart, J. A. (1959). Plant Physiol. 34,457-460. Lockhart, J. A. (1962). Plant Physiol. 37, 759-764. Lorenzi, R., Horgan, R. and Heald, J. K. (1976). Phytochern. 15,789-790. Lorenzi, R., Saunders, P. F., Heald, J. K . and Horgan, R. (1977). Plant Sci. Letters 8, 179-1 82. Loveys, B. R. and Wareing, P. F. (1971a). Planta 98, 109-116.

146

ALAN CROZIER

Loveys, B . R. and Wareing, P. F. (1971b). Planta 98, 117-127. Luckwill, L. W. and Whyte, P. (1968). In “Plant Growth Regulators”, pp. 87-101. SOC. Chem. Ind. Monograph No. 31. Staples, London. MacMillan, J. (1972). In “Plant Growth Substances 1970” (D. J. Cam, Ed.), pp. 790-797. Springer-Verlag, Heidelberg. MacMillan, J. (1974). In “Biochemistry and Chemistry of Plant Growth Regulators” (K. Schreiber, H. R. Schutte and G. Sembdner, Eds), pp. 3347. Acad. Sci. German Democratic Republic Institute Plant Biochem., Halle (Saale). MacMillan, J. and Pryce, R. J. (1973). In “Phytochemistry” (L. P. Miller, Ed.), Vol. 3, pp. 283-326. Van Nostrand-Reinhold, New York. MacMillan, J. and Suter, P. J. (1958). Naturwissenschaften 46,46. MacMillan, J. and Suter, P. J . (1963). Nature 197, 190. MacMillan, J. and Takahashi, N. (1968). Nature 217, 17&171. MacMillan, J. and Wels, C. M. (1973). J. Chromatogr. 87,271-276. MacMillan, J. and Wels, C. M. (1974). Phytochern. 13, 1413-1417. McFadden, W. H. (1979). J. Chromogr. Sci.17,2-17. McFadden, W. H., Bradford, D. C., Gaines, D. E. and Gower, J . L. (1977). International Laboratory Oct., 5544. McInnes, A. G., Smith, D. G., Durley, R. C., Pharis, R. P., Arsenault, G. P., MacMillan, J., Gaskin, P. and Vining, L. C. (1977). Canad. J . Biochem. 55,728-735. Majors, R. E. (1975). International Laboratory. Nov./Dec., 11-35. Majors, R. E. and MacDonald, F. R. (1973). J . Chromatogr. 83, 169-179. Metzger, J. D. and Zeevaart, J. A. D. (1980). Plant Physiol. 66,844-846. Mishra, D. and Pradham, G. C. (1968). Curr. Sci.,pp. 263-264. Moore, T. C. and Coolbaugh, R. C. (1976). Phytochem. 15,1241-1247. Morris, R. 0.and Zaerr, J. B. (1978). Anafyt. Letters. A l l (l), 73-83. Murakami, Y. (1968). Bot. Mag. (Tokyo) 79,3343. Murakami, Y. (1970a). Jap. Agric. Res. Quart. 5, 5-9. Murakami, Y. (1970b). Bot. Mag. (Tokyo) 83,211-213. Murofushi, N., Irinchijima, S., Takahashi, N., Tamura, S., Kato, J., Wada, Y., Watanabe, E., Aoyama, T. (1966). Agric. Bid. Chem. 30,917-924. Murofushi, N., Nagura, N. and Takahashi, N. (1979). Agric. Bid. Chem. 43, 11591161. Murphy, G. J. P. and Briggs, D. E. (1973). Phytochem. 12,1299-1308. Murphy, G. J. P., and Briggs, D. E. (1975). Phytochem. 14, 429433. Murphy, P. J . and West, C. A. (1969). Arch. Biochem. Biophys. 133,395407. Musgrave, A. and Kende, H. (1970). Plant Physiol. 45,53-61. Nadeau, R. and Rappaport, L. (1972). Phytochem. 11, 1611-1616. Nadeau, R., Rappaport, L. and Stolp, C. F. (1972). Planta 107,315-324. Nakano, H., Sato, H. and Tamoahi, B. H. (1968). Biochim. Biophys. Acta, 164,585-595. Nash, L. J. (1976). Ph.D. thesis, University of Glasgow. Nash, L. J. and Crozier, A. (1975). Planta 127,221-231. Nash, L. J., Jones, R. L. and Stoddart, J. L. (1978). Planta 140, 143-150. Nash, L. J., Reeve, D. R. and Crozier, A. (1982). In preparation. Nicholls, P. B. and Paleg, L. G. (1963). Nature 199,823-824. Ogawa, Y. (1967). Bot. Mag. (Tokyo) 80,27-32. Oster, M. 0. and West, C. A. (1968). Arch. Biochem. Biophys. 127, 112-123. Paleg, L. G. (1960). Plant Physiol. 35,902-906. Patterson, R. J. and Rappaport, L. (1974). Planta 119, 183-191. Patterson, R. J., Rappaport, L. and Breidenbach, R. W. (1975). Phytochem. 14, 363-368. Pharis, R. P. and Kuo, C. C. (1977). Canad. J . For. Res. 7,299-325.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

147

Phillips, I. D. J. (1971). Planta 107, 277-282. Phillips, I. D. J. and Jones, R. L. (1964). Planta 63, 269-278. Phinney, B. 0. (1956). Proc. Natl. Acad. Sci. U.S.A.42, 185-189. Phinney, B. 0.(1961). In “Plant Growth Regulation” (R. M. Klein, Ed.), pp. 489-501. Iowa State College Press, Ames. Phinney, B. 0. (1979). In “Plant Growth Substances” (B. Mandova, Ed.), pp. 57-78. ACS Symposium Series 111, Amer. Chem. SOC.,Washington, D.C. Phinney, B. 0. and West, C. A. (1961). In “Encyclopedia of Plant Physiology” (W. Ruhland, Ed.), Vol. 14, pp. 1185-1227. Springer-Verlag, Heidelberg. Phinney, B. O., West, C. A,, Ritzel, M. B. and Neeley, P. M. (1957). Proc. Nut. Acad. Sci. U.S.A. 43,398-404. Pitel, D. W., Vining, L. C. and Arsenault, G.P. (1971a). Canad. J . Biochem.49,185-193. Pitel, D. W.,Vining, L. C. and Arsenault, G. P. (1971b). Canad. J . Biochem.49,194200. Powell, L. E. (1963). Nature 200,79. Powell, L. E. and Tautvydas, K. J. (1967). Nature 213,292-293. Pryce, R. J. (1973). Phytochem. 12,507-514. Pryde, A. and Gilbert, M. T. (1979). “Applications of High Performance Liquid Chromatography”. Chapman and Hall, London. Rademacher, W. and Graebe, J. E. (1979). Biochem. Biophys. Res. Comm. 91,3540. Radley, M. (1956). Nature 178, 1070-1071. Radley, M. (1967). Planta 75, 164-171. Railton, I. D. (1974a). Plant Sci. Letters 3, 207-212. Railton, I. D. (1974b). Plantn 120, 197-200. Railton, I. D. (1976). S. Afr. J . Sci. 72, 371-377. Railton, I . D. (1977). Z. PJanzenphysiol. 81, 323-329. Railton, I . D. (1979). Z . PJanzenphysiol. 91,283-290. Railton, I. D. and Reid, D. M. (1974a). PLant Sci. Letters 2, 157-163. Railton, I. D. and Reid, D. M. (1974b). Plant Sci. Letters 3,303-308. Railton, I. D. and Rechav, M. (1979). Plant Sci.Letters 14,75-78. Railton, I . D., Durley, R. C. and Pharis, R. P. (1973). Phytochem. 12,2351-2352. Railton, I . D., Durley, R. C. and Pharis, R. P. (1974a). In “Plant Growth Substances 1973”, pp. 294-304. Hirokawa Publishing Co., Tokyo. Railton, I . D., Durley, R. C. and Pharis, R. P. (1974b). Plant Physiol. 5 4 , 6 1 2 . Railton, I. D., Murofushi, N., Durley, R. C. and Pharis, R. P. (1974~).Phytochem. 13, 793-796. Rappaport, L. and Adams, D. (1978). Phil. Trans R. Soc. London B.284,521-539. Reeve, D. R. and Crozier, A. (1974). J . Expt. Bot. 25,431-445. Reeve, D. R. and Crozier, A. (1975). In “Gibberellins and Plant Growth” (H. N. Krishnamoorthy, Ed.), pp. 35-64. Wiley Eastern Ltd., New Delhi. Reeve, D. R. and Crozier, A. (1976). Phytochem. 15,791-793. Reeve, D. R. and Crozier, A. (1977). J . Chromatogr. 137,271-282. Reeve, D. R. and Crozier, A. (1978). In “Isolation of Plant Growth Substances” (J. R. Hillman, Ed.), pp. 41-77. Society for Experimental Biology Seminar Series No. 4. Cambridge University Press, Cambridge. Reeve, D. R. and Crozier, A. (1980). In “Hormonal Regulation of Development I. Molecular Aspects of Plant Hormones” (J. MacMillan, Ed.). Encyclopedia of Plant Physiology, New Series Vol. 9, pp. 203-280. Springer-Verlag, Heidelberg. Reeve, D. R., Yokota, T., Nash, L. J. and Crozier, A. (1976). J. exp. Bot. 27,1243-1258. Reid, D. M. and Burrows, W. J . (1968). Experimentia 24,189-190. Rkid, D. M. and Carr, D. J. (1967). Planta 73, 1-1 1. Reid, D. M . and Crozier, A. (1970). PLanta 94,95-106. Reid, D. M. and Crozier, A. (1971). J . Expt. Bot. 22, 3948.

148

ALAN CROZIER

Reid, D. M. and Crozier, A. (1972). In “Plant Growth Substances 1970” (D. J. Carr, Ed.), pp. 420-427. Springer-Verlag, Heidelberg. Reid, D. M., Clements, J. B. and Carr, D. J. (1968). Nature 217, 580-582. Reid, D. M., Crozier, A. and Harvey, B. R. M. (1969). Plunta 89,37&379. Robinson, D . R. and West, C. A. (1970a). Biochem. 9,7&79. Robinson, D. R. and West, C.A. (1970b). Biochem. 9,8&89. Rogers, L. J . , Shah, S. P. J. and Goodwin, T. W. (1966). Biochem. J . 99,381-388. Ropers, H. J., Graebe, J. E., Gaskin, P. and MacMillan, J . (1978). Biuckem. Biophys. Res. Comun. 80,69&697. Ruddat, M., Lang, A. and Mosettig, E. (1963). Narurwissetischrlften 50,23. Ruddat, M., Heftman, E. and Lang, A . (1965). Arch. Biuchem. Biophys. 111, 187- 190. Rudich, J., Sell, H. M. and Baker, L. R. (1976). Plant Physiol. 57, 734-737. Sawada, K. (1912). Formosan Agr. Rev. 36, 10-16. Schreiber, K . , Weiland, J., and Sembdner, G. (1970). Phytochem. 9, 189-198. Sembdner, G. and Schreiber, K. (1965). Phytochem. 4, 49-56. Sembdner, G., Weiland, J., Aurich, 0. and Schreiber, K. (1968). In “Plant Growth Regulators”, pp. 70-86. SOC.Chem. Ind. Monograph No. 31. Staples, London. Sembdner, G., Borgmann, E., Schneider, G., Liebisch, H. W., Miersch, O., Adam, G., Lishchewski, M. and Schreiber, K. (1976). Planta 132,249-257. Shechter, I. and West, C. A. (1969). J. Biol. Chem. 244, 3200-3209. Silk, W. K., Jones, R. L. and Stoddart, J. L. (1977). Plant Physiol. 59,211-216. Simcox, P. D., Dennis, D. T. and West, C. A. (1975). Biochem. Biophys. Res. Comm. 66, 166172. Simpson, T. H. (1968). J . Chromatog. 38,23-34. Sitton, D., Richmond, A. and Vaadia, Y. (1967). Phytochem. 6,1101-1105. Skene, K. G. M. (1967). Planta 74,250-262. Smith, L. L. (1974). Terpenoid Steroids 4 , 394-530. Spector, C. (1964). Ph.D. thesis. University of California, Los Angles. Spector, C. and Phinney, B. 0. (1966). Science 153, 1397-1398. Spector, C. and Phinney, B. 0. (1968). Physiol. Plant 21, 127-136. Sponsel, V. M. and MacMillan, J. (1977). Planta 135, 129-136. Sponsel, V. M. and MacMillan, J. (1978). Planta 144, 69-78. Sponsel, V . M., Hoad, G. V. and Beeley, L. J. (1977). Plunta 135, 143-147. Sponsel, V. M., Gaskin, P. and MacMillan, J. (1979). Planta 146, 101-105. Stoddart, J. L. (1969). Phytochern. 8, 831-837. Stoddart, J. L. (1976). Nature 261,454455. Stoddart, J. L., Breidenbach, R. W., Nadeau, R. and Rappaport, L. (1974). Proc. Nut. Acad. Sci.USA 71,3255-3259. Stodola, F. H., Raper, K. B., Fennell, D. I., Conway, H. F., Sohns, V. E., Langford, C. T. and Jackson, R. W. (1955). Arch. Biochem. 54,240-245. Takahashi, N., Kitamura, H., Kawarada, A., Seta, Y., Takai, M., Tamura, S. and Sumiki, Y. (1955). Bull. Agr. Chem. Soc. Japan 19,267-277. Upper, C. D. and West, C. A. (1967). J . Biol. Chem. 242, 3285-3292. Van Bragt, J. (1969). Neth. 1.Agric. Sci. 17, 183-188. Vining, L. C. (1971). J . Chromatogr. 60, 141-143. Watanabe, N., Yokota, T. and Takahashi, N. (1978). Plant and Cell Physiol. 19, 1263-1 270. Weaver, R. J. (1972). “Plant Growth Substances in Agriculture”. W. H. Freeman and Co., San Francisco. Wellburn, A. R. and Hampp, R. (1976). Biochem. J . 158,231-233.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

149

West, C . A . (1973). In “The Control of Biosynthesis in Higher Plants”(B. V. Milborrow, Ed.), pp. 143-169. Academic Press, New York and London. 81, 24242427. West, C. A. and Phinney, B. 0. (1958). J . A m . Chen7. SOC. Whyte, P. and Luckwill, L. C. (1966). Nuture 210. 1360. Wiinsche, V . (1969) Planta 85, 108-1 10. Yabuta, T. (1935). Agr.. and Hort. 10, 17-22. Yabuta, T. and Hayashi, T. (1939). J . Agric. Chem. Soc. (Japan) 15,257-266. Yabuta, T. and Sumiki, Y . (1938). J . Agric. Chem. Soc. Japan 14, 1526. Yamaguchi, I., Yokota, T., Murofushi, N., Ogawa, Y. and Takahashi, N. (1970). Agric. Biol. Chrm. 34, 1439-1441. Yamane, H.. Yamaguchi, I., Yokota, N., Murofushi, N., Takahashi, N. and Katsumi, K. (1973). Phytochem. 12, 255-261. Yamane, H., Murofushi, N. and Takahashi, N. (197.5). Phytochrni. 14, 1195-1200. Yamane, H., Murofushi, N . Osada, H. and Takahashi, N . (1977). Ph)Toc/wni. 16, 83 1-835. Yamane, H., Takahashi, N., Takeno, K. and Furuya, M. (1979). Plutiru 147,251-256. Yokota. T., Murofushi, N., Takahashi, N . and Katsumi, M. (1971). Phyrochem. 10. 2943-2949. Yokota, T., Yamazaki, S., Takahashi, N. and Iitaka, Y. (1974). Tetrahedron Letters pp. 2957-2960. Yokota, T., Yamane, H. and Takahashi, N . (1976). Agric. Biol. Cheni. 40, 2507-2508. Yomo. H. (1960). Hukko Kyoktri Shi 18, 603-606. Yomo, H. and Iinuma, H. (1966). Pllrr7t~i71. 113-1 18. Zeevaart, J. A . D. (1966). Plunr Physiol. 41, 856862. NOTE A D D E D I N P R O O F Recent studies by Hedden and Graebe (1981) reveal that in cell-free preparations from Cucurbita maxima liquid endosperm 7b-hydroxykaurenolide is not formed from enf7a-hydroxykaurenoic acid as illustrated in Fig. 24. Instead the pathway branches at ent-kaurenoic acid and proceeds to 7P-hydroxykaurenolide via ent-kauradienoic acid ( m f - A 6 . ’ kaurenoic acid). In turn, 7b-hydroxykaurenolide undergoes 12a-hydroxylation to yield 7p, 12a-dihydroxykaurenolide. Cell-free preparations derived from the suspensor of the embryo of Phasrolus coccinetu seed at a very early stage of development have been shown to convert (1) mevalonic acid to ent-kaurene (Ceccarelli et a / . , 1979), (ii) ent-kaurene to ent-kaurenol, ent-kaurenol, ent-kaurenoic acid and ent-7a-hydroxykaurenoic acid (Ceccarelli ef a/., 1981a) and (iii) ent-7a-hydroxykaurenoic acid to G A L ,G A Sand GAS (Ceccarelli et al., 1981b). G A I is an established endogenous component of the Phaseolus cotcineus suspensor (Alpi et al., 1979). Alpi, A,, Lorenzi, R., Cionini, P. G., Bennici, A. and D’Amato, F. (1979). Planta 147, 225-228. Ceccarelli, N., Lorenzi, R. and Alpi, A. (1979). Phytochem. 18, 1657-1658. Ceccarelli, N., Lorenzi, R. and Alpi, A. (1981a). Planr Sci. Letters 21. 325-332. Ceccarelli, N., Lorenzi, R. and Alpi, A. (1981b). 2. Pflanxnphysiol. 102, 37-44. Hedden, P. and Graebe, J . E. (1981). Phyrochem. 20, 1011-1015.