General Features of Plant Hormones, Their Analysis, and Quantitation

C H A P T E R

5 General Features of Plant Hormones, Their Analysis, and Quantitation

several scientists over almost 50 Years established that the bending was due to a natural substance, indole acetic acid (lAA), that was produced in the tip and diffused downward, causing uneven growth on the two sides (Fig. 5-1). Because it was a diffusible substance, it could be collected and, when reapplied to plant systems, could give a measurable response, thus creating the first bioassay for a plant hormone. In the 1950s and 1960s, several other naturally occurring substances, gibberellins (GAs), cytokinins (CKs), abscisic acid (ABA), and ethylene, were shown to influence many plant responses (although some of them had been discovered earlier). Along with auxin, they are among the major plant hormones, sometimes referred to as the "classical five/' Note that we use the expression "auxins,'' "gibberellins," and "cytokinins." This is because there are several naturally occurring or synthetic compounds that show auxin-, or gibberellin-, or cytokinin-like activity. In contrast, ABA and ethylene are single compounds, although some of their metabolites may also show activity. More recently, brassinosteroids and jasmonic acid have also come to be recognized as hormones, and several other substances, salicylic acid, polyamines, oligosaccharins, and a small peptide (systemin), have been shown to act as growth regulators or signaling molecules. During World War II and the years following, many synthetic substances were also discovered and found to affect plant growth and development, thus adding to the list of plant growth regulators. This list continues to increase.

1. 2. 3. 4.

DISCOVERY OF AUXIN AND OTHER HORMONES 141 CHARACTERISTICS OF PLANT HORMONES 143 HORMONE VS PLANT GROWTH REGULATOR 143 HORMONAL RESPONSES ARE SPECIFIC TO A PHYSIOLOGICAL STATE 144 5. BIOASSAYS 145 5.1. Bioassays Serve Many Useful Functions 145 5.2. Limitations of Bioassays 145 6. HORMONE EXTRACTION, ANALYSIS, AND QUANTITATION 145 6.1. Diffusible vs Extractable Amounts 145 6.2. Analytical Methodologies 146 7. DETERMINATION OF HORMONE SYNTHETIC PATHWAYS 150 7.1. Use of Labeled Precursors 150 7.2. Synthesis Mutants 150 7.3. Inhibitors of Hormone Synthesis 151 8. REGULATION OF HORMONE LEVELS (HORMONAL HOMEOSTASIS) 151 9. CHAPTER SUMMARY 152 REFERENCES 152

1. DISCOVERY OF AUXIN AND OTHER HORMONES It is a common observation that aerial parts of plants bend toward a unidirectional source of light. This bending is caused by uneven growth on the two sides of the stem, with the side away from the light source growing faster than the side facing the light source. A series of ingenious experiments by

141

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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 Darwin (1880)

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F I G U R E 5-1 A summary of important experiments that established the existence of auxin in plants. Coleoptiles of grass seedlings were used in all experiments. Darwin (1880) illuminated the coleoptile of canary grass {Phalaris canariensis) from one side and noted that the coleoptile bent toward the light source. If the tip was cut off or capped, there was no response, indicating that light perception occurred in the tip. Since the bend occurred in the subterminal part, he correctly deduced that bending was due to the diffusion of some "influence" from the tip to the subterminal regions. Boysen Jensen (1913) placed thin mica or gelatin strips between the cut tip and the stump and noted that the "influence" was diffusible through gelatin, but not through the mica strip and thus established that the influence was chemical in nature. Paal (1919) showed that the coleoptile tip cut and placed on one side of the stump caused bending of the stump in the absence of unidirectional light. Went (1926) placed cut coleoptile tips on an agar plate and collected the diffusate; small blocks of agar with the diffusate were placed on cut stumps and elicited the

143

5. General Features of Plant Hormones 2. CHARACTERISTICS OF PLANT H O R M O N E S Each of the major classes of hormones brings about a variety of growth and morphogenetic responses, and each is pleiotropic in its effects. For example, auxins are involved in the regulation of cell division, cell growth, apical dominance, responses to directional stimuli (tropic response), and fruit setting, responses that are very different from each other. Not all responses are stimulatory. Auxins promote shoot growth, but at similar concentrations inhibit root growth. Second, several hormones may affect the same response; for instance, cell elongation is affected by auxins, gibberellins, and brassinosteroids; cell division is affected by auxins, cytokinins, and gibberellins. Thus, there is an apparent redundancy in control of the same response. Whether the same signaling pathway is involved and whether even the mechanics of the response are the same in each case is not known, but the phenomenon of redundancy is common. Third, plant hormones are active at small concentrations, usually in the nanomolar range, although some responses begin at even lower concentrations (10 to 100 picomolar, pM). Up to a certain concentration of the hormone, the response increases and then saturates (Fig. 5.2). For some hormones, higher concentrations may even become inhibitory (see Fig 5.1). Plant hormones are often reported to be active over 4 or 5, rather than 2 or 3, decades of concentration. These results from a response in vivo, or bioassays, do not necessarily reflect the concentration of the hormone at the target site because of problems in uptake, transport, and/or metabolism of the hormone in question (see Section 5 below). As more precise measurements of hormone concentration at the target site become possible, this conclusion may change. Fourth, transport of hormones from the site of synthesis to the site of action (target site) occurs in some cases, e.g., basipetal transport of auxin affecting lateral bud dormancy, but in others, the site of production and the site of action may be the same, e.g., ethylene production in fruit ripening. Moreover, plant hormones are synthesized at several different sites in the plant, not in any one specific gland or tissue. Thus, lAA is synthesized in young apical buds, young leaves, immature fruits, developing seeds, and soon.

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F I G U R E 5-2 Avena coleoptile extension growth as a function of auxin concentration. Subapical segments (5 mm in length) from oat coleoptiles were depleted of endogenous auxin by floating in buffer solution (pH 4.7) containing sucrose (20 g • liter"^) for 120 min, then different concentrations of lAA were added, and growth over the next 150 min was measured. Data are plotted as growth vs log lAA concentration. The minimum concentration of lAA that elicits a growth response varies from experiment to experiment, but usually ranges between 10 and 30 nM. The growth rate then increases proportionally to the logarithm of auxin concentration, reaching an optimal rate at about 1.0|JLM. Auxin concentrations in excess of 10 |JLM are superoptimal. Adapted from Cleland (1972).

3. H O R M O N E V S PLANT GROWTH REGULATOR Because animal hormones^ are usually produced in a specific gland or tissue, are transported to the target site, are usually more specific in their biological action, and are active in a more narrow range of concentrations, it has been suggested that the word "hormone'' is inappropriate for plants and should be replaced with a term more suitable. Also, many synthetic ^"Hormone," as applied to animal systems, refers to a naturally occurring-or^anic molecule that occurs in small concentrations, is produced in one site and transported to another site (target region), and brings about a specific biochemical or morphogenetic response.

curvature response. This response could be measured, thus giving rise to the first bioassay. The diffusate was called ''auxin" and was shown chemically to be indole-3-acetic acid (lAA). (Bottom) The oat {Avena sativa) coleoptile curvature response plotted against the number of coleoptile tips stood on agar (left) and against the concentration of lAA (right; note that supraoptimal concentrations of lAA can cause inhibition). Curiously, the first chemical determination of indole-3-acetic acid was made from human urine (Haagen-Smit, 1934), and only in 1941 was it proven unequivocally that lAA was also present in higher plants. Adapted from Taiz and Zeiger (1998).

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11. Structure and M e t a b o l i s m of Plant Hormones

compounds are equally, at times even more, active than the naturally occurring compound. For both these reasons, it has been suggested that it is better to call these substances plant growth regulators (PGRs) and include in that term both naturally occurring and synthetic compounds with growth-promoting activity. However, that is putting too restrictive a definition on the term hormone. Plants have evolved differently from animals in several important ways, e.g., rooted habit, open form of growth, open differentiation, presence of cell walls, and autotrophic habit. It can be argued that having multiple sites of production or having the site of production and target site the same has selective advantage for plants that are subject to predation, even wanton damage, by a host of animals, including humans. Also, it is not necessary to assume that plant hormones must satisfy all criteria for hormones sensu strictu the animal world. The term hormone needs to be interpreted more loosely. Furthermore, the term plant growth regulator or substance also has exceptions; many responses brought on by PGRs, e.g., stomatal closure, are not growth related. Accordingly, in this book, the term hormone is used in a broad sense to include those plant substances that occur naturally, in small quantities, and bring about a variety of specific responses. In contrast, the term PGR, is used to include hormones, as well as the various synthetic compounds with ''hormonal'' properties.

4. H O R M O N A L RESPONSES ARE SPECIFIC TO A PHYSIOLOGICAL STATE It should be emphasized that plant hormone responses are tissue and time specific. Each response occurs at a certain time in plant development and is seen only in a specific tissue or organ. An example is gibberellin-induced elongation of lettuce {Lactuca sativa) hypocotyls. If lettuce seedlings are germinated and then transferred to light, the hypocotyls grow to about 3-4 mm in 72 h. However, if gibberellic acid (GA3) is supplied to these light-grown lettuce seedlings, the hypocotyls elongate 400- to 500-fold over the control (Fig. 5-3). The response is shown by hypocotyls and petioles of cotyledons, not by roots. Moreover, if GA3 is given in 4-h pulses, it becomes clear that hypocotyls are most sensitive to GA3 application between 8 and 12 h after the start of experiment. Later applications of GA3, after 24 h, have very little effect.

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The term "sensitivity'' is used to denote the responsiveness of a tissue to a hormone. For a specific response, the sensitivity is maximal at a certain stage in development; it is less before and declines after that stage. The saturating concentration of a hormone for a particular response bears an inverse relationship to sensitivity of the tissue. If the sensitivity is declining or not at maximal, higher concentrations of the hormone may be needed to bring about the same magnitude of response. Much of our information on hormone-mediated responses comes from bioassays or exogenous applica-

5. General Features of Plant Hormones tions of hormones. The endogenous content of the hormone(s) in the response site is often not known, and very few studies on hormonal responses in vivo have tried to make this correlation. However, this situation is changing as better and more sensitive techniques of hormone detection and quantification have become available and are being used to reexamine some well-known physiological responses. Intriguing questions in this context are how does a response come to an end and what feedback mechanisms are operative? Beginnings are being made in elucidation of this phenomenon as well (see Chapter 19, Section II).

5. BIOASSAYS A bioassay is the use of a plant, or plant organ or tissue, to measure its response to a specific plant growth regulator. Because there are many naturally occurring hormones and literally hundreds of their synthetic analogues and because these PGRs affect a wide spectrum of phenomena in plant growth and development, the number of bioassays is very large (see Yopp et al, 1986). 5.1. Bioassays Serve Many Useful Functions Bioassays are useful in many ways: (i) They are one of the major methods to determine the endogenous levels of a hormone in a plant organ or tissue with sensitivities of detection ranging from 1 to 1000 ng • g fw"^. (ii) They are essential for monitoring the biological activity of a fraction during a hormone (or PGR) purification protocol, (iii) They are invaluable for testing new compounds—the range of responses elicited by the compound and its potency—in relation to natural hormones, (iv) They are useful for determining the relationship between the molecular structure of a compound and its biological activity, i.e., structure-activity relationships, and in devising new compounds. The usefulness of a bioassay depends on its specificity and sensitivity, i.e., the response is given by only one class of PGRs and not others, and that a measurable response is obtained at relatively low concentrations of the PGR (e.g., < lOng • g fw~^). It also depends on the precision with which the response can be measured and the ease with which the bioassay can be performed. For instance, the precision of measuring a flowering response is much less than that of measuring the elongation response, which in turn is much less than measuring the induction of an enzyme. The ease with which a bioassay can be performed includes such

145

factors as availability of a reasonably uniform plant source, ease of setting up the experiment in a laboratory, and costs of chemicals and apparatus. 5.2. Limitations of Bioassays Bioassays suffer from three major limitations, (i) The actual concentration of the PGR at the site of action may be vastly different from that supplied because of conditions affecting the uptake of the PGR a n d / o r its transport to the site of action, (ii) The PGR may be raetabolized by living tissue, thus affecting its concentration; also, an inactive form of the PGR may be metabolized to an active form by the living tissues, or vice versa, (iii) Bioassay responses are subject to statistical error and usually are not accurate within 1 log of response. For these reasons, for qualitative and quantitative determination of specific hormones and PGRs, bioassays have been mostly replaced by newer, more precise analytical techniques (see Section 6). Bioassays remain indispensable, however, because they provide biological meaning to testing of new compounds and for determination of structure-activity relationships.

6. H O R M O N E EXTRACTION, ANALYSIS, A N D QUANTITATION 6.1. Diffusible vs Extractable Amounts A distinction is sometimes made between diffusible and extractable amounts of a hormone. The diffusible amounts are those that diffuse out from a cut surface and that can be collected in an agar block or some other receiver. They usually represent the fraction that is freely mobile. The first Avena coleoptile bioassay was performed using a diffusible hormone (see Fig. 5-1). In contrast, the extractable amounts represent those extracted by one or more solvents from a tissue homogenate. They include both the mobile fraction and one that may be bound to some other moiety t)r compartmentalized and, hence, immobile. The concept of diffusible hormone is not very useful because its relationship to endogenous content remains unknown. Henceforth, in this book, the term diffusible hormone is not used. The terms "ixee" and "bound" hormones are used, however. Bound hormone means that the hormone is chemically linked, or conjugated, to another moiety, often a sugar residue or an amino acid or a peptide. Conjugates may be hydrolyzed to release the free hormone (for lAA, see Chapter 6, Section 5.1.1).

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11. Structure a n d M e t a b o l i s m of Plant H o r m o n e s

6.2. Analytical Methodologies 6.2.1, Physicochemical

Methods

Analytical methodologies for fractionation of a tissue homogenate and the subsequent identification and quantitation of hormone levels have undergone pro-

BOX 5-1

found changes since the 1980s. These methodologies utilize the high resolving power of high-pressure liquid chromatography (HPLC) and gas chromatography (GC), and identification of the molecular species by mass spectroscopy (MS).

HORMONE ANALYSIS: TOOLS OF THE TRADE

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IGH-PRESSURE LIQUID CHROMATOGRAPHY, LIKE Other chromatographic methods, relies on the separation of different molecular species on the basis of their relative affinity to a stationary matrix, coated inside a column, and the carrier solvent that is used to elute them (Fig. 5-4). The higher the affinity of a molecule for the solid matrix, the greater the elution time (known as retention time). Eluates are monitored by a detector as they emerge out of the column and appear as peaks on the chromatogram. The height and size of the peak are measures of the relative amount of the eluate. Gas chromatography also uses a column, a very long, narrow-bore, capillary column, which is coated on the inside by the matrix material, but in this case, the sample is volatalized when injected into the column. The volatiles so produced are eluted by a carrier gas, usually an inert gas such as helium. Their retention time in the column depends on both their affinity for the matrix material (the greater the affinity, the longer the retention time) and their volatility. Their emergence is monitored by a detector and plotted as peaks on a chromatogram. Most compounds are not easily volatalized. Hence, samples for GC are usually derivatized with methyl groups before injection into GC. GC has a much higher resolving capacity than HPLC, but because the samples are usually destroyed and cannot be recovered, it is used mostly for identification

20 Time (min) Column

FIGURE 5-4 (A) Flow diagram of high-pressure liquid chromatography. The sample is injected into the column, coated on the inside by a stationary matrix (see cut through), and eluted by the solvent at a defined flow rate. Different molecular species in the sample pass through and exit the column in reverse order of their affinity for the matrix; the lesser the affinity, the shorter the retention time in the column. On exiting, they pass through a detector connected to a recording device, which plots the retention time as well as the quantities passing through. The samples are collected in a fraction collector for further analysis. (B) Separation of [^HjGAs by reverse-phase HPLC. The mixture of standards was injected onto an ODS Hypersil column (25 x 0.46 cm) and eluted at 1 m l / m i n over 40 min in a gradient of 28-100% methanol in water containing 50 jxl/liter acetic acid. Radioactivity was determined by counting an aliquot of each 1-min fraction in a liquid scintillation counter. (Courtesy of Peter Hedden).

147

5. G e n e r a l Features of P l a n t H o r m o n e s

purposes (special columns have been designed for use in purification). Many different kinds of columns both HPLC and GC. Likewise, many kinds of detectors, suited for specific classes of compounds, are available on the market. In a mass spectrometer, the sample is ionized and molecular ions (M^) are separated according to their mass and displayed as mass peaks. Under certain modes of operation, the sample is bombarded with high-energy particles, such that the molecules are fragmented. These fragments are also charged ions, they can be collected on the basis of their mass and displayed as peaks. Since each molecule fragments in a specific way, it produces a characteristic array of ions called a mass spectrum, very much like a fingerprint (Fig. 5-5). Molecular ions, as well as fragment ions, provide an unambiguous identification of compounds. Because unknown samples may have hundreds of different compounds, samples for MS are usually fractionated and purified by previous HPLC, or GC connected on-line to MS, such that only a small number of molecular species are present in any sample. Still, a full scan of the different ionic species is usually essential for knowing the range of compounds present in the unknown sample. Greater quantitative information can be obtained by a modification in which only a selected group of ions is monitored (selected ion monitoring or SIM). The combination of these techniques, fractionation of tissue samples by HPLC and identification by GC and GC-MS, provides as complete a picture as possible on the molecular species and metabolites of a hormone, and their respective amounts, in a sample.

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148

11. Structure and Metabolism of Plant Hormones

Usually the tissues are frozen in liquid nitrogen, ground to a fine powder, homogenized, and extracted with one or more solvents to recover as much of the hormone a n d / o r its metabolites as possible. The extract is partitioned against solvents (liquid-liquid or solidliquid partitioning) to remove undesirable compounds, such as pigments, lipids, and phenolics, and the hormone extract is reduced to or dried and taken up in a small, defined volume. This crude extract is fractionated using HPLC. Fractions showing biological activity (by bioassays) may be further fractionated before being taken for identification on a GC or GC-MS. For ease of operation, precision and high resolution, and unambiguous identification of individual compounds, these methods far surpass the earlier thin-layer or open column chromatographic methods for fractionation and bioassays for identification and quantitation. The sensitivities and speed of these techniques are such that picogram quantities of hormones can be detected in a few milligram of plant tissues (Table 5-1), and the whole operation can be concluded within a day. To appreciate the elegance of these methods, it is fitting to remember that the isolation and identification of the first natural cytokinin, zeatin, required 60 kg of maize kernels and many liters of solvents (Letham et al., 1964). 6.2.1.1. Internal Standards The efficacy of these methods relies on the availability of chemically pure, but labeled, standards, which can be added to the plant tissue during extraction. These labeled standards have to be such that they are chemically identical, or nearly so, to the hormone being extracted so that it can be assumed that the labeled compound will behave similarly to the unlabeled endogenous compound throughout the isolation and analytical procedures. It is also very important that the labeled standards be prepared in such a way that they are stable under the extraction and analytical procedures. Two types of standards are used. Radioactively labeled standards, prepared by adding to or substituting ^H or ^^C in a precursor, are useful in the extraction of hormones from plant tissues and their purification. Since radioactivity TABLE 5-1

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can be detected in very small quantities, only a few milliliters of each fraction, from an HPLC column, need to be counted in a scintillation counter; 300-400 counts per minute (cpm) over a background count of 30-40 cpm provide adequate differentiation to determine which fractions should be pursued further. The ratio of radioactivity added before extraction to that recovered in a fraction reveals the recovery of the hormone and, thus, the efficiency of the extraction protocol. ^H-labeled standards have a higher specific radioactivity than ^^C-labeled products and are preferred for most purification protocols. Radioactively labeled compounds contaminate instruments and glassware and require a great deal of caution in their use. Accordingly, for most analytical work, pure standards labeled with heavy, but nonradioactive isotopes, such as ^H, ^^N, and ^^C, are preferred and have proven invaluable for quantitative determinations. Their ions have a heavier mass than those of the natural hormone and can be separated in the MS. Usually, several heavy atoms are substituted in a molecule to improve the efficiency of mass separation. Stable, heavy isotope-labeled standards have been developed for most classes of plant hormones (e.g., [^^CeJIAA, [^HsJABA, pH2]GAs), but their preparation requires great technical skill and hence they are still available only in selected laboratories. Ethylene does not require an internal standard because it can be identified and quantified using GC alone equipped with a flame ionization detector. For quantitative work, internal standards are added in known amounts to the sample. Because the amount of labeled compound is known, determining the ratio of heavy isotope-labeled to unlabeled hormone reveals the amount of endogenous unlabeled compound, usually by use of a calibration curve (Croker et ah, 1994). These measurements are usually made in the SIM mode, with the MS set to monitor one or two ions of the endogenous compound and the equivalent massshifted ions of the internal standard. 6,22. Immunochemical

Methods

Since the mid-1980s, immunological methods have also been established for the determination of several plant hormones. For immunological assays, an antibody must be prepared first. Because plant hormones are small molecules, unable to elicit an antigenic response in an animal (rabbit, mice, sheep), they are first conjugated to a protein [e.g., bovine serum albumin (BSA), ovalbumin, haemocyanin], injected into the animal, and antisera collected. As to which part of a hormone molecule is used for such attachment is an important consideration. For production of antibodies

149

5. General Features of Plant Hormones

that recognize a specific part of the hormone molecule, it is important not to use that part of the hormone for attachment. Antibodies come from different cells in the spleen; hence, they are polyclonal antibodies (PAbs) and show varying degrees of specificity toward different parts of the hormone-protein conjugate. These parts recognized by specific antibodies are referred to as "epitopes.'' PAbs may be partly purified to provide greater specificity toward one or another epitope. PAbs, however, are unsuitable for large-scale quantitative work because that type of work requires an assured supply of well-characterized antibodies. To get this supply, it is essential to make monoclonal antibodies (MAbs). MAbs are produced by macerating the spleen and fusing the cells with tumor or myeloma cells. Spleen cells, by themselves, do not grow in the culture medium, but myeloma cells do. Thus, hybridization of spleen cells with myeloma cells produces a hybrid cell or a "hybridoma" that can grow in culture and also produce the antibody specific to the spleen cell. MAbs are purified by a combination of dilution and growth in culture, repeated several times, and their specificity to a specific epitope determined. They can be kept indefinitely in culture or frozen and stored for subsequent use. 6.2.2.1. Radioimmunoassay (RIA) Radioimmunoassay requires a radioactively labeled hormone. A defined concentration of the radioactive hormone is incubated with a known quantity of antibody to which it binds, and then the free or unbound hormone is separated from that bound to the antibody by a suitable method (e.g., by dialysis or precipitation of the antibody by ammonium sulfate or by an antiantibody followed by filtration or centrifugation). The radioactivity bound to the antibody is counted in a scintillation counter (Fig. 5-6A). This is the control (cpm 1). Another mixture includes the same quantity of antibody and the same concentration of the radioactive hormone, plus the plant extract with the unknown amount of hormone. The radioactivity bound to the antibody in this mixture is also counted (cpm 2). The more unlabeled hormone in the plant extract, the less radioactivity is bound to the antibodies. Thus, the difference in radioactivity between the control vs the sample is proportional to the amount of the unlabeled hormone in the sample. The advantages of RIA are its relative simplicity and the high sensitivity provided by the use of radioactive compounds. Fiowever, there are several disadvantages as well: high specific activity-radiolabeled hormones and a scintillation counter are required, and they may not be easily available. Also, care is required in handling radioactive compounds.

6.2.2.2. Enzymeimmunoassays (EIA) In enzymeimmunoassays, an enzyme replaces the radiolabel and the bound enzyme activity is measured, usually by a color reaction produced when a suitable substrate is presented (Fig. 5-6B). Monoclonal antibodies rather than polyclonal antisera are preferred because they are better characterized and can be produced in large quantities. The enzyme reactions are usually catalyzed by alkaline phosphatase or peroxidase. EIA requires that the enzyme be linked to the MAbs, which have been prepared against the hormone-antigen. The linkage can be accomplished in either of two ways: the enzyme can be covalently linked to the MAb (direct EIA) or it can be linked to a second antibody that specifically recognizes the MAb (indirect EIA). In direct EIA, the enzyme-coated MAb is incubated with a known quantity of hormone, p l u s / minus the plant extract. Subsequently, the substrate is presented, and the intensity of color is measured. The difference in color between the two assays provides a measure of the hormone content in the sample. In the indirect method, incubation mixtures consist of the same ingredients, plus the second antibody carrying the enzyme. An EIA in which the antibody against the hormoneantigen is immobilized by adsorption onto a solid substrate is called an enzyme-linked immunosorbent

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11. Structure and Metabolism of Plant Hormones

assay (ELISA). ELISAs have become the preferred method for quantitation of a hormone in plant material. Commercial assay kits are available for some hormones, such as ABA, lAA, and several cytokinins, which have microtiter plates with many small wells that are precoated with MAbs prepared against the hormone-antigen. Both RIA and ELISAs are relatively easy to use and do not require costly equipment. Yet, the sensitivity of detection can be as low as 50 fmol, or about 10 pg of a hormone, such as ABA. In practice, however, these detection limits are often not reached because of impurities in the sample. Consequently, samples to be tested are purified as far as possible, preferably using HPLC, but purification leads to losses and therefore inaccuracies. For these reasons, results from immunological methods are usually verified by or calibrated against data obtained by GC and GC-MS. 6.2.2.3. Purification Protocols Using Immunological Methods MAbs prepared against a specific antigen provide a very powerful tool for fast, reliable, and large-scale purification of the antigen or a close analog from an impure mixture. For such purification, MAbs are prepared as described earlier against the antigen of interest (if the antigen is a protein, it does not need to be conjugated to another protein, such as BSA). These MAbs are then covalently bound to a suitable solid matrix, usually a Sephadex bead. These beads come in standard sizes, and some are designed specially to provide linkage groups for binding to specific groups on MAbs. These special types of beads are referred to as affinity gels or simply Affi-Gels. It is important to ensure that the MAbs are bound to the Affi-Gel in a stable manner, a procedure that usually involves checking the stability of binding by the use of a radiolabeled antigen. The Affi-Gel bound to the MAbs can be packed in a column and then the impure mixture (or extract) containing the antigen is passed through the column. The antigen in the mixture adheres to the MAbs, while the rest of the mixture goes through. After a few washes to remove nonspecifically adsorbed proteins, the bound antigen can be eluted from the column by a suitable ionic buffer. In an alternate procedure, known as a batch method, the impure mixture may simply be mixed with a certain amount of MAbs bound to Affi-Gel, washed, and centrifuged and then the antigen is eluted from the MAbs as described earlier. Irrespective of whether a column or batch method is used, immunoaffinity purification provides a fast and easy method to purify large amounts of an antigen in one single step.

7. DETERMINATION OF H O R M O N E SYNTHETIC PATHWAYS Three approaches are used to establish synthetic pathways for hormones. 7.1. U s e of Labeled Precursors The most common method is to feed labeled precursors to the plant or plant tissues in vivo or to a plant extract in vitro and follow the label in the products after varying periods of incubation. If the label is seen moving progressively from the precursor to A to B to C and so on, and finally the active hormone, one has a fairly good idea of the synthetic pathway. For example, precursor ^ A ^ B - ^ C - ^ D ^ active hormone Initial studies are usually carried out using intact plants or plant parts in vivo and, later, extended to crude extracts from the same parts. Crude extracts, which are clear of cellular debris, i.e., cell-free systems, can contain all the enzymes and cosubstrates/ cofactors necessary for biosynthesis in vitro. In vitro synthesis offers two advantages over in vivo work: (i) Before the addition of labeled precursors, the extract is usually dialyzed, which removes the endogenous hormone and thus prevents isotopic dilution. This is important because, as explained earlier, isotopically labeled hormones or analogs are added during an extraction protocol to facilitate the fractionation and identification of compounds of interest, (ii) It allows for eventual purification and characterization of the metabolizing enzymes, which can lead to cloning of their genes. Use of an in vitro synthesis system, coupled with the enormous analytical capabilities of fractionation by HPLC and the identification of compounds by GC and GC-MS, has proven invaluable for the elucidation of biosynthetic pathways for several hormones. These studies can be combined with the use of hormone synthesis mutants and synthesis inhibitors (see below), which can point out with great accuracy the precise steps in biosynthesis.

7.2. Synthesis Mutants Each of the steps in biosynthesis is catalyzed by an enzyme. So another method relies on the use of mutants, natural or artificially created, that have a lesion in one of the enzymes in the biosynthetic pathway, and hence are deficient in the hormone. Of course, in order to recognize these mutants, they have

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5. General Features of Plant Hormones to show a phenotype that suggests a deficiency of the hormone. These are called synthesis mutants, and they have become a very powerful tool in determining the biosynthetic pathways of hormones. For example, a mutant that has a defective enzyme for the step C to D in the scheme given earlier would show a lack of, or deficiency in, the endogenous content of the hormone; it would also show an accumulation of products C and maybe others, before the blocked step as shown: precursor -^ A —»B

[C],

/ / -^ [Dldecr

—y [hormone] deer If a series of such mutants blocked in different steps is available, it is a wonderful aid in building up the biosynthetic pathway for the hormone in question. As explained in Appendix 1, the identification of mutant alleles allows cloning of the wild-type gene. Such cloning bypasses the requirement for purification of the enzyme protein. 7.3. Inhibitors of Hormone Synthesis Still another method is to use inhibitors of biosynthesis, chemicals that block one or another step in the biosynthetic pathway. The result is the same as in the case of synthesis mutants, but because inhibitors often have side effects, which may be undesirable or may complicate results, the use of synthesis mutants, if available, is preferred. Note: In addition to synthesis mutants, there are response mutants, which do not have a deficiency of the hormone, but where hornione signaling is affected such that the expected response to the hormone is not obtained. Similarly, there are chemicals that inhibit the action of a hormone, not its synthesis. Response mutants and inhibitors of hormone action are valuable tools in deciphering the mode of action of a hormone. They are referred to in many parts of the book, but mostly in Section IV.

than is actually required. Evidence comes from synthesis mutants that are leaky, i.e., the mutated allele is not a null allele, but is still able to produce a partly functional enzyme. Such leaky mutants often produce enough hormone to carry out many responses, although perhaps not all. Thus, the regulation of endogenous levels of bioactive hormones, or hormone homeostasis, is of prime importance to normal growth and development of plants. Plants use three mechanisms to regulate endogenous levels of hormone: (i) regulation of the rate of hormone synthesis, (ii) inactivation of the hormone by conjugation with carbohydrates, amino acids, or peptides, and (iii) an irreversible breakdown of the hormone. Other means of regulating the levels of free hormone include transport to other parts of the plant a n d / o r inactivation and storage in some compartment (Fig. 5-7). Inactivation or breakdown of hormones and compartmentation in an inactive form are strategies that are regularly utilized. Similar inactivation or breakdown is also seen if plant tissues are presented with exogenous hormone in unnaturally large quantities or if the plant produces an excessive amount of the hormone as a result of a mutation or genetic transformation. Before leaving this section, it is important to emphasize that mutants deficient in a particular hormone, or mutants or plants that have been transformed to overproduce a hormone, are invaluable tools in deciphering the physiological and/or biochemical roles of that hormone in plant growth and development. They point out with great specificity the particular roles a hormone plays and far surpass in accuracy the conclusions drawn from supplying the hormone to a whole plant or plant tissues and noting the effect(s).

Hormone homeostasis

8. REGULATION OF H O R M O N E LEVELS (HORMONAL HOMEOSTASIS)

Synthesis

[Hormone]

Hormones are required for specific actions at specific times in growth and development, and it is important for the plant, not only to be able to synthesize the hormone, but also to inactivate it when not needed. Furthermore, hormones are required in small amounts, picomolar to micromolar quantities, and plants often produce far more bioactive hormone

Transport compartmentation

Hormone breakdown

Hormone conjugates

F I G U R E 5-7 Summary diagram showing regulation of endogenous levels of a hormone.

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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

9. CHAPTER SUMMARY The discovery of indoleacetic acid as a natural hormone in plants in the 1930s was followed by the identification of several other natural hormones, gibberellins, cytokinins, abscisic acid, and ethylene in the 1950s and of brassinosteroids and jasmonates in the 1980s. In addition, many other natural and synthetic compounds are known that have roles in growth regulation or signaling. Plant hormones show several features that set them somewhat apart from hormones in animal systems. Each hormone generally brings about several responses that are distinct from each other, i.e., each hormone is pleiotropic in its effects. Moreover, several hormones may bring about the same response; thus, there is an apparent redundancy in their functions. Although plant hormones are active in small concentrations, they are usually active over several decades of concentration, which allows a certain flexibility in their use. Finally, plant hormones are not synthesized in a specific gland or tissue, but at many locations in the plant. These features of plant hormones are of importance to the survival of plants with their rooted habit. Hormones are required for specific actions in a highly time- and tissue-specific manner. The sensitivity of a tissue to a hormone is maximal at such times; it is less before, and declines after the stage is past. A bioassay measures a biological response to a hormone. Bioassays serve many useful purposes, but for quantitation of a hormone in biological samples, they have largely been replaced by physicochemical and immunochemical methods. The newer analytical methods, especially fractionation of samples by highpressure liquid chromatography and identification and quantitation by gas chromatography, combined with mass spectrometry, allow unambiguous identification of hormones in plant tissues and their precise quantitation. Improvements in GC-MS techniques allow determination of hormone amounts in a single grain of cereal, or a single coleoptile tip. Immunochemical methods allow processing of large numbers of samples in a short time, as well as rapid methods of hormone purification. Synthetic pathways for hormones are determined by three methods; incubating plant tissues with labeled precursors and following the incorporated label in various products, use of mutants that are defective in one or another enzyme in the synthetic pathway, and use of inhibitors of biosynthesis. These methods, in combination with analysis and quantitation of products by GC-MS, have proven invaluable in defining the synthetic pathways for many hormones. Hormonal homeostasis is crucial to the orderly growth and development of plants. It is

maintained by a combination of several mechanisms, control over hormone synthesis, inactivation of bioactive hormone by conjugation, and hormonal breakdown.

References Section 1-4 Cleland, R. E. (1972). The dosage response curve for auxin-induced cell elongation: A reevaluation. Planta 104,1-9. Davies, P. J. (1995). The plant hormone concept: Concentration, sensitivity and transport. In "Plant Hormones, Physiology, Biochemistry and Molecular Biology" (P. J. Davies, ed.), pp. 13-38. Kluwer, Dordrecht. Sawhney, V. K., and Sivastava, L. M. (1974). Gibberellic acid induced elongation of lettuce hypocotyl and its inhibition by colchicine. Can. }. Bot. 52, 259-264. Taiz, L., and Zeiger, E. (1998). "Plant Physiology," 2nd ed. Sinauer Associates, Inc., Publishers, Sunderland, MA. Trewavas, A. J. (1982). Growth substance sensitivity: The limiting factor in plant development. Physiol Plant 55, 60-72. Wareing, P. F., and Phillips, I. D. J. (1981). "Growth and Differentiation in Plants," 3rd Ed. Pergamon Press, Oxford. 5. Bioassays Yopp, J. H., Aung, L. H., and Steffens, G. L. (eds.) (1986). "Bioassays and Other Special Techniques for Plant Hormones and Plant Growth Regulators." Plant Growth Regulator Society of America. 6.1. Analytical Methodologies Albrecht, T., Kehlen, A., Stahl, K., Knofel, H.-D., Sembdner, G., and Weiler, E. W. (1993). Quantification of rapid, transient increases in jasmonic acid in wounded plants using a monoclonal antibody. Planta 191, 86-94. Caruso, J. L., Pence, V. C , and Leverone, L. A. (1995). Immunoassay methods of plant hormone analysis. In "Plant Hormones, Physiology, Biochemistry and Molecular Biology" (P. J. Davies, ed.), pp. A33-447. Kluwer, Dordrecht. Croker, S. J., Gaskin, P., Hedden, P., MacMillan, J., and MacNeil, K. A. G. (1994). Quantitative analysis of gibberellins by isotope dilution mass spectrometry: A comparison of the use of calibration curves, an isotope dilution fit program and arithmetical correction of isotope ratios. Phytochem. Anal. 5, 74-80. Edlund, A., Eklof, S., Sundberg, B., Moritz, T., and Sandberg, G. (1995). A microscale technique for gas chromatography-mass spectrometry measurements of picogram amounts of indole-3acetic acid in plant tissues. Plant Physiol. 108, 1043-1047. Hedden, P. (1993). Modern methods for the quantitative analysis of plant hormones. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 107-129. Horgan, R. (1995). Instrumental methods of plant hormone analysis. In "Plant Hormones, Physiology, Biochemistry and Molecular Biology" (P. J. Davies, ed.), pp. 415-432. Kluwer, Dordrecht. Knox, J. P., Beale, M. H., Butcher, G. W., and MacMillan, J. (1987). Preparation and characterization of monoclonal antibodies which recognize different gibberellin epitopes. Planta 170, 86-91. Letham, D. S., Shannon, J. C , and MacDonald, I. R. C. (1964). The structure of zeatin, a factor inducing cell division. Proc. Chem. Soc. Lond. 230-231. Moritz, T., and Olsen, J. E. (1995). Comparison between high resolution selected ion monitoring, selected reaction monitoring, and four sector tandem mass spectrometry in quantitative analysis of

5. G e n e r a l F e a t u r e s of P l a n t H o r m o n e s gibberellins in milligram amounts of plant tissue. Anal. Chem. 67, 1711-1716. Morris, R. O., Jameson, P. E., Laloue, M., and Morris, J. W. (1991). Rapid identification of cytokinins by an immunological method. Plant Physiol. 95, 1156-1161. Nakajima, M., Yamaguchi, L, Nagatani, A., Kizawa, S., Murofushi, N., Furuya, M., and Takahashi, N. (1991). Monoclonal antibodies specific for non-derivatized gibberellins. I. Preparation of monoclonal antibodies against GA4 and their use in immunoaffinity column chromatography. Plant Cell. Physiol. 32, 515-521. Ribinicki, D. M., Cooke, T. J., and Cohen, J. D. (1998). A microtechnique for the analysis of free and conjugated indole-3-acetic acid

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in milligram amounts of plant tissue using a benchtop gas chromatograph-mass spectrometer. Planta 204,1-7. Walker-Simmons, M. (1987). ABA levels and sensitivity in developing wheat embryos of sprouting resistant and susceptible cultivars. Plant Physiol. 84, 61-66. Weiler, E. W. (1986). Plant hormone immunoassays based on monoclonal and polyclonal antibodies. In 'Immunology in Plant Science: Modern Methods of Plant Analysis'' (H. F. Linskens and J. F. Jackson eds.). Vol. 4, pp. 1-17. Springer-Verlag, Berlin. Zhang, S. Q., Hite, D. R. C , and Outlaw, W. H., Jr. (1991). Modification required for abscisic acid microassay (enzyme-amplified ELISA). Physiol. Plant, 83, 304-306.