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Cytokinins
C H A P T E R
8 Cytokinins
1. 2. 3. 4.
DISCOVERY 191 BIOLOGICAL FUNCTIONS AND BIO ASSAYS 192 STRUCTURE OF CYTOKININS 192 CYTOKININS OCCUR FREE IN THE CYTOPLASM AS WELL AS COMPONENTS OF tRNA 192 5. RELATIVE DISTRIBUTION OF NATURAL CYTOKININS AMONG PLANTS 193 6. BIOSYNTHESIS IN HIGHER PLANTS 195 6.1. The Postulated Pathway for Free Cytokinins 195 6.2. Synthetic Enzymes 195 6.3. Formation of Cytokinins from tRNA 197 6.4. Sites of Synthesis 197 7. REGULATION OF CYTOKININ LEVELS 197 7.1. Regulation of Cytokinin Biosynthesis 198 7.2. Reversible Inactivation 198 7.3. Irreversible Inactivation 199 8. SYNTHETIC COMPOUNDS WITH CYTOKININ-LIKE ACTIVITY 200 8.1. Phenylurea Derivatives 201 9. CYTOKININ ANTAGONISTS (ANTICYTOKININS) 201 10. CHAPTER SUMMARY 202 REFERENCES 202
In the 1940s and 1950s, the techniques of plant tissue culture were being developed, and it was noted that callus cultures from tobacco pith explants or carrot roots, both favorite objects for study, placed on agar blocks soaked with nutrients and auxin still showed very little cell division. They required other substances, e.g., coconut milk (liquid endosperm from coconut), or extracts from vascular tissues, yeast extract, autoclaved DNA, or even adenine. In 1956, Carl Miller in Folke Skoog's laboratory at Wisconsin University, Madison, discovered that a substituted adenine, 6 furfuryl amino purine, obtained from autoclaved herring sperm DNA was far more potent than adenine in promoting cell division in tobacco pith explants. This substance was given the name kinetin (Fig. 8-1) [for a personal and interesting account of this discovery, see Skoog (1994)]. Kinetin does not occur naturally in plants (but see Section 8). In the search for natural substances, it was expected that endosperm tissue, which provides nutrition for the growth of embryo during its early
1. DISCOVERY Fiaberlandt, a German plant physiologist, noted as far back as 1913 that certain diffusible factors from phloem tissue, e.g., phloem exudate, could cause cell division in potato tubers. Later in 1921 he discovered that healing of cut plant tissues by cell division was prevented if the cut surfaces were washed with water. Both observations suggested the presence of a soluble factor in plant tissues that promoted cell division.
FIGURE 8-1 Kinetin or N^-(furfurylamino)purine. The side chain is shaded for comparison with side chains in Fig. 8-2.
191
192
II. Structure and Metabolism of Plant Hormones
development or, postgerminatively, for growth of young seedling (see Chapters 18 and 19), might be a promising material to look for hormones that promote cell division. Liquid endosperm from coconut, maize, horse chestnut, and immature fruits of banana were favorite objects for study. Thus, in 1963, D. S. Letham at CSIRO, Canberra, Australia, isolated a substance from kernels of sweet corn that had high cell division-promoting capacity in callus cultures. The material was a N^-substituted adenine [6-(4-hydroxy-3methyl-frflns-2-enylamino) purine] and was given the name zeatin, since it was isolated from maize {Zea mays). Since the discovery of zeatin, several naturally occurring cytokinins and some synthetic substances with similar biological activities have been discovered. These substances, while they all carry an adenine moiety, differ in the structures of their side chains. Thus, like auxins, cytokinins are defined more on the basis of their biological functions than their structure.
2. BIOLOGICAL FUNCTIONS A N D BIOASSAYS Cytokinins are defined as compounds that promote cell division in callus and tissue culture. In combination with auxins, they regulate the ratio of shoot bud vs root growth in tissue culture and in stem cuttings; in intact plants, they regulate apical dominance and lateral root initiation. They also retard senescence and chlorophyll degradation in aging leaf tissues and, in combination with ethylene and light, regulate the growth of dicot seedlings in dark. These responses are covered in Sections III and V of this book. Bioassays for cytokinin activity include (1) promotion of growth in tobacco pith culture or soybean callus tissue and (2) expansion of excised radish cotyledons in culture. These assays are based on the promotion of cell division and expansion activity by cytokinins. Another common bioassay is inhibition of senescence, as measured by a reduction in loss of chlorophyll from leaf tissues. Cytokinins, like auxins and gibberellins, have a wide occurrence in plants and are known besides vascular plants from mosses and algae. In mosses they regulate bud growth in protonema. They are also produced by several strains of soil-living and phytopathogenic bacteria, such as Agrobacterium and Pseudomonas, which cause galls or tumors in plants, and by certain phytopathogenic fungi, such as Helminthosporium and Ustilago (see Appendix 2). Interestingly, an example of the inhibition of senescence by cytokinins is provided
by these fungi: they cause the formation of "green islands" on leaves of plants, which they infect.
3. STRUCTURE OF CYTOKININS All naturally occurring cytokinins have an adenine ring structure with a 5 carbon isopentenyl side chain from N^ of the adenine molecule (Fig. 8-2). Some authors refer to these as the isoprenoid class of cytokinins. In addition to zeatin, they include isopentenyladenine (or dimethylallyladenine) and the reduced form of zeatin, the dihydrozeatin. Because the side chain in zeatin possesses a double bond, geometric isomers are possible. These occur either as transzeatin or c/s-zeatin. In the cis isomer, the hydroxyl group of the isopentenyl side chain is oriented toward the N^ position of the purine ring, whereas in the trans isomer the hydroxyl group is oriented away from the purine ring. Adenine occurs in DNA and RNA, and its ribose and ribose plus phosphate derivatives, adenosine and adenosine 5'-monophosphate, respectively, are common in plants. The ribose is always attached to N^ in the purine ring. Thus, natural cytokinins also occur as their sugar derivatives, N^ ribosides and ribotides (sugar plus phosphate). Figure 8-3 shows the structures for zeatin riboside and zeatin ribotide; others have similar structures. In addition to ribosides and ribotides, conjugates of cytokinins with glucose, xylose, and amino acids are also known. Some authors treat all of these compounds together as natural cytokinins. Thus, there can be a bewildering array of naturally occurring cytokinins or cytokinin-type compounds. In this text, the term cytokinin is used, as far as possible, for natural cytokinins with an adenine base. Ribosyl derivatives are referred to as ribosylated derivatives, and conjugates are referred to as conjugates.
4. CYTOKININS OCCUR FREE IN THE CYTOPLASM AS WELL A S C O M P O N E N T S OF tRNA Natural cytokinins and their ribosides and ribotides occur free in the cytoplasm, but they also occur as integral components of certain transfer RNAs (tRNAs). As components of these tRNAs, they are always in the form of ribosylated derivatives, not as adenine bases. Many such ribosylated cytokinins are known from tRNAs (e.g., isopentenyladenine-9-riboside, cisand frflns-zeatin-9-riboside, 2-methylthioisopentenyl-
193
8. C y t o k i n i n s 4
1
HNH
HN
Adenine (6-aminopurJne)
H. HN
CH2
k
\ ^N H
CH2
3 / CH3
CH3
fi 2 N -(A -lsopentenyl)adenine
>CH20H CH3
\ 2 /
H\ HN
CH2
k
\
/CH3 CH2OH
^N H c/s-Zeatin
frans-Zeatin
H H H. I I ,CH20H
)c-c( HN—CH^ N N^ \
^CH3
Dihydrozeatin F I G U R E 8-2 Structure of the isoprenoid class of cytokinins. (Top left) Adenine (6-aminopurine) with the ring numbering system. Natural cytokinins are all derivatives of adenine, with substitution of a five carbon chain in the amino group (shaded area). Carbon atoms in the side chain are numbered 1-5. Hydroxylation of the side chain at C-4 creates asymmetry and gives rise to trans- or cis-zeatin.
ademne-9-riboside, cis- and trans-1 methylthiozeatin9-riboside). Such tRNA-bound ribosylated cytokinins occur widely in a variety of organisms: plants, phytopathogenic bacteria, yeast, and animals. Transfer RNAs are rich in substitutions (e.g., methylations), which occur all over the molecule, but the cytokinin ribosides occupy a precise location—they occur exclusively as the base adjacent to the 3' end of the anticodon (Fig. 8-4). The function of such exclusive localization is obscure, but it has been suggested that it may play a regulatory role in protein synthesis, probably by promoting the codon-anticodon interaction a n d / o r increasing the binding affinity of aminoacyl tRNA to the ribosomes. However, there is no direct proof for this assumption. The supply of exogenous cyto-
kinins to plant tissues is known to promote protein synthesis, but it is considered doubtful that such promotion is mediated via cytokinin derivatives bound to tRNA.
5. RELATIVE DISTRIBUTION OF NATURAL CYTOKININS A M O N G PLANTS It is likely that different plants show lesser or greater abundance of one or the other types of natural cytokinins. Among the plants that have been investigated, trans-zea.tm (unless necessary, hereafter referred to as zeatin only) seems to be the most widely distributed.
194
II. Structure a n d M e t a b o l i s m of Plant H o r m o n e s
Hv
)c=c(
HN
CH2^
CH2OH
H
CH3
HN
Ribosylzeatin
CH3
Ribosylzeatin-5-monophosphate
(zeatin riboside) FIGURE 8-3
CH2
CH2OH
(zeatin ribotide)
Structures of zeatin riboside and zeatin ribotide (zeatin is shown in trans configuration).
A—O—Tyrosyl I
C C I pC = I U= I C=
G I I A I G / I U G \ I C=G \ A I I Me A G=C \ Me^\/"U / ^G=C^C-C-C-G-C C-C-G
UHp UH2 / GOMe
I
G \
A Me \ G
G-G-G-C-G \ T^x^^ \ C —Me G-C = G ^UH2 Me^/ I I^A Me A= ^G-A I A = I I G= C I I / A = ¥ ^ C A I I U A—IP(iPAdo) \ ^ " / Anticodon - ^ G - \ | / — A ^ C — A —U-^mRNA
G-G-C. UH2
./ UHo
•UHp
t^
_
J
Codon FIGURE 8-4 The structure of yeast tRNA for tyrosine. Note that the isopentenyladenosine (iPAdo) is the base next to the 3' end of the anticodon. From Hall (1970).
195
8. Cytokinins Zeatin is also one of the most biologically active cytokinins known. In contrast, c/s-zeatin has much less activity. Until recently, c/s-zeatin was thought to be present mainly in tRNA as a ribosylated derivative, and there were only sporadic reports of its occurrence as a free cytokinin. Studies using GC-MS for identification reveal, however, that c/s-zeatin, as well as its ribosides and ribotides, in free form are the predominant cytokinins in chick pea seeds, whereas trans isomers are minor constituents. As more plants are investigated, it may turn out that both forms of zeatin occur in free form and that their relative abundance varies in different plants, or plant parts. Dihydrozeatin and its ribosyl derivatives and conjugates also occur frequently, but isopentenyladenine seems to have much less free occurrence.
6. BIOSYNTHESIS IN HIGHER PLANTS 6.1. The Postulated Pathway for Free Cytokinins In Chapter 7, we noted that isopentenyl diphosphate (IPP) is the starting point for several plant hormones, gibberellins, brassinosteroids, and, via carotenoids, abscisic acid (see Fig. 7-5 in Chapter 7). It is also the starting point for the synthesis of the isoprenoid class of cytokinins. IPP isomerizes with dimethylallyl diphosphate (DMAPP or A2 isopentenyl diphosphate). DMAPP, in turn, is believed to condense with adenosine-5'-monophosphate (AMP) to give rise to isopentenyladenosine-5'-monophosphate (iPMP) (Fig. 8-5). iPMP is the precursor for all naturally occurring cytokinins. It is stereospecifically hydroxylated at C-4 of the side chain to form zeatin ribotide (ZMP). In a subsequent step, the ribose plus phosphate are cleaved to give zeatin. The hydroxylation step seems to occur very quickly, because iPMP, isopentenyladenosine (iPA), and isopentenyladenine (iP) are rarely found as free compounds in most plants. Zeatin, once formed, is stable and may be reduced to dihydrozeatin at a later step. The scheme just given is based on feeding ^^C-labeled adenine to plant tissues or extracts, and analysis of products. It is difficult to be certain of the pathway because the products occur in low abundance and their radioactivity has to be measured against a
background of radioactivity in precursors that occur in large abundance, a situation very similar to that for lAA (see Chapter 6). Moreover, there are no known cytokinin synthesis mutants or inhibitors of cytokinin biosynthesis, and the enzymes associated with these reactions have not been completely purified (but see below).
6.2. Synthetic E n z y m e s Several phytopathogenic bacteria that cause tumors in plants synthesize cytokinins via transfer of DMAPP to N^ of 5' AMP, and the bacterial genes encoding these isopentenyl transferases, have been cloned and characterized (see Appendix 2). Thus, it is likely that the first step in cytokinin biosynthesis in higher plants is catalyzed by a similar protein. However, efforts to purify the key enzyme, an isopentenyl transferase, also known as cytokinin synthase, have proven difficult, partly because it seems to be highly unstable, and partly because the substrates IPP/DMAPP and AMP, as well as the reaction product iPMP are targets for attack by phosphatases in plant tissues. In this dilemma, molecular techniques have come to rescue. An analysis of Arabidopsis genome has revealed nine gene sequences that encode putative isopentenyl transferases. Eight of these sequences expressed in E. coli yield recombinant proteins that show isopentenyl transferase activity. Among these, one gene, AtIPT4, gives a recombinant protein which catalyzes in vitro the transfer of the isopentenyl moiety from DMAPP to ATP with K^ values comparable to those for similar enzymes in bacteria. The recombinant AtIPT4 can utilize ADP as well, but significantly does not utilize AMP, as a substrate. The enzyme is biologically significant because an overexpression of the sequence in transgenic Arabidopsis induces callus cultures to show cytokinin responses constitutively, that is, form shoots in the absence of added cytokinins. Thus, it seems that a major enzyme catalyzing the key step in cytokinin biosynthesis is a DMAPP:ATP/ADP isopentenyl transferase, rather than a DMAPPiAMP isopentenyl transferase. Another gene sequence. AtlPTl, encodes a similar enzyme, which utilizes AMP but with a much higher K^ value. Based on these findings, a model for cytokinin synthesis in plants has been proposed (Fig. S-5). The model postulates utilization of ATP, ADP as well as AMP, as substrates together with DMAPP. The
196
II. Structure and Metabolism of Plant Hormones NH2
NH2
(EKEKE^OyoJ
(EhO-|/oJ
(EKE)-OYOJ
A T P HO OH
DMAPP
A D P HO OH
A M P HO OH
©
N
(EKgKE^OyoJ
sopentenyl adenine (iP)
(EHE^OYOJ HO OH
HO OH
isopentenyl ATP (iPTP)
N
1
isopentenyl ADP 1
I
(iPDP)
/
iPMP
HO OH
\
/ N
N
1 (EKE)-
w
HO OH
zeatin triphosphate? (ZTP)
1 (EKE^On/oJ
W
cx> u > cx> (EHDyoJ
frans-zeatin
HO OH
HO OH
ZMP
ZR
HO OH
zeatin diphosphate? (ZDP)
F I G U R E 8-5 A model for the cytokinin biosynthetic pathway in plants. The model visualizes the transfer of the isopentenyl moiety of DMAPP to ATP, ADP, or AMP by isopentenyl transferases (step 1) to give rise to corresponding isopentenyladenosine-tri-, di- or monophosphates (iPTP, iPDP, or iPMP). The tri- or diphophates are thought to funnel into the iPMP. In step (2), iPMP is hydroxylated at C-4 in the side chain to give rise to zeatin monophosphate (ZMP). Similar hydroxy la tions can also occur in iPTP or iPDP to yield corresponding zeatin tri- or diphosphates (ZTP or ZDP), which on subsequent dephosphorylations can give rise to ZMP. In step (3), ZMP is converted to zeatin riboside which is then cleaved to give rise to zeatin and sugar, or cleaved directly to give zeatin and sugar phosphate. Similarly iPMP gives rise to iPA and iP, which can also be hydroxylated to ZR and zeatin, respectively. The products that are conjectural are indicated by question marks. From Kakimoto (2001).
precise steps and reaction products are still unknown, but the pathway is thought to progress via iPMP to ZMP to zeatin. The hydroxylation at C-4 of the side chain of iP and iPA and iP to ZR and zeatin, respectively, is catalyzed by a membrane-bound cytochrome P450 monooxygenase. It is likely that the same enzyme also catalyzes the conversion of iPMP to ZMP in step 2 of
the synthesis of zeatin (see Fig. 8-5). The conversion of ZMP to ZR and zeatin and similar conversions of iPMP to IPA and iP in step 3 (Fig. 8-5) are probably accomplished by generic enzymes catalyzing the conversion of nucleotides to nucleosides and nitrogen bases. An alternate view has also been advanced that in Arabidopsis zeatin-type cytokinins are produced inde-
197
8. Cytokinins pendently of iPMP. In the model shown in Fig. 8-5, if one of the methyl groups in iPTP or iPDP are hydroxylated to produce zeatin-ribosyl-5'-tri- or diphosphate (ZTP or ZDP) followed by dephosphorylation, an iPMP-independent origin of zeatin can occur. 6.3. Formation of Cytokinins from tRNA Because tRNAs of plants contain ribosylated cytokinins (see Section 4 and Fig. 8-4), there has been a persistent theme for a long time that free cytokinins arise from the degradation of tRNAs. While it is possible that small amounts of free cytokinins arise in this manner, the consensus is that this is not the major route for production of free cytokinins. There are several reasons. Some are outlined below: i. In the tRNA of plants and plant-associated bacteria, the cis isomer of zeatin generally predominates over the trans isomer by a ratio of almost 40:1. The ratios of the two isomers in the free form in cytoplasm are the reverse, where trans-zeeitm predominates and the cis form is scarce. ii. Certain tissue culture lines of tobacco require an exogenous supply of cytokinins for growth, whereas other lines are cytokinin autonomous. In lines that are cytokinin dependent, ribosylated derivatives of cytokinins still occur in the tRNAs. If the tRNAs were a major source of free cytokinins, cytokinin dependence of such cultures is hard to explain. Also, in lines that are cytokinin autonomous, the tRNA pool does not seem to turn over at a high enough rate to account for the pool of free cytokinins in the cytoplasm. As mentioned earlier, c/s-zeatin and its ribosides/ ribotides have been shown to be the predominant form of free cytokinins in chick pea seeds. The synthesis of these cis forms is still a mystery. In summary, there is little support for the origin of free cytokinins from degradation of tRNAs. Instead, it has been thought that the synthesis of cytokinins occurs via transfer of DMAPP to AMP in a manner analogous to that seen in many phytopathogenic bacteria. This view has been challenged recently by the isolation of gene sequences in Arabidopsis that encode isopentenyl transferases that can catalyze in vitro the transfer of DMAPP to ATP/ADP or to a lesser extent AMP. Moreover, an overexpression of the gene for DMAPP:ATP/ADP isopentenyl transferase in transgenic tissue gives cytokinin responses constitutively. Thus, plants seem to utilize a novel type of isopentenyl transferase that has greater affinity for ATP/ADP than AMP. Since many gene sequences in Arabidopsis encode isopentenyl transferases, it also seems
likely that multiple isoforms of the enzyme occur in plants. The details of reaction products and steps involved in biosynthesis of zeatin starting from ATP/ ADP and DMAPP are still unknown, but are thought to progress via iPMP. There is also evidence that zeatin may be formed independently of the iPMP pathway. 6.4. Sites of Synthesis Cytokinins are believed to be synthesized in young or meristematic tissues, e.g., root apices, developing shoot buds, cambial tissue, developing seeds, especially liquid endosperm, and young fruits; in short, in areas where cell divisions are occurring at a high frequency. The cloning of the isopentenyl transferase genes is likely to provide definitive information as to the sites of cytokinin synthesis and environmental factors that regulate such synthesis. The cytokinins produced in roots tips are known to migrate upward through the xylem, as can be shown by collecting xylem sap from cut stem stumps and analyzing the sap. Likewise, phloem exudate coming from apical buds can be collected and shown to contain cytokinins (see Chapter 13). There is not much reliable information on endogenous levels because very few plants/parts have been investigated using GC, GC-MS. From bioassays, levels of 1-100 |jLg • g fw~^ are reported, lesser in vegetative parts, and more in seeds and storage tissues, but in these latter tissues, they occur mostly in conjugated forms (see Section 7.2).
7. REGULATION OF CYTOKININ LEVELS As in the case of lAA and GAs, regulation of the free cytokinin content is critical for orderly plant growth and development. This is shown clearly by plants that overproduce cytokinins either because of a mutation or because they have been transformed by the ipt gene from Agrobacterium. As mentioned earlier, mutants in cytokinin biosynthesis have not been identified, but a cytokinin overproducing mutant, ampl (for altered meristem program), is known from Arabidopsis. This mutant was isolated in a screen for altered morphologies in seedling growth and proved to contain five to six times higher concentrations of cytokinins. The mutant plants show several abnormalities, including production of multiple cotyledons in the embryo, enhanced lateral branching, retarded root development, and delayed
198
II. Structure a n d M e t a b o l i s m of Plant H o r m o n e s
senescence of leaves. Except for polycotyly, similar effects are seen in plants transformed with and overexpressing the bacterial ipt gene. These cytokininoverproducing plants also show enhanced levels of cytokinin metabolites. 7.1. Regulation of Cytokinin Biosynthesis Factors that trigger or modulate the synthesis of cytokinins are still mostly unknown, but with the cloning of the IPT genes in Arabidopsis, rapid progress is expected. However, there is considerable information on cytokinin metabolism. This information comes mainly from feeding labeled zeatin or dihydrozeatin to plant tissues or organs and analysis of labeled products. Because cytokinins are known to be synthesized in roots and translocated to aboveground parts, one common method of feeding exogenous cytokinins is via cut ends of seedlings with roots removed. Other favorite materials for feeding experiments are immature seeds, fruits such as kernels or pods, or cut leaves. 7.2. Conjugation of Cytokinins 7.2.1. Formation of Ribosyl
Derivatives
Tritiated zeatin and dihydrozeatin fed to plant tissues are rapidly converted to their N^-ribosides and ribotides as shown in Fig. 8-6 (for structures of these molecules, see Fig. 8-3). Free cytokinins and their ribosides, and ribotides are interconvertible. In a two-step process, zeatin can be converted to zeatin riboside by adenosine phosphorylase, and zeatin riboside can be converted to the ribotide form by an adenosine kinase. Zeatin can also be directly converted to its ribotide by an adenosine phosphoribosyltransferase (APRT). Enzymes involved in these interconversions are generic enzymes, which catalyze adenine to
zeatin-5-ribotide ^^ f^^'
zeatin
zeatin riboside adenosine phosphorylase
F I G U R E 8-6 Schematic illustration of interrelationships among zeatin, zeatin riboside, and zeatin ribotide. APRT, adenosine phosphoribosyl transferase.
nucleotide conversions. For example, an APRT, isolated and characterized from A. thaliana, can catalyze phosphoribosylation of not only free cytokinin bases, such as zeatin, but also adenine as a substrate. The ribosides and ribotides show biological activity in bioassays, but most likely they are not active per se and it is the free N bases released after hydrolysis that are active. As mentioned in Section 4, zeatin ribotide is also formed during the synthesis of zeatin (see Fig. S-5) and can give rise to zeatin riboside. Thus, ribosides and ribotides are intermediates in the biosynthesis of cytokinins and also represent forms that scavenge excess active cytokinins. The regulation of these pools of cytokinins and their ribosyl derivatives is little understood but likely involves different enzymes in the two sets of reactions and probably different cellular compartments. There also seems to be a homeostatic control over their levels. For instance, after continuous feeding of cytokinins or ribosyl derivatives to plant tissues, excess ribosides and ribotides may be irreversibly converted to N-glucosylated derivatives or be broken down by side chain cleavage (see Section 7.3). 7.2.2. Glycosylation
of the Side Chain
Sugars such as glucose or xylose can be conjugated via the -OH group in the side chain forming what are known as O-glycosides (Fig. 8-7). These reactions are reversible. O-glycosides, like ribosides and ribotides, show activity in bioassays, but it is generally accepted that free bases provide the activity. O-glycosides are very common and seem to be the principal form in which the cytokinins are stored in storage tissues and developing seeds, where they may accumulate to surprisingly high levels. Their synthesis involves specific glycosyl transferases, and genes encoding a zeatin-O-glucosyl transferase (ZOGl) and a zeatin-0-xylosyl transferase (ZOXI) have been cloned from Phaseolus species. With cloning of these genes, more information about the sites of glycosyl conjugation and factors that regulate it is likely to accumulate. Because of their increased polarity, O-glycosylated conjugates can be sequestered into vacuoles for possible use later. The hydrolysis of O-glycosyl conjugates occurs readily, and several p-glycosidases, are known to cleave the O-glycosyl group. In the absence of p-glycosidases, O-glycosides are relatively stable in plant tissues. They are protected against oxidative cleavage of the side chain by the main degradative enzyme cytokinin oxidase (see later) because of the presence of the glycosyl residue in the side chain.
199
8. Cytokinins CH2O
CH2OH
CH2OH
LI> *
. ^ I
N ^ N - ^ ^ N ^
OH
p-D-glucose
zeatin-O-p-D-glucoside
FIGURE 8-7 Glycosylation of the side chain in zeatin.
7.3. Irreversible Inactivation Cytokinins are irreversibly inactivated by certain modifications in the purine ring structure or by cleavage of the N^ side chain. 7,3,1. Conjugation of Glucose or Amino Acid Residues with Adenine Glucose conjugates at the N^ or N^ position in the adenine ring (e.g., [7Glc]Z or [9Glc]diHZ) are formed readily if zeatin or dihydrozeatin is supplied to plant tissues (Fig. 8-8A). N^-glucosides are also formed but appear to be less common. Such position-specific glucosylations are probably catalyzed by specific glucosyltransferases, not the ones involved in 0-glycosylations, but the details are obscure. Amino acid conjugates with alanine at the N^ position are also formed (Fig. 8-8B). They were first isolated as minor metabolites in lupin {Lupinus luteus) and, hence, were named lupinic acid (conjugate with zeatin ([9Ala]Z) and dihydrolupinic acid (conjugate with dihydrozeatin (diH[9Ala]Z). An enzyme, a P-(6-allylaminopurine-9-yl) adenine syn-
thase, has been characterized from developing seeds of lupin. N-glucosylated or amino acid conjugates are stable over long periods and seem to be irreversibly inactivated products. Enzymes capable of hydrolyzing these products have not been investigated. These compounds are much more polar than the original cytokinins, which may facilitate their sequestration in the vacuole. 7,3,2, Oxidative Cleavage of the N^ Side Chain Cytokinin oxidase is the enzyme that catalyzes the cleavage of the N^ side chain from adenine. The enzyme requires the A2 double bond in the side chain for its activity. Hence, its natural substrates are isopentenyladenine and zeatin and their ribosylated derivatives (Fig. 8-9). The enzyme is unable to cleave side chains that lack the double bond, as in dihydrozeatin and its derivatives. It is also inactive against O-glycosylated conjugates, where the side chains have a glucosyl or xylosyl residue (see Fig. 8-7), and against synthetic cytokinins (e.g., benzyladenine, kinetin), where the side chains have an aromatic ring (see later).
CH2 HC — COOH
I
NH2
zeatin-7-p-D-glucoside [7G]Z
p-(zeatin-9-yl)-alanine [9Ala] Z
FIGURE 8-8 N-conjugation of the adenine ring by glucose (A) or alanine (B)
200
II. Structure a n d Metabolisin of Plant H o r m o n e s
CH3
cytokinin oxidase Isopentenyladenine
i;i:> • "^ Adenine
CH3
3-Methyl-2-butenal
F I G U R E 8-9 Cleavage of the side chain of isopentenyladenine by cytokinin oxidase. The products from isopentenyladenine are adenine and 3-methyl-2-butenal.
8.3.2.1. Cytokinin Oxidase Activity Is Enhanced by Its Substrate As mentioned before, the endogenous levels of free cytokinins are strictly regulated. Cytokinin oxidase activity is increased severalfold if plant tissues are incubated with large quantities of cytokinins in vivo. Similarly, an increase in cytokinin oxidase activity is seen in tobacco plants transformed with the ipt gene from Agrobacterium. These data indicate that cytokinin oxidase activity is induced by its own substrate and provide an elegant example of a negative feedback control over the endogenous levels of bioactive hormones. 8.3.2.2. Cytokinin Oxidase Activity Varies in Different Tissues and Is Developmentally Regulated The N-glucosylation of the purine ring and side chain cleavage are two methods for irreversibly lowering the intracellular levels of biologically active cytokinins, but the degree to which they are practiced in different plants, or plant parts, seems to vary. For instance, in radish seedlings, cytokinin oxidase activity is minimal, and more N^- than N^-glucosylation is favored. In corn seedlings and embryos in culture, N^glucosides are more common. Cytokinins are abundant in embryo and endosperm and in fruit pericarp during early phases of seed and fruit development (see Chapters 17 and 18). These levels are gradually reduced and there is a progressive increase in cytokinin oxidase activity in tissues as cell divisions cease and tissues mature. For instance, in developing fruits (pods) of Phaseolus sp., the degradation of zeatin by cytokinin oxidase was more rapid in seed coat tissue (where cell divisions cease earlier) than in the embryo or fruit tissue. In corn kernels, cytokinin oxidase activity increased as maturation progressed. 8.3.3.3. The Gene for Cytokinin Oxidase Has Been Cloned CK oxidase was purified to homogeneity from immature maize kernels, and the sequence used to clone
its gene called CKXl (for cytokinin oxidasel, the authors use the designation ckxl). The enzyme is an oxidoreductase and requires flavin adenine dinucleotide (FAD) and molecular O2 for activity. It can use trans-zeatin, isopentenyladenine, and isopenteneyladenosine, as substrates, but not, as expected, dihydrozeatin. Several genes from Arabidopsis have been cloned also, and detailed studies on the enzyme kinetics are in progress using recombinant protein. The cloning of cytokinin oxidase genes in maize and Arabidopsis and O-glycosylating enzymes in Phaseolus spp are important first steps in our understanding of cytokinin metabolism in plants. More work is likely to follow, and show whether these genes are regulated at the transcriptional or translational level, whether there are different isoforms with tissue a n d / o r substrate specificities, and what specific factors cause their induction.
8. SYNTHETIC C O M P O U N D S WITH CYTOKININ-LIKE ACTIVITY Kinetin was originally obtained from herring sperm DNA, and its discovery led to a search for synthetic compounds that could mimic cytokinin activity. Several compounds were isolated that had high activity in promoting cell division in tobacco pith culture. One of the most potent compounds isolated was 6-benzylaminopurine or benzyladenine (BA) (Fig. 8-10). Like all other cytokinins, BA has an adenine ring with an N^ side chain, but the side chain carries an aromatic ring, as in kinetin. Other synthetic compounds include several hydroxylated benzyl derivatives (Fig. 8-10). Benzyladenine and kinetin are two of the most widely used cytokinins. Benzyladenine has the added advantage that it is easily available and is relatively inexpensive. Both are stable compounds because, unlike zeatin and isopentenyladenine, their
201
8. C y t o k i n i n s
HN-CH2^Q
- C H 2 ^ }
H Benzyladenine (BA) HNrc
- C H 2 ^ Q
Metatoponin Orthotoponin (two hydroxylated derivatives of BA)
H2^j>
Tetrahydropyranobenzyladenine F I G U R E 8-10
Structure of benzyladenine and some other synthetic cytokinins.
side chains are immune to attack by cytokinin oxidase. The glucosylated conjugates of the side chain are also not formed because of the differences in the side chain. However, the enzymes that catalyze the modifications of the purine ring, i.e., formation of ribosylated derivatives and N-substituted glucosides and amino acid conjugates, are able to use BA and kinetin as substrates. Thus, BA administered exogenously is known to give rise to its N^-ribosyl derivatives, as well as N^glucoside and [9Ala] conjugates. Some authors refer to these cytokinins as aromatic cytokinins. Although aromatic cytokinins are still considered as of nonplant origin, reports suggest that they occur naturally in plants. Thus, benzyladenine and its hydroxylated derivatives have been reported in several plant tissues on the basis of immunoassays. These reports need to be confirmed by GC-MS analysis. Kinetin has also been reported to occur naturally and be formed via oxidative degradation of DNA in vivo.
9. CYTOKININ A N T A G O N I S T S (ANTICYTOKININS) Several synthetic compounds are known that show high anticytokinin activity. The most potent and specific of these appear to be substituted pyroUo [2,3d]-pyrimidines and 7-substituted-3-methylpyrazolo [4,3-d]pyrimidines (Fig. 8-12). At low doses (^ 1 fxM), these molecules specifically inhibit cytokinin-induced growth in cytokinin-dependent tobacco tissue cultures. Although their mode of action is not known, they seem to act as competitive inhibitors because
A/, A/^-Dlphenylurea O
8.1 Phenylurea Derivatives Some other substances, such as modified diphenyl and pyridyl-phenyl urea derivatives (Fig. 8-11), display significant cytokinin activity in several bioassays. They stimulate cell division in tobacco pith culture, promote shoot formation in callus tissue, and retard leaf senescence; however even those derivatives that have the highest activity in cell division assay still are only 1/100th as active as the adenine cytokinins, such as benzyladenine. Their mode of action is not known, but they probably function by inhibiting endogenous cytokinin oxidase, thereby having a "sparing" effect on cytokinins present in the tissue by inhibiting their breakdown.
/
y—NH—C—HN—^ Forchlorofenuron
r^^.,_" NH
•HN
NN CI
\L^N
Thidiazuron F I G U R E 8-11 Some phenylurea derivatives with cytokinin activity: N,N'-Diphenylurea; N-(2-chloro-4-pyridyl)-N'-phenylurea (trade name, Forchlorofenuron); and N-phenyl-]V'-(l,2,3-thiadiazol-5yl)urea (Thidiazuron). From Shudo (1994).
202
II. Structure a n d M e t a b o l i s m of Plant H o r m o n e s CH3 CH2 — C H CH3
H3CS
3-Methyl-7-(3-methylbutylamino) pyrazolo [4,3-c/] pyrimidine
4-(Cyclopentylamino)-2-methylthiopyrrolo [2,3-d] pyrimidine
FIGURE 8-12 Two potent anticytokinins: 3-methyl-7-(3-methylbutylamino) pyrazolo [4,3-rf]pyrimidine and 4-(cyclopentylamino)-2-methylthiopyrrolo[2,3-rf]pyrimidine.
inhibition can be reversed by increasing the cytokinin concentration in the medium.
10. CHAPTER SUMMARY Cytokinins serve many important functions in plant development and morphogenesis. They are involved in the regulation of cell division; they interact with auxins in the control of apical dominance and lateral branching and the root-shoot ratio in intact plants and in tissue culture. They retard the senescence of leaves and promote the light-independent deetiolation response, including greening, of dark-grown seedlings. Several cytokinins occur naturally in plants. They have an adenine base and a five carbon isopentenyl side chain. Among these, zeatin, specifically trans-zeatin, is the most abundant. The synthesis of cytokinins in higher plants has been unclear and controversial for a long time, but progress finally seems to be achieved with the cloning of genes encoding isopentenyl transferases (IPTs) in Arabidopsis. These IPTs seem to use ATP/ADP, rather than AMP, together with DMAPP, to yield isopentenyladenine monophosphate (iPMP), which ultimately gives rise to zeatin and other naturally occurring adenine cytokinins. The possibility that cytokinins may arise from the degradation of tRNAs is not considered likely. Ribosylated derivatives, with a ribose or ribose 5'-monophosphate attached to the adenine moiety, of cytokinins are common. Conjugates of cytokinins where a glycosyl moiety is attached to the OH group in the side chain are also common. Both ribosylated derivatives and glycosyl conjugates show activity in bioassays, but it is generally believed that they do so after hydrolysis to free bases. Irreversible deactivation of cytokinins occurs in two ways. The side chain may be cleaved by cytokinin oxidase, an enzyme that has specific requirements for an -OH
group and a A2,3 bond in the side chain. Significantly, this enzyme is induced by high concentrations of cytokinin in plant tissues. Modifications to the adenine ring, by substitution of glucose or amino acids (particularly alanine) to the nitrogen atoms, also cause irreversible deactivation. Genes encoding cytokinin oxidase and glycosylating enzymes have been cloned from several plants and rapid progress in our understanding of regulation of cytokinin metabolism is expected. Several synthetic cytokinins (e.g., benzyladenine, kinetin) are known. They have the adenine base, but have an aromatic ring in the side chain. Supplied to plant tissues, these cytokinins form ribosylated derivatives and glucosyl or amino acid substituents in the adenine ring, but they are more stable (than zeatin) in plant tissues because they are not subject to side chain cleavage by cytokinin oxidase. There are some reports that benzyladenine and kinetin occur naturally. Some synthetic compounds, which inhibit cytokinin promotion of cell division in tissue/ cell culture, probably as competitive inhibitors, are known.
References Books and Reviews Binns, A. N. (1994). Cytokinin accumulation and action: Biochemical, genetic, and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 173-196. Letham, D. S., and Palni, L. M. S. (1983). The biosynthesis and metabolism of cytokinins. Annu. Rev. Plant Physiol. 34, 163197. McGaw, B. A. and Burch, L. R. (1995). Cytokinin biosynthesis and metabolism. In "Plant Hormones, Physiology, Biochemistry and Molecular Biology" (P. J. Davies, ed.), pp. 98-117. Kluwer, Dordrecht. Mok, D. W. S., and Mok, M. C. (eds.) (1994). "Cytokinins: Chemistry, Activity, and Function." CRC Press, Boca Raton, FL. Morris, R. O. (1995). Genes specifying auxin and cytokinin biosynthesis in prokaryotes. In "Plant Hormones, Physiology, Biochem-
8. C y t o k i n i n s istry and Molecular Biology" (P. J. Davies, ed.), pp. 318-339. Kluwer, Dordrecht. Skoog, F., and Miller, C. O. (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol. 11, 118-131.
203
Prinsen, E., Kaminek, M. and van Onckelen, H. A. (1997). Cytokinin biosynthesis: A black box? Plant Growth Regul. 23, 3-15. Takei, K., Sakakibara, H., and Sugiyama, T. (2001). Identification of genes encoding adenylate isopentenyltransferase, a cytokinin bisynthesis enzyme, in Arabidopsis thaliana. J. Biol. Chem. 276, 26405-26410.
1. Discovery Haberlandt, G. (1913). Zur physiologie der zellteilungen. Sitzungsber. K. Preuss. Akad. Wiss., 318-345. Letham, D. S. (1963). Zeatin, a factor inducing cell division from Zea mays. Life Sci. 8, 569-573. Miller, C. O., Skoog, F., Okomura, F. S., Von Saltza, M. H., and Strong, F. M. (1956). Isolation, structure and synthesis of kinetin, a substance promoting cell division. /. Am. Chem. Soc. 78, 1345-1350. Skoog, F. (1994). A personal history of cytokinin and plant hormone research. In "Cytokinins: Chemistry, Activity, and Function" (D. W. S. Mok and M. C , Mok, eds.), pp. 1-14. CRC Press, Boca Raton, FL. 3. Structure of Cytokinins Angra, R., Mandahar, C. L., and Gulati, A. (1990). The possible involvement of cytokinins in the pathogenecity of Helminthosporium maydis. Mycopathologica 109, 177-182. Burrows, W. J., Skoog, F., and Leonard, N. J. (1971). Isolation and identification of cytokinins located in the transfer ribonucleic acid of tobacco callus grown in the presence of 6-benzylaminopurine. Biochemistry 10, 2189-2194. Emery, R. J. N., Leport, L., Barton, J. E., Turner, N. C , and Atkins, C. A. (1998). Cfs-isomers of cytokinins predominate in chickpea seeds throughout their development. Plant Physiol. 117, 1515-1523. Hall, R. H. (1970). N^-(D2-isopentenyl) adenosine: Chemical reactions, biosynthesis, metabolism and significance to the structure and function of tRNA. Prog. Nucleic Acid Res. Mol. Biol. 10, 57086. Letham, D. S., Shannon, J. C , and MacDonald, I. R. C. (1964). The structure of zeatin, a (kinetin like) factor inducing cell division. Proc. Chem. Soc. Lond., 230-231 Palni, L. M. S., and Horgan, R. (1983). Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea. Planta 159, 178-181. 6. Biosynthesis in Higher Plants Astot, C , Dolezal, K., Nordstrom, A., Wang, Q., Kunkel, T., Moritz, T., Chua, N.-H., and Sandberg, G. (2000). An alternative cytokinin biosynthesis pathway. Proc. Natl. Acad. Sci. USA 97, 14778-14783. Bassil, N. v., Mok, D. W. S., and Mok, M. C. (1993). Partial purification of zeatin O-xylosyltransferase in Phaseolus. Plant Physiol. 102, 867-872. Blackwell, J. R., and Horgan, R. (1994). Cytokinin biosynthesis by extracts of Zea mays. Phytochemistry 35, 339-342. Chen, C.-M. (1982). Cytokinin biosynthesis in cell-free systems. In "Plant Growth Substances" (P. F. Wareing, ed.), pp. 155-164. Academic Press, London. Chen C.-M. (1997). Cytokinin biosynthesis and interconversion. Physiol. Plant 101, 665-673. Chen, C.-M., and Ertl, J. R. (1994). Cytokinin biosynthetic enzymes in plants and slime mold. In "Cytokinins: Chemistry, Activity, and Function" (D. W. S. Mok and Mok, M. C. eds.), pp. 81-85. CRC Press, Boca Raton, FL. Kakimoto, T. (2001). Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP / ADPisopentenyltransferases. Plant Cell Physiol. 42, 677-685.
7. Regulation of Cytokinin Levels Bilyeu, K. D., Cole, J. L., Loskey, J. G., Riekhof, W. R., Esparza, T. J., Kramer, M. D., and Morris, R. O. (2001). Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol, lis, 378-386. Brzobohaty, B., Moore, I., Kristofferson, P., Bako, L., Campos, N., Schell, J., and Palme, K. (1993). Release of active cytokinin by pglucosidase localized to the maize root meristem. Science 161, 1051-1054. Burch, L. R., and Stutchbury, T. (1987). Activity and distribution of enzymes that interconvert purine bases, ribosides for cytokinin metabolism. Physiol. Plant 69, 283-288. Chaudhury, A. M., Letham, D. S., Craig, S., and Dennis, E. S. (1993). ampl-a mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering. Plant J. 4, 907-916. Dixon, S. C , Martin, R. C , Mok, M. C , Shaw, G., and Mok, D. W. S. (1989). Zeatin glucosylation enzymes in Phaseolus: Isolation of O-glucosyltransferase from P. lunatus and comparison to O-xylosyltransferase from P. vulgaris. Plant Physiol. 90, 13161321. Kaminek, M., and Armstrong, D. J. (1990). Genotypic variation in cytokinin oxidase from Phaseolus callus cultures. Plant Physiol. 93, 1530-1538. Martin, R. C , Mok, M. C , and Mok, D. W. S. (1993). Cytolocalization of zeatin O-xylosyltransferase in Phaseolus. Proc. Natl. Acad. Sci. USA 90, 953-957. Martin, R. C , Mok, M. C. and Mok, D. W. S. (1999a). A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris. Plant Physiol. 120, 353-357. Martin, R. C , Mok, M. C , and Mok, D. W. S. (1999b). Isolation of a cytokinin gene, ZOGl, encoding zeatin O-glucosyltransferase from Phaseolus lunatus. Proc. Natl. Acad. Sci. USA 96, 284-289. McGaw, B. A., Horgan, R., and Heald, J. K. (1985). Cytokinin metabolism and the modification of cytokinin activity in radish. Phytochemistry 24, 9-13. Medford, J. I., Horgan, R., El-Sawi, Z., and Klee, H. J. (1989). Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. Plant Cell 1, 403-413. Moffat, B., Pethe, C , and Laloue, M. (1991) Metabolism of benzyladenine is impaired in a mutant of Arabidopsis thaliana lacking adenine phosphoribosyltransferase activity. Plant Physiol. 95, 900-908. Mok, D. W. S., and Martin, R. C. (1994). Cytokinin metabolic enzymes. In "Cytokinins: Chemistry, Activity, and Function" (D. W. S. Mok and M. C. Mok, eds.), pp. 129-137. CRC Press, Boca Raton, FL. Morris, R. C , Bilyeu, K. D., Laskey, J. G., and Cheikh, N. N. (1999). Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem. Biophys. Res. Commun. 255, 328-333. Motyka, V., Faiss, M., Strnad, M., Kaminek, M., and Schmiilling, T. (1996). Changes in cytokinin content and cytokinin oxidase activity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol. Ill, 10351043.
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11. Structure and Metabolism of Plant Hormones
8. Synthetic Compounds with Cytokinin-like Activity Barciszewski, J., Siboska, G. E., Pedersen, B. O., Clark, B. F. C , and Rattan, S. I. S. (1997). A mechanism for the in vivo formation of N^-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA. FEES Lett. 393, 197-200. Hare, P. D., and Van Staden, J. (1994). Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell Physiol. 35, 1121-1125. Shudo, K. (1994). Chemistry of phenylurea cytokinins. In "Cytokinins: Chemistry, Activity and Function" (D. W. S. Mok and M. C. Mok, eds.), pp. 35-42. CRC Press, Boca Raton, FL. Strnad, M. (1997). The aromatic cytokinins. Physiol. Plant 101, 674-688.
9. Cytokinin Antagonists (Anticytokinins) Gregorini, G., and Laloue, M. (1980). Biological effects of cytokinin antagonists 7-(pentylamino) and 7-(benzylamino)-3-methylpyrazolo (4,3-rf) pyrimidines on suspension-cultured tobacco cells. Plant Physiol 65, 363-367. Skoog, F., Schmitz, R. Y., Bock, R. M., and Hecht, S. M. (1973). Cytokinin antagonists: Synthesis and physiological effects of 7substituted 3-methylpyrazolo [4,3-^] pyrimidines. Phytochemistry 12, 25-37. Skoog, F., Schmitz, R. Y., Hecht, S. M., and Frye, R. B. (1975). Anticytokinin activity of substituted pyrrolo [2,3-rf]pyrimidines. Proc. Natl. Acad. Sci. USA 72, 3508-3512.
8 Cytokinins
1. 2. 3. 4.
DISCOVERY 191 BIOLOGICAL FUNCTIONS AND BIO ASSAYS 192 STRUCTURE OF CYTOKININS 192 CYTOKININS OCCUR FREE IN THE CYTOPLASM AS WELL AS COMPONENTS OF tRNA 192 5. RELATIVE DISTRIBUTION OF NATURAL CYTOKININS AMONG PLANTS 193 6. BIOSYNTHESIS IN HIGHER PLANTS 195 6.1. The Postulated Pathway for Free Cytokinins 195 6.2. Synthetic Enzymes 195 6.3. Formation of Cytokinins from tRNA 197 6.4. Sites of Synthesis 197 7. REGULATION OF CYTOKININ LEVELS 197 7.1. Regulation of Cytokinin Biosynthesis 198 7.2. Reversible Inactivation 198 7.3. Irreversible Inactivation 199 8. SYNTHETIC COMPOUNDS WITH CYTOKININ-LIKE ACTIVITY 200 8.1. Phenylurea Derivatives 201 9. CYTOKININ ANTAGONISTS (ANTICYTOKININS) 201 10. CHAPTER SUMMARY 202 REFERENCES 202
In the 1940s and 1950s, the techniques of plant tissue culture were being developed, and it was noted that callus cultures from tobacco pith explants or carrot roots, both favorite objects for study, placed on agar blocks soaked with nutrients and auxin still showed very little cell division. They required other substances, e.g., coconut milk (liquid endosperm from coconut), or extracts from vascular tissues, yeast extract, autoclaved DNA, or even adenine. In 1956, Carl Miller in Folke Skoog's laboratory at Wisconsin University, Madison, discovered that a substituted adenine, 6 furfuryl amino purine, obtained from autoclaved herring sperm DNA was far more potent than adenine in promoting cell division in tobacco pith explants. This substance was given the name kinetin (Fig. 8-1) [for a personal and interesting account of this discovery, see Skoog (1994)]. Kinetin does not occur naturally in plants (but see Section 8). In the search for natural substances, it was expected that endosperm tissue, which provides nutrition for the growth of embryo during its early
1. DISCOVERY Fiaberlandt, a German plant physiologist, noted as far back as 1913 that certain diffusible factors from phloem tissue, e.g., phloem exudate, could cause cell division in potato tubers. Later in 1921 he discovered that healing of cut plant tissues by cell division was prevented if the cut surfaces were washed with water. Both observations suggested the presence of a soluble factor in plant tissues that promoted cell division.
FIGURE 8-1 Kinetin or N^-(furfurylamino)purine. The side chain is shaded for comparison with side chains in Fig. 8-2.
191
192
II. Structure and Metabolism of Plant Hormones
development or, postgerminatively, for growth of young seedling (see Chapters 18 and 19), might be a promising material to look for hormones that promote cell division. Liquid endosperm from coconut, maize, horse chestnut, and immature fruits of banana were favorite objects for study. Thus, in 1963, D. S. Letham at CSIRO, Canberra, Australia, isolated a substance from kernels of sweet corn that had high cell division-promoting capacity in callus cultures. The material was a N^-substituted adenine [6-(4-hydroxy-3methyl-frflns-2-enylamino) purine] and was given the name zeatin, since it was isolated from maize {Zea mays). Since the discovery of zeatin, several naturally occurring cytokinins and some synthetic substances with similar biological activities have been discovered. These substances, while they all carry an adenine moiety, differ in the structures of their side chains. Thus, like auxins, cytokinins are defined more on the basis of their biological functions than their structure.
2. BIOLOGICAL FUNCTIONS A N D BIOASSAYS Cytokinins are defined as compounds that promote cell division in callus and tissue culture. In combination with auxins, they regulate the ratio of shoot bud vs root growth in tissue culture and in stem cuttings; in intact plants, they regulate apical dominance and lateral root initiation. They also retard senescence and chlorophyll degradation in aging leaf tissues and, in combination with ethylene and light, regulate the growth of dicot seedlings in dark. These responses are covered in Sections III and V of this book. Bioassays for cytokinin activity include (1) promotion of growth in tobacco pith culture or soybean callus tissue and (2) expansion of excised radish cotyledons in culture. These assays are based on the promotion of cell division and expansion activity by cytokinins. Another common bioassay is inhibition of senescence, as measured by a reduction in loss of chlorophyll from leaf tissues. Cytokinins, like auxins and gibberellins, have a wide occurrence in plants and are known besides vascular plants from mosses and algae. In mosses they regulate bud growth in protonema. They are also produced by several strains of soil-living and phytopathogenic bacteria, such as Agrobacterium and Pseudomonas, which cause galls or tumors in plants, and by certain phytopathogenic fungi, such as Helminthosporium and Ustilago (see Appendix 2). Interestingly, an example of the inhibition of senescence by cytokinins is provided
by these fungi: they cause the formation of "green islands" on leaves of plants, which they infect.
3. STRUCTURE OF CYTOKININS All naturally occurring cytokinins have an adenine ring structure with a 5 carbon isopentenyl side chain from N^ of the adenine molecule (Fig. 8-2). Some authors refer to these as the isoprenoid class of cytokinins. In addition to zeatin, they include isopentenyladenine (or dimethylallyladenine) and the reduced form of zeatin, the dihydrozeatin. Because the side chain in zeatin possesses a double bond, geometric isomers are possible. These occur either as transzeatin or c/s-zeatin. In the cis isomer, the hydroxyl group of the isopentenyl side chain is oriented toward the N^ position of the purine ring, whereas in the trans isomer the hydroxyl group is oriented away from the purine ring. Adenine occurs in DNA and RNA, and its ribose and ribose plus phosphate derivatives, adenosine and adenosine 5'-monophosphate, respectively, are common in plants. The ribose is always attached to N^ in the purine ring. Thus, natural cytokinins also occur as their sugar derivatives, N^ ribosides and ribotides (sugar plus phosphate). Figure 8-3 shows the structures for zeatin riboside and zeatin ribotide; others have similar structures. In addition to ribosides and ribotides, conjugates of cytokinins with glucose, xylose, and amino acids are also known. Some authors treat all of these compounds together as natural cytokinins. Thus, there can be a bewildering array of naturally occurring cytokinins or cytokinin-type compounds. In this text, the term cytokinin is used, as far as possible, for natural cytokinins with an adenine base. Ribosyl derivatives are referred to as ribosylated derivatives, and conjugates are referred to as conjugates.
4. CYTOKININS OCCUR FREE IN THE CYTOPLASM AS WELL A S C O M P O N E N T S OF tRNA Natural cytokinins and their ribosides and ribotides occur free in the cytoplasm, but they also occur as integral components of certain transfer RNAs (tRNAs). As components of these tRNAs, they are always in the form of ribosylated derivatives, not as adenine bases. Many such ribosylated cytokinins are known from tRNAs (e.g., isopentenyladenine-9-riboside, cisand frflns-zeatin-9-riboside, 2-methylthioisopentenyl-
193
8. C y t o k i n i n s 4
1
HNH
HN
Adenine (6-aminopurJne)
H. HN
CH2
k
\ ^N H
CH2
3 / CH3
CH3
fi 2 N -(A -lsopentenyl)adenine
>CH20H CH3
\ 2 /
H\ HN
CH2
k
\
/CH3 CH2OH
^N H c/s-Zeatin
frans-Zeatin
H H H. I I ,CH20H
)c-c( HN—CH^ N N^ \
^CH3
Dihydrozeatin F I G U R E 8-2 Structure of the isoprenoid class of cytokinins. (Top left) Adenine (6-aminopurine) with the ring numbering system. Natural cytokinins are all derivatives of adenine, with substitution of a five carbon chain in the amino group (shaded area). Carbon atoms in the side chain are numbered 1-5. Hydroxylation of the side chain at C-4 creates asymmetry and gives rise to trans- or cis-zeatin.
ademne-9-riboside, cis- and trans-1 methylthiozeatin9-riboside). Such tRNA-bound ribosylated cytokinins occur widely in a variety of organisms: plants, phytopathogenic bacteria, yeast, and animals. Transfer RNAs are rich in substitutions (e.g., methylations), which occur all over the molecule, but the cytokinin ribosides occupy a precise location—they occur exclusively as the base adjacent to the 3' end of the anticodon (Fig. 8-4). The function of such exclusive localization is obscure, but it has been suggested that it may play a regulatory role in protein synthesis, probably by promoting the codon-anticodon interaction a n d / o r increasing the binding affinity of aminoacyl tRNA to the ribosomes. However, there is no direct proof for this assumption. The supply of exogenous cyto-
kinins to plant tissues is known to promote protein synthesis, but it is considered doubtful that such promotion is mediated via cytokinin derivatives bound to tRNA.
5. RELATIVE DISTRIBUTION OF NATURAL CYTOKININS A M O N G PLANTS It is likely that different plants show lesser or greater abundance of one or the other types of natural cytokinins. Among the plants that have been investigated, trans-zea.tm (unless necessary, hereafter referred to as zeatin only) seems to be the most widely distributed.
194
II. Structure a n d M e t a b o l i s m of Plant H o r m o n e s
Hv
)c=c(
HN
CH2^
CH2OH
H
CH3
HN
Ribosylzeatin
CH3
Ribosylzeatin-5-monophosphate
(zeatin riboside) FIGURE 8-3
CH2
CH2OH
(zeatin ribotide)
Structures of zeatin riboside and zeatin ribotide (zeatin is shown in trans configuration).
A—O—Tyrosyl I
C C I pC = I U= I C=
G I I A I G / I U G \ I C=G \ A I I Me A G=C \ Me^\/"U / ^G=C^C-C-C-G-C C-C-G
UHp UH2 / GOMe
I
G \
A Me \ G
G-G-G-C-G \ T^x^^ \ C —Me G-C = G ^UH2 Me^/ I I^A Me A= ^G-A I A = I I G= C I I / A = ¥ ^ C A I I U A—IP(iPAdo) \ ^ " / Anticodon - ^ G - \ | / — A ^ C — A —U-^mRNA
G-G-C. UH2
./ UHo
•UHp
t^
_
J
Codon FIGURE 8-4 The structure of yeast tRNA for tyrosine. Note that the isopentenyladenosine (iPAdo) is the base next to the 3' end of the anticodon. From Hall (1970).
195
8. Cytokinins Zeatin is also one of the most biologically active cytokinins known. In contrast, c/s-zeatin has much less activity. Until recently, c/s-zeatin was thought to be present mainly in tRNA as a ribosylated derivative, and there were only sporadic reports of its occurrence as a free cytokinin. Studies using GC-MS for identification reveal, however, that c/s-zeatin, as well as its ribosides and ribotides, in free form are the predominant cytokinins in chick pea seeds, whereas trans isomers are minor constituents. As more plants are investigated, it may turn out that both forms of zeatin occur in free form and that their relative abundance varies in different plants, or plant parts. Dihydrozeatin and its ribosyl derivatives and conjugates also occur frequently, but isopentenyladenine seems to have much less free occurrence.
6. BIOSYNTHESIS IN HIGHER PLANTS 6.1. The Postulated Pathway for Free Cytokinins In Chapter 7, we noted that isopentenyl diphosphate (IPP) is the starting point for several plant hormones, gibberellins, brassinosteroids, and, via carotenoids, abscisic acid (see Fig. 7-5 in Chapter 7). It is also the starting point for the synthesis of the isoprenoid class of cytokinins. IPP isomerizes with dimethylallyl diphosphate (DMAPP or A2 isopentenyl diphosphate). DMAPP, in turn, is believed to condense with adenosine-5'-monophosphate (AMP) to give rise to isopentenyladenosine-5'-monophosphate (iPMP) (Fig. 8-5). iPMP is the precursor for all naturally occurring cytokinins. It is stereospecifically hydroxylated at C-4 of the side chain to form zeatin ribotide (ZMP). In a subsequent step, the ribose plus phosphate are cleaved to give zeatin. The hydroxylation step seems to occur very quickly, because iPMP, isopentenyladenosine (iPA), and isopentenyladenine (iP) are rarely found as free compounds in most plants. Zeatin, once formed, is stable and may be reduced to dihydrozeatin at a later step. The scheme just given is based on feeding ^^C-labeled adenine to plant tissues or extracts, and analysis of products. It is difficult to be certain of the pathway because the products occur in low abundance and their radioactivity has to be measured against a
background of radioactivity in precursors that occur in large abundance, a situation very similar to that for lAA (see Chapter 6). Moreover, there are no known cytokinin synthesis mutants or inhibitors of cytokinin biosynthesis, and the enzymes associated with these reactions have not been completely purified (but see below).
6.2. Synthetic E n z y m e s Several phytopathogenic bacteria that cause tumors in plants synthesize cytokinins via transfer of DMAPP to N^ of 5' AMP, and the bacterial genes encoding these isopentenyl transferases, have been cloned and characterized (see Appendix 2). Thus, it is likely that the first step in cytokinin biosynthesis in higher plants is catalyzed by a similar protein. However, efforts to purify the key enzyme, an isopentenyl transferase, also known as cytokinin synthase, have proven difficult, partly because it seems to be highly unstable, and partly because the substrates IPP/DMAPP and AMP, as well as the reaction product iPMP are targets for attack by phosphatases in plant tissues. In this dilemma, molecular techniques have come to rescue. An analysis of Arabidopsis genome has revealed nine gene sequences that encode putative isopentenyl transferases. Eight of these sequences expressed in E. coli yield recombinant proteins that show isopentenyl transferase activity. Among these, one gene, AtIPT4, gives a recombinant protein which catalyzes in vitro the transfer of the isopentenyl moiety from DMAPP to ATP with K^ values comparable to those for similar enzymes in bacteria. The recombinant AtIPT4 can utilize ADP as well, but significantly does not utilize AMP, as a substrate. The enzyme is biologically significant because an overexpression of the sequence in transgenic Arabidopsis induces callus cultures to show cytokinin responses constitutively, that is, form shoots in the absence of added cytokinins. Thus, it seems that a major enzyme catalyzing the key step in cytokinin biosynthesis is a DMAPP:ATP/ADP isopentenyl transferase, rather than a DMAPPiAMP isopentenyl transferase. Another gene sequence. AtlPTl, encodes a similar enzyme, which utilizes AMP but with a much higher K^ value. Based on these findings, a model for cytokinin synthesis in plants has been proposed (Fig. S-5). The model postulates utilization of ATP, ADP as well as AMP, as substrates together with DMAPP. The
196
II. Structure and Metabolism of Plant Hormones NH2
NH2
(EKEKE^OyoJ
(EhO-|/oJ
(EKE)-OYOJ
A T P HO OH
DMAPP
A D P HO OH
A M P HO OH
©
N
(EKgKE^OyoJ
sopentenyl adenine (iP)
(EHE^OYOJ HO OH
HO OH
isopentenyl ATP (iPTP)
N
1
isopentenyl ADP 1
I
(iPDP)
/
iPMP
HO OH
\
/ N
N
1 (EKE)-
w
HO OH
zeatin triphosphate? (ZTP)
1 (EKE^On/oJ
W
cx> u > cx> (EHDyoJ
frans-zeatin
HO OH
HO OH
ZMP
ZR
HO OH
zeatin diphosphate? (ZDP)
F I G U R E 8-5 A model for the cytokinin biosynthetic pathway in plants. The model visualizes the transfer of the isopentenyl moiety of DMAPP to ATP, ADP, or AMP by isopentenyl transferases (step 1) to give rise to corresponding isopentenyladenosine-tri-, di- or monophosphates (iPTP, iPDP, or iPMP). The tri- or diphophates are thought to funnel into the iPMP. In step (2), iPMP is hydroxylated at C-4 in the side chain to give rise to zeatin monophosphate (ZMP). Similar hydroxy la tions can also occur in iPTP or iPDP to yield corresponding zeatin tri- or diphosphates (ZTP or ZDP), which on subsequent dephosphorylations can give rise to ZMP. In step (3), ZMP is converted to zeatin riboside which is then cleaved to give rise to zeatin and sugar, or cleaved directly to give zeatin and sugar phosphate. Similarly iPMP gives rise to iPA and iP, which can also be hydroxylated to ZR and zeatin, respectively. The products that are conjectural are indicated by question marks. From Kakimoto (2001).
precise steps and reaction products are still unknown, but the pathway is thought to progress via iPMP to ZMP to zeatin. The hydroxylation at C-4 of the side chain of iP and iPA and iP to ZR and zeatin, respectively, is catalyzed by a membrane-bound cytochrome P450 monooxygenase. It is likely that the same enzyme also catalyzes the conversion of iPMP to ZMP in step 2 of
the synthesis of zeatin (see Fig. 8-5). The conversion of ZMP to ZR and zeatin and similar conversions of iPMP to IPA and iP in step 3 (Fig. 8-5) are probably accomplished by generic enzymes catalyzing the conversion of nucleotides to nucleosides and nitrogen bases. An alternate view has also been advanced that in Arabidopsis zeatin-type cytokinins are produced inde-
197
8. Cytokinins pendently of iPMP. In the model shown in Fig. 8-5, if one of the methyl groups in iPTP or iPDP are hydroxylated to produce zeatin-ribosyl-5'-tri- or diphosphate (ZTP or ZDP) followed by dephosphorylation, an iPMP-independent origin of zeatin can occur. 6.3. Formation of Cytokinins from tRNA Because tRNAs of plants contain ribosylated cytokinins (see Section 4 and Fig. 8-4), there has been a persistent theme for a long time that free cytokinins arise from the degradation of tRNAs. While it is possible that small amounts of free cytokinins arise in this manner, the consensus is that this is not the major route for production of free cytokinins. There are several reasons. Some are outlined below: i. In the tRNA of plants and plant-associated bacteria, the cis isomer of zeatin generally predominates over the trans isomer by a ratio of almost 40:1. The ratios of the two isomers in the free form in cytoplasm are the reverse, where trans-zeeitm predominates and the cis form is scarce. ii. Certain tissue culture lines of tobacco require an exogenous supply of cytokinins for growth, whereas other lines are cytokinin autonomous. In lines that are cytokinin dependent, ribosylated derivatives of cytokinins still occur in the tRNAs. If the tRNAs were a major source of free cytokinins, cytokinin dependence of such cultures is hard to explain. Also, in lines that are cytokinin autonomous, the tRNA pool does not seem to turn over at a high enough rate to account for the pool of free cytokinins in the cytoplasm. As mentioned earlier, c/s-zeatin and its ribosides/ ribotides have been shown to be the predominant form of free cytokinins in chick pea seeds. The synthesis of these cis forms is still a mystery. In summary, there is little support for the origin of free cytokinins from degradation of tRNAs. Instead, it has been thought that the synthesis of cytokinins occurs via transfer of DMAPP to AMP in a manner analogous to that seen in many phytopathogenic bacteria. This view has been challenged recently by the isolation of gene sequences in Arabidopsis that encode isopentenyl transferases that can catalyze in vitro the transfer of DMAPP to ATP/ADP or to a lesser extent AMP. Moreover, an overexpression of the gene for DMAPP:ATP/ADP isopentenyl transferase in transgenic tissue gives cytokinin responses constitutively. Thus, plants seem to utilize a novel type of isopentenyl transferase that has greater affinity for ATP/ADP than AMP. Since many gene sequences in Arabidopsis encode isopentenyl transferases, it also seems
likely that multiple isoforms of the enzyme occur in plants. The details of reaction products and steps involved in biosynthesis of zeatin starting from ATP/ ADP and DMAPP are still unknown, but are thought to progress via iPMP. There is also evidence that zeatin may be formed independently of the iPMP pathway. 6.4. Sites of Synthesis Cytokinins are believed to be synthesized in young or meristematic tissues, e.g., root apices, developing shoot buds, cambial tissue, developing seeds, especially liquid endosperm, and young fruits; in short, in areas where cell divisions are occurring at a high frequency. The cloning of the isopentenyl transferase genes is likely to provide definitive information as to the sites of cytokinin synthesis and environmental factors that regulate such synthesis. The cytokinins produced in roots tips are known to migrate upward through the xylem, as can be shown by collecting xylem sap from cut stem stumps and analyzing the sap. Likewise, phloem exudate coming from apical buds can be collected and shown to contain cytokinins (see Chapter 13). There is not much reliable information on endogenous levels because very few plants/parts have been investigated using GC, GC-MS. From bioassays, levels of 1-100 |jLg • g fw~^ are reported, lesser in vegetative parts, and more in seeds and storage tissues, but in these latter tissues, they occur mostly in conjugated forms (see Section 7.2).
7. REGULATION OF CYTOKININ LEVELS As in the case of lAA and GAs, regulation of the free cytokinin content is critical for orderly plant growth and development. This is shown clearly by plants that overproduce cytokinins either because of a mutation or because they have been transformed by the ipt gene from Agrobacterium. As mentioned earlier, mutants in cytokinin biosynthesis have not been identified, but a cytokinin overproducing mutant, ampl (for altered meristem program), is known from Arabidopsis. This mutant was isolated in a screen for altered morphologies in seedling growth and proved to contain five to six times higher concentrations of cytokinins. The mutant plants show several abnormalities, including production of multiple cotyledons in the embryo, enhanced lateral branching, retarded root development, and delayed
198
II. Structure a n d M e t a b o l i s m of Plant H o r m o n e s
senescence of leaves. Except for polycotyly, similar effects are seen in plants transformed with and overexpressing the bacterial ipt gene. These cytokininoverproducing plants also show enhanced levels of cytokinin metabolites. 7.1. Regulation of Cytokinin Biosynthesis Factors that trigger or modulate the synthesis of cytokinins are still mostly unknown, but with the cloning of the IPT genes in Arabidopsis, rapid progress is expected. However, there is considerable information on cytokinin metabolism. This information comes mainly from feeding labeled zeatin or dihydrozeatin to plant tissues or organs and analysis of labeled products. Because cytokinins are known to be synthesized in roots and translocated to aboveground parts, one common method of feeding exogenous cytokinins is via cut ends of seedlings with roots removed. Other favorite materials for feeding experiments are immature seeds, fruits such as kernels or pods, or cut leaves. 7.2. Conjugation of Cytokinins 7.2.1. Formation of Ribosyl
Derivatives
Tritiated zeatin and dihydrozeatin fed to plant tissues are rapidly converted to their N^-ribosides and ribotides as shown in Fig. 8-6 (for structures of these molecules, see Fig. 8-3). Free cytokinins and their ribosides, and ribotides are interconvertible. In a two-step process, zeatin can be converted to zeatin riboside by adenosine phosphorylase, and zeatin riboside can be converted to the ribotide form by an adenosine kinase. Zeatin can also be directly converted to its ribotide by an adenosine phosphoribosyltransferase (APRT). Enzymes involved in these interconversions are generic enzymes, which catalyze adenine to
zeatin-5-ribotide ^^ f^^'
zeatin
zeatin riboside adenosine phosphorylase
F I G U R E 8-6 Schematic illustration of interrelationships among zeatin, zeatin riboside, and zeatin ribotide. APRT, adenosine phosphoribosyl transferase.
nucleotide conversions. For example, an APRT, isolated and characterized from A. thaliana, can catalyze phosphoribosylation of not only free cytokinin bases, such as zeatin, but also adenine as a substrate. The ribosides and ribotides show biological activity in bioassays, but most likely they are not active per se and it is the free N bases released after hydrolysis that are active. As mentioned in Section 4, zeatin ribotide is also formed during the synthesis of zeatin (see Fig. S-5) and can give rise to zeatin riboside. Thus, ribosides and ribotides are intermediates in the biosynthesis of cytokinins and also represent forms that scavenge excess active cytokinins. The regulation of these pools of cytokinins and their ribosyl derivatives is little understood but likely involves different enzymes in the two sets of reactions and probably different cellular compartments. There also seems to be a homeostatic control over their levels. For instance, after continuous feeding of cytokinins or ribosyl derivatives to plant tissues, excess ribosides and ribotides may be irreversibly converted to N-glucosylated derivatives or be broken down by side chain cleavage (see Section 7.3). 7.2.2. Glycosylation
of the Side Chain
Sugars such as glucose or xylose can be conjugated via the -OH group in the side chain forming what are known as O-glycosides (Fig. 8-7). These reactions are reversible. O-glycosides, like ribosides and ribotides, show activity in bioassays, but it is generally accepted that free bases provide the activity. O-glycosides are very common and seem to be the principal form in which the cytokinins are stored in storage tissues and developing seeds, where they may accumulate to surprisingly high levels. Their synthesis involves specific glycosyl transferases, and genes encoding a zeatin-O-glucosyl transferase (ZOGl) and a zeatin-0-xylosyl transferase (ZOXI) have been cloned from Phaseolus species. With cloning of these genes, more information about the sites of glycosyl conjugation and factors that regulate it is likely to accumulate. Because of their increased polarity, O-glycosylated conjugates can be sequestered into vacuoles for possible use later. The hydrolysis of O-glycosyl conjugates occurs readily, and several p-glycosidases, are known to cleave the O-glycosyl group. In the absence of p-glycosidases, O-glycosides are relatively stable in plant tissues. They are protected against oxidative cleavage of the side chain by the main degradative enzyme cytokinin oxidase (see later) because of the presence of the glycosyl residue in the side chain.
199
8. Cytokinins CH2O
CH2OH
CH2OH
LI> *
. ^ I
N ^ N - ^ ^ N ^
OH
p-D-glucose
zeatin-O-p-D-glucoside
FIGURE 8-7 Glycosylation of the side chain in zeatin.
7.3. Irreversible Inactivation Cytokinins are irreversibly inactivated by certain modifications in the purine ring structure or by cleavage of the N^ side chain. 7,3,1. Conjugation of Glucose or Amino Acid Residues with Adenine Glucose conjugates at the N^ or N^ position in the adenine ring (e.g., [7Glc]Z or [9Glc]diHZ) are formed readily if zeatin or dihydrozeatin is supplied to plant tissues (Fig. 8-8A). N^-glucosides are also formed but appear to be less common. Such position-specific glucosylations are probably catalyzed by specific glucosyltransferases, not the ones involved in 0-glycosylations, but the details are obscure. Amino acid conjugates with alanine at the N^ position are also formed (Fig. 8-8B). They were first isolated as minor metabolites in lupin {Lupinus luteus) and, hence, were named lupinic acid (conjugate with zeatin ([9Ala]Z) and dihydrolupinic acid (conjugate with dihydrozeatin (diH[9Ala]Z). An enzyme, a P-(6-allylaminopurine-9-yl) adenine syn-
thase, has been characterized from developing seeds of lupin. N-glucosylated or amino acid conjugates are stable over long periods and seem to be irreversibly inactivated products. Enzymes capable of hydrolyzing these products have not been investigated. These compounds are much more polar than the original cytokinins, which may facilitate their sequestration in the vacuole. 7,3,2, Oxidative Cleavage of the N^ Side Chain Cytokinin oxidase is the enzyme that catalyzes the cleavage of the N^ side chain from adenine. The enzyme requires the A2 double bond in the side chain for its activity. Hence, its natural substrates are isopentenyladenine and zeatin and their ribosylated derivatives (Fig. 8-9). The enzyme is unable to cleave side chains that lack the double bond, as in dihydrozeatin and its derivatives. It is also inactive against O-glycosylated conjugates, where the side chains have a glucosyl or xylosyl residue (see Fig. 8-7), and against synthetic cytokinins (e.g., benzyladenine, kinetin), where the side chains have an aromatic ring (see later).
CH2 HC — COOH
I
NH2
zeatin-7-p-D-glucoside [7G]Z
p-(zeatin-9-yl)-alanine [9Ala] Z
FIGURE 8-8 N-conjugation of the adenine ring by glucose (A) or alanine (B)
200
II. Structure a n d Metabolisin of Plant H o r m o n e s
CH3
cytokinin oxidase Isopentenyladenine
i;i:> • "^ Adenine
CH3
3-Methyl-2-butenal
F I G U R E 8-9 Cleavage of the side chain of isopentenyladenine by cytokinin oxidase. The products from isopentenyladenine are adenine and 3-methyl-2-butenal.
8.3.2.1. Cytokinin Oxidase Activity Is Enhanced by Its Substrate As mentioned before, the endogenous levels of free cytokinins are strictly regulated. Cytokinin oxidase activity is increased severalfold if plant tissues are incubated with large quantities of cytokinins in vivo. Similarly, an increase in cytokinin oxidase activity is seen in tobacco plants transformed with the ipt gene from Agrobacterium. These data indicate that cytokinin oxidase activity is induced by its own substrate and provide an elegant example of a negative feedback control over the endogenous levels of bioactive hormones. 8.3.2.2. Cytokinin Oxidase Activity Varies in Different Tissues and Is Developmentally Regulated The N-glucosylation of the purine ring and side chain cleavage are two methods for irreversibly lowering the intracellular levels of biologically active cytokinins, but the degree to which they are practiced in different plants, or plant parts, seems to vary. For instance, in radish seedlings, cytokinin oxidase activity is minimal, and more N^- than N^-glucosylation is favored. In corn seedlings and embryos in culture, N^glucosides are more common. Cytokinins are abundant in embryo and endosperm and in fruit pericarp during early phases of seed and fruit development (see Chapters 17 and 18). These levels are gradually reduced and there is a progressive increase in cytokinin oxidase activity in tissues as cell divisions cease and tissues mature. For instance, in developing fruits (pods) of Phaseolus sp., the degradation of zeatin by cytokinin oxidase was more rapid in seed coat tissue (where cell divisions cease earlier) than in the embryo or fruit tissue. In corn kernels, cytokinin oxidase activity increased as maturation progressed. 8.3.3.3. The Gene for Cytokinin Oxidase Has Been Cloned CK oxidase was purified to homogeneity from immature maize kernels, and the sequence used to clone
its gene called CKXl (for cytokinin oxidasel, the authors use the designation ckxl). The enzyme is an oxidoreductase and requires flavin adenine dinucleotide (FAD) and molecular O2 for activity. It can use trans-zeatin, isopentenyladenine, and isopenteneyladenosine, as substrates, but not, as expected, dihydrozeatin. Several genes from Arabidopsis have been cloned also, and detailed studies on the enzyme kinetics are in progress using recombinant protein. The cloning of cytokinin oxidase genes in maize and Arabidopsis and O-glycosylating enzymes in Phaseolus spp are important first steps in our understanding of cytokinin metabolism in plants. More work is likely to follow, and show whether these genes are regulated at the transcriptional or translational level, whether there are different isoforms with tissue a n d / o r substrate specificities, and what specific factors cause their induction.
8. SYNTHETIC C O M P O U N D S WITH CYTOKININ-LIKE ACTIVITY Kinetin was originally obtained from herring sperm DNA, and its discovery led to a search for synthetic compounds that could mimic cytokinin activity. Several compounds were isolated that had high activity in promoting cell division in tobacco pith culture. One of the most potent compounds isolated was 6-benzylaminopurine or benzyladenine (BA) (Fig. 8-10). Like all other cytokinins, BA has an adenine ring with an N^ side chain, but the side chain carries an aromatic ring, as in kinetin. Other synthetic compounds include several hydroxylated benzyl derivatives (Fig. 8-10). Benzyladenine and kinetin are two of the most widely used cytokinins. Benzyladenine has the added advantage that it is easily available and is relatively inexpensive. Both are stable compounds because, unlike zeatin and isopentenyladenine, their
201
8. C y t o k i n i n s
HN-CH2^Q
- C H 2 ^ }
H Benzyladenine (BA) HNrc
- C H 2 ^ Q
Metatoponin Orthotoponin (two hydroxylated derivatives of BA)
H2^j>
Tetrahydropyranobenzyladenine F I G U R E 8-10
Structure of benzyladenine and some other synthetic cytokinins.
side chains are immune to attack by cytokinin oxidase. The glucosylated conjugates of the side chain are also not formed because of the differences in the side chain. However, the enzymes that catalyze the modifications of the purine ring, i.e., formation of ribosylated derivatives and N-substituted glucosides and amino acid conjugates, are able to use BA and kinetin as substrates. Thus, BA administered exogenously is known to give rise to its N^-ribosyl derivatives, as well as N^glucoside and [9Ala] conjugates. Some authors refer to these cytokinins as aromatic cytokinins. Although aromatic cytokinins are still considered as of nonplant origin, reports suggest that they occur naturally in plants. Thus, benzyladenine and its hydroxylated derivatives have been reported in several plant tissues on the basis of immunoassays. These reports need to be confirmed by GC-MS analysis. Kinetin has also been reported to occur naturally and be formed via oxidative degradation of DNA in vivo.
9. CYTOKININ A N T A G O N I S T S (ANTICYTOKININS) Several synthetic compounds are known that show high anticytokinin activity. The most potent and specific of these appear to be substituted pyroUo [2,3d]-pyrimidines and 7-substituted-3-methylpyrazolo [4,3-d]pyrimidines (Fig. 8-12). At low doses (^ 1 fxM), these molecules specifically inhibit cytokinin-induced growth in cytokinin-dependent tobacco tissue cultures. Although their mode of action is not known, they seem to act as competitive inhibitors because
A/, A/^-Dlphenylurea O
8.1 Phenylurea Derivatives Some other substances, such as modified diphenyl and pyridyl-phenyl urea derivatives (Fig. 8-11), display significant cytokinin activity in several bioassays. They stimulate cell division in tobacco pith culture, promote shoot formation in callus tissue, and retard leaf senescence; however even those derivatives that have the highest activity in cell division assay still are only 1/100th as active as the adenine cytokinins, such as benzyladenine. Their mode of action is not known, but they probably function by inhibiting endogenous cytokinin oxidase, thereby having a "sparing" effect on cytokinins present in the tissue by inhibiting their breakdown.
/
y—NH—C—HN—^ Forchlorofenuron
r^^.,_" NH
•HN
NN CI
\L^N
Thidiazuron F I G U R E 8-11 Some phenylurea derivatives with cytokinin activity: N,N'-Diphenylurea; N-(2-chloro-4-pyridyl)-N'-phenylurea (trade name, Forchlorofenuron); and N-phenyl-]V'-(l,2,3-thiadiazol-5yl)urea (Thidiazuron). From Shudo (1994).
202
II. Structure a n d M e t a b o l i s m of Plant H o r m o n e s CH3 CH2 — C H CH3
H3CS
3-Methyl-7-(3-methylbutylamino) pyrazolo [4,3-c/] pyrimidine
4-(Cyclopentylamino)-2-methylthiopyrrolo [2,3-d] pyrimidine
FIGURE 8-12 Two potent anticytokinins: 3-methyl-7-(3-methylbutylamino) pyrazolo [4,3-rf]pyrimidine and 4-(cyclopentylamino)-2-methylthiopyrrolo[2,3-rf]pyrimidine.
inhibition can be reversed by increasing the cytokinin concentration in the medium.
10. CHAPTER SUMMARY Cytokinins serve many important functions in plant development and morphogenesis. They are involved in the regulation of cell division; they interact with auxins in the control of apical dominance and lateral branching and the root-shoot ratio in intact plants and in tissue culture. They retard the senescence of leaves and promote the light-independent deetiolation response, including greening, of dark-grown seedlings. Several cytokinins occur naturally in plants. They have an adenine base and a five carbon isopentenyl side chain. Among these, zeatin, specifically trans-zeatin, is the most abundant. The synthesis of cytokinins in higher plants has been unclear and controversial for a long time, but progress finally seems to be achieved with the cloning of genes encoding isopentenyl transferases (IPTs) in Arabidopsis. These IPTs seem to use ATP/ADP, rather than AMP, together with DMAPP, to yield isopentenyladenine monophosphate (iPMP), which ultimately gives rise to zeatin and other naturally occurring adenine cytokinins. The possibility that cytokinins may arise from the degradation of tRNAs is not considered likely. Ribosylated derivatives, with a ribose or ribose 5'-monophosphate attached to the adenine moiety, of cytokinins are common. Conjugates of cytokinins where a glycosyl moiety is attached to the OH group in the side chain are also common. Both ribosylated derivatives and glycosyl conjugates show activity in bioassays, but it is generally believed that they do so after hydrolysis to free bases. Irreversible deactivation of cytokinins occurs in two ways. The side chain may be cleaved by cytokinin oxidase, an enzyme that has specific requirements for an -OH
group and a A2,3 bond in the side chain. Significantly, this enzyme is induced by high concentrations of cytokinin in plant tissues. Modifications to the adenine ring, by substitution of glucose or amino acids (particularly alanine) to the nitrogen atoms, also cause irreversible deactivation. Genes encoding cytokinin oxidase and glycosylating enzymes have been cloned from several plants and rapid progress in our understanding of regulation of cytokinin metabolism is expected. Several synthetic cytokinins (e.g., benzyladenine, kinetin) are known. They have the adenine base, but have an aromatic ring in the side chain. Supplied to plant tissues, these cytokinins form ribosylated derivatives and glucosyl or amino acid substituents in the adenine ring, but they are more stable (than zeatin) in plant tissues because they are not subject to side chain cleavage by cytokinin oxidase. There are some reports that benzyladenine and kinetin occur naturally. Some synthetic compounds, which inhibit cytokinin promotion of cell division in tissue/ cell culture, probably as competitive inhibitors, are known.
References Books and Reviews Binns, A. N. (1994). Cytokinin accumulation and action: Biochemical, genetic, and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 173-196. Letham, D. S., and Palni, L. M. S. (1983). The biosynthesis and metabolism of cytokinins. Annu. Rev. Plant Physiol. 34, 163197. McGaw, B. A. and Burch, L. R. (1995). Cytokinin biosynthesis and metabolism. In "Plant Hormones, Physiology, Biochemistry and Molecular Biology" (P. J. Davies, ed.), pp. 98-117. Kluwer, Dordrecht. Mok, D. W. S., and Mok, M. C. (eds.) (1994). "Cytokinins: Chemistry, Activity, and Function." CRC Press, Boca Raton, FL. Morris, R. O. (1995). Genes specifying auxin and cytokinin biosynthesis in prokaryotes. In "Plant Hormones, Physiology, Biochem-
8. C y t o k i n i n s istry and Molecular Biology" (P. J. Davies, ed.), pp. 318-339. Kluwer, Dordrecht. Skoog, F., and Miller, C. O. (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol. 11, 118-131.
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Prinsen, E., Kaminek, M. and van Onckelen, H. A. (1997). Cytokinin biosynthesis: A black box? Plant Growth Regul. 23, 3-15. Takei, K., Sakakibara, H., and Sugiyama, T. (2001). Identification of genes encoding adenylate isopentenyltransferase, a cytokinin bisynthesis enzyme, in Arabidopsis thaliana. J. Biol. Chem. 276, 26405-26410.
1. Discovery Haberlandt, G. (1913). Zur physiologie der zellteilungen. Sitzungsber. K. Preuss. Akad. Wiss., 318-345. Letham, D. S. (1963). Zeatin, a factor inducing cell division from Zea mays. Life Sci. 8, 569-573. Miller, C. O., Skoog, F., Okomura, F. S., Von Saltza, M. H., and Strong, F. M. (1956). Isolation, structure and synthesis of kinetin, a substance promoting cell division. /. Am. Chem. Soc. 78, 1345-1350. Skoog, F. (1994). A personal history of cytokinin and plant hormone research. In "Cytokinins: Chemistry, Activity, and Function" (D. W. S. Mok and M. C , Mok, eds.), pp. 1-14. CRC Press, Boca Raton, FL. 3. Structure of Cytokinins Angra, R., Mandahar, C. L., and Gulati, A. (1990). The possible involvement of cytokinins in the pathogenecity of Helminthosporium maydis. Mycopathologica 109, 177-182. Burrows, W. J., Skoog, F., and Leonard, N. J. (1971). Isolation and identification of cytokinins located in the transfer ribonucleic acid of tobacco callus grown in the presence of 6-benzylaminopurine. Biochemistry 10, 2189-2194. Emery, R. J. N., Leport, L., Barton, J. E., Turner, N. C , and Atkins, C. A. (1998). Cfs-isomers of cytokinins predominate in chickpea seeds throughout their development. Plant Physiol. 117, 1515-1523. Hall, R. H. (1970). N^-(D2-isopentenyl) adenosine: Chemical reactions, biosynthesis, metabolism and significance to the structure and function of tRNA. Prog. Nucleic Acid Res. Mol. Biol. 10, 57086. Letham, D. S., Shannon, J. C , and MacDonald, I. R. C. (1964). The structure of zeatin, a (kinetin like) factor inducing cell division. Proc. Chem. Soc. Lond., 230-231 Palni, L. M. S., and Horgan, R. (1983). Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea. Planta 159, 178-181. 6. Biosynthesis in Higher Plants Astot, C , Dolezal, K., Nordstrom, A., Wang, Q., Kunkel, T., Moritz, T., Chua, N.-H., and Sandberg, G. (2000). An alternative cytokinin biosynthesis pathway. Proc. Natl. Acad. Sci. USA 97, 14778-14783. Bassil, N. v., Mok, D. W. S., and Mok, M. C. (1993). Partial purification of zeatin O-xylosyltransferase in Phaseolus. Plant Physiol. 102, 867-872. Blackwell, J. R., and Horgan, R. (1994). Cytokinin biosynthesis by extracts of Zea mays. Phytochemistry 35, 339-342. Chen, C.-M. (1982). Cytokinin biosynthesis in cell-free systems. In "Plant Growth Substances" (P. F. Wareing, ed.), pp. 155-164. Academic Press, London. Chen C.-M. (1997). Cytokinin biosynthesis and interconversion. Physiol. Plant 101, 665-673. Chen, C.-M., and Ertl, J. R. (1994). Cytokinin biosynthetic enzymes in plants and slime mold. In "Cytokinins: Chemistry, Activity, and Function" (D. W. S. Mok and Mok, M. C. eds.), pp. 81-85. CRC Press, Boca Raton, FL. Kakimoto, T. (2001). Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP / ADPisopentenyltransferases. Plant Cell Physiol. 42, 677-685.
7. Regulation of Cytokinin Levels Bilyeu, K. D., Cole, J. L., Loskey, J. G., Riekhof, W. R., Esparza, T. J., Kramer, M. D., and Morris, R. O. (2001). Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol, lis, 378-386. Brzobohaty, B., Moore, I., Kristofferson, P., Bako, L., Campos, N., Schell, J., and Palme, K. (1993). Release of active cytokinin by pglucosidase localized to the maize root meristem. Science 161, 1051-1054. Burch, L. R., and Stutchbury, T. (1987). Activity and distribution of enzymes that interconvert purine bases, ribosides for cytokinin metabolism. Physiol. Plant 69, 283-288. Chaudhury, A. M., Letham, D. S., Craig, S., and Dennis, E. S. (1993). ampl-a mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering. Plant J. 4, 907-916. Dixon, S. C , Martin, R. C , Mok, M. C , Shaw, G., and Mok, D. W. S. (1989). Zeatin glucosylation enzymes in Phaseolus: Isolation of O-glucosyltransferase from P. lunatus and comparison to O-xylosyltransferase from P. vulgaris. Plant Physiol. 90, 13161321. Kaminek, M., and Armstrong, D. J. (1990). Genotypic variation in cytokinin oxidase from Phaseolus callus cultures. Plant Physiol. 93, 1530-1538. Martin, R. C , Mok, M. C , and Mok, D. W. S. (1993). Cytolocalization of zeatin O-xylosyltransferase in Phaseolus. Proc. Natl. Acad. Sci. USA 90, 953-957. Martin, R. C , Mok, M. C. and Mok, D. W. S. (1999a). A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris. Plant Physiol. 120, 353-357. Martin, R. C , Mok, M. C , and Mok, D. W. S. (1999b). Isolation of a cytokinin gene, ZOGl, encoding zeatin O-glucosyltransferase from Phaseolus lunatus. Proc. Natl. Acad. Sci. USA 96, 284-289. McGaw, B. A., Horgan, R., and Heald, J. K. (1985). Cytokinin metabolism and the modification of cytokinin activity in radish. Phytochemistry 24, 9-13. Medford, J. I., Horgan, R., El-Sawi, Z., and Klee, H. J. (1989). Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. Plant Cell 1, 403-413. Moffat, B., Pethe, C , and Laloue, M. (1991) Metabolism of benzyladenine is impaired in a mutant of Arabidopsis thaliana lacking adenine phosphoribosyltransferase activity. Plant Physiol. 95, 900-908. Mok, D. W. S., and Martin, R. C. (1994). Cytokinin metabolic enzymes. In "Cytokinins: Chemistry, Activity, and Function" (D. W. S. Mok and M. C. Mok, eds.), pp. 129-137. CRC Press, Boca Raton, FL. Morris, R. C , Bilyeu, K. D., Laskey, J. G., and Cheikh, N. N. (1999). Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem. Biophys. Res. Commun. 255, 328-333. Motyka, V., Faiss, M., Strnad, M., Kaminek, M., and Schmiilling, T. (1996). Changes in cytokinin content and cytokinin oxidase activity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol. Ill, 10351043.
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8. Synthetic Compounds with Cytokinin-like Activity Barciszewski, J., Siboska, G. E., Pedersen, B. O., Clark, B. F. C , and Rattan, S. I. S. (1997). A mechanism for the in vivo formation of N^-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA. FEES Lett. 393, 197-200. Hare, P. D., and Van Staden, J. (1994). Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell Physiol. 35, 1121-1125. Shudo, K. (1994). Chemistry of phenylurea cytokinins. In "Cytokinins: Chemistry, Activity and Function" (D. W. S. Mok and M. C. Mok, eds.), pp. 35-42. CRC Press, Boca Raton, FL. Strnad, M. (1997). The aromatic cytokinins. Physiol. Plant 101, 674-688.
9. Cytokinin Antagonists (Anticytokinins) Gregorini, G., and Laloue, M. (1980). Biological effects of cytokinin antagonists 7-(pentylamino) and 7-(benzylamino)-3-methylpyrazolo (4,3-rf) pyrimidines on suspension-cultured tobacco cells. Plant Physiol 65, 363-367. Skoog, F., Schmitz, R. Y., Bock, R. M., and Hecht, S. M. (1973). Cytokinin antagonists: Synthesis and physiological effects of 7substituted 3-methylpyrazolo [4,3-^] pyrimidines. Phytochemistry 12, 25-37. Skoog, F., Schmitz, R. Y., Hecht, S. M., and Frye, R. B. (1975). Anticytokinin activity of substituted pyrrolo [2,3-rf]pyrimidines. Proc. Natl. Acad. Sci. USA 72, 3508-3512.