Modern Methods for the Quantitative Analysis of Plant Hormones

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MODERN METHODS FOR THE

QUANTITATIVE ANALYSIS OF

PLANT HORMONES

Peter Hedden

Department of Agricultural Science, University of Bristol, AFRC-Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, UK

KEYWORDS: phytohormone, combined gas chromatography-mass spectrometry, immunoassay

CONTENTS

INTRODUCTION ... 107

PURIFICATION PROCEDURES ... 108

Solid-Phase Extraction ... 108

High-Performance Liquid Chromatography ... 109

Immunoaffinity Chromatography ... 110 PHYSICOCHEMICAL ASSAYS ... 111 HPLC-BasedMethods ... 111 GC-Based Methods ... 113 Quantification of Ethylene... 118 IMMUNOASSAYS ... 119 Radioimmunoassays ... 120

Enzyme-Linked Immunosorbent Assays ... ... 121

CONCLUSIONS ... 122

INTRODUCTION

Since the last article in this series on plant hormone analysis (12), the most significant development has been the steady increase in the use of im munoassays. Physicochemical methods continue to be important, particularly combined gas chromatography-mass spectrometry (GC-MS), for which the sensitivity and ease of use have improved substantially in recent years, with a

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ANNUAL REVIEWS

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108 HEDDEN

simultaneous decrease in cost of the "bench-top" models. Except for ethylene, plant hormone analysis is now dominated by GC-MS, used in the selected ion monitoring (SIM) mode, and immunoassays.

There has been a great deal of discussion on the reliability of analytical

techniques with regard to plant hormones (12, 74, 75, 102, 103, 117, 118),

resulting in an acute awareness of the limitations of analytical methods and of the criteria necessary to establish the accuracy of plant hormone assays. This discussion will not be continued here, except to point out perceived limitations in the reliability of particular methods. One of the most serious potential sources of inaccuracy in hormone analysis is their extraction from plant tis sues, the efficiency of which is difficult to determine. Although extractable hormones may be released from tissues relatively quickly (128), it is not possible to determine how much of the hormone pool has been recovered. As pointed out by Sandberg et al (112), a solution to this conundrum may have to await the development of in situ assays.

In view of the large number of papers incorporating plant hormone analy ses published in the last ten years, a comprehensive review of the literature is impractical in the space available. Since 1980 the subject has been reviewed frequently (49, 50, 102, 105, 121, 151) and there have been several more specialized articles covering analysis of auxins (80), brassinosteroids (131),

gibberellins (GAs) (6, 44, 46), cytokinins (52, 58), and abscisic acid (ABA)

(47, 79, 93). This review concentrates on recent advances, with particular emphasis on quantitative analysis.

The trend in plant honnone quantification has been towards speed and convenience, both facets benefiting from the ever increasing sensitivity of modem analytical methods. Even for immunoassays, sample preparation ac counts for a large proportion of the time and effort expended in performing an analysis. Simplified procedures for plant hormone purification have been de veloped, particularly for GC-MS analysis, and are discussed below.

PURIFICATION PROCEDURES

Solid-phase extraction

Improvements in the convenience and speed of plant hormone purification prior to physicochemical analyses have resulted primarily from replacing sol vent-solvent partition steps with solid-liquid extraction procedures. The use of solid phases packed into small disposable columns (99) has led to rapid sample preparation, good recoveries, and a requirement for small solvent volumes. Such procedures, which usually involve combinations of reverse phase (usually silica-bonded CI8), ion-exchange and silica gel matrices, have been developed for most hormone classes.

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PLANT HORMONE ANALYSIS 109 Prinsen et al (101) developed a method for IAA and ABA, in which the extract, diluted to 50% aqueous methanol, is passed sequentially through C18, cation-exchange, and anion-exchange columns. The acidic hormones bind to the last column and are eluted with 6% formic acid. They are then concen trated on a second CI8 column before high-performance liquid chromatogra phy (HPLC). In a simpler procedure developed for IAA, the extract in isopropanol-imidazole buffer is passed through an amino anion-exchange col umn, which is washed sequentially with solvents of increasing polarity (17). Elution of IAA is effected with 2% acetic acid in methanol. Although such protocols are designed for general applicability, modification will often be necessary to meet the particular demands of the plant material under investiga

tion. The simple purification procedure described by Dunlap & Guinn (28), in

which extracts in aqueous solution were passed through a nylon filter and then partitioned into dichloromethane, was found to be inadequate preparation for quantitative GC-MS of IAA and ABA in the hands of Li et al (71), who preferred to use a protocol based on solvent-solvent partition. However, the

Dunlap & Guinn method was used successfully in quantifying ABA by radio

immunoassay (146).

Purification of cytokinins and GAs is complicated by the broad range of polarities encountered within these groups. Nevertheless, a rapid purification procedure for cytokinins using a combination of CI8 and anion-exchange cartridges has been described (39). Small columns of CI8 have been used to separate GAs and their glucosyl conjugates from less polar kaurenoid precur sors (63), although it is difficult by this method to obtain substantial purifica tion and maintain the GAs as a group. Purification on the strong anion-exchanger QAE-Sephadex followed by concentration on CI8 cartridges have proved valuable steps between solvent-solvent partition and HPLC in preparing GAs for GC-MS analysis (23). Silica gel columns have been used widely to purify and separate GAs and their conjugates by partition chroma tography (63) and to separate GA precursors by adsorption chromatography (130). Group separation of GAs by adsorption on silica gel cartridges gives substantial purification and, in combination with anion-exchange and CI8 cartridges, can form the basis of a quick and convenient purification scheme.

High-performance liquid chromatography

Preparative HPLC is now incorporated into almost all physicochemical assays for plant hormones and into many immunoassays. Reverse-phase chromatog raphy on CI8 bonded to microparticulate silica (ODS) has become the standard technique for all classes of hormones, although normal-phase HPLC using chemically-bonded polar phases, adsorption, ion-exchange, and gel-permea tion chromatography have been used as alternative or supplementary steps when sample purification is difficult. Although there have been numerous

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110 HEDDEN

publications on HPLC purification of plant hormones, significant new devel opments are few. Consequently, the technique will not be covered in detail here and the reader is referred to Rivier & Crozier (lOS) for a thorough review of the subject. Automated purification using valve-switching between car tridges and HPLC columns, such as has been described for ABA (15), should improve the speed and convenience of sample preparation.

Immunoaffinity chromatography

Although the potential of immobilized antibodies for providing a quick and efficient isolation procedure for plant hormones is considerable, there are still relatively few reports of the application of this technique. Immunoaffinity columns have been prepared for most classes of plant hormones. Monoclonal antibodies (McAbs) or the IgG fraction from antisera are coupled usually to cyanogen bromide-activated Sepharose 4B or cellulose, although a number of other supports have been assessed (26). In order to obtain efficient binding of antigens to the columns, some pre-purification of plant extracts is usually necessary. This is achieved most conveniently by passing them through anion exchange and/or reverse-phase cartridges (73, 83, 85, 114). Columns of im mobilized fetal calf serum, which remove components that would bind non specifically to the immunoaffinity matrix, were found to be a particularly effective pre-treatment, enhancing the efficiency of the immunoaffinity col umn and prolonging its effective life (124). Extracts are applied to the im munoaffinity columns in aqueous buffer or buffered saline and the analytes eluted with methanol or methanol-water (73, 135), chaotropic agents such as SCN- (62, 85), or low pH (129). In one instance, where the McAb belonged to the IgM class, GAs could be eluted with water (124). The columns are remark ably robust, withstanding many applications of protein-denaturing reagents (27, 73). Capacities of 0.1-5 Ilg antigen per ml bed volume are typical, al though 27llg (+)-ABA per ml has been reported (59). In general, McAbs give higher capacities than antisera.

The efficient purification that can be achieved by immunoaffinity chroma tography is of particular advantage in quantitative analysis. Following this procedure it may be possible to use nonspecific methods of detection that are not normally appropriate. Examples are the quantification of IAA from Pinus

sylvestris (129) or Pisum sativum (135) by HPLC with fluorimetric detection

and of ABA from conifers using HPLC and UV detection (59). The integrity of the measured peak should be verified by a method other than that used for the quantification and the identity of the analyte confirmed by mass spectro metry. Sundberg et al (129) validated their quantification of IAA by succes sive approximation (102), in which the measured IAA concentration remained constant after serial analyses with different HPLC conditions. Immunoaffinity purification followed by immunoassay, of which there are several examples in

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PLANT HORMONE ANALYSIS 111 the literature, would seem guaranteed to produce misleading results, particu larly if the same antibodies are employed in both procedures.

The production of immunoaffinity matrices for purifying groups of related compounds is an important consideration. By selecting McAbs of broad speci ficity (85, 123) or producing antiserum against a mixture of immunogens (29) it was possible to produce immunoaffinity columns with which several GAs of interest could be co-purified. Smith et al (124) used an antibody raised against GAl coupled via C-3 to keyhole limpet hemocyanin (61) to purify 13-hydroxy GAs. In an alternative approach, Sayavedra-Soto et al (114) mixed batches of immobilized McAbs with different specificities to prepare an immunoaffinity column that bound a broad range of cytokinins.

PHYSICOCHEMICAL ASSAYS

Most current physicochemical assay systems for plant hormones incorporate a high-resolution chromatographic step, usually HPLC or capillary gas chroma tography (GC), and an on-line detector of high selectivity. Because capillary GC columns have the superior resolving power, GC-based methods are now the most commonly used in plant hormone analysis. HPLC is used only with highly selective detectors or extensively purified samples.

HPLC-based methods

Quantitative analysis by HPLC is important mainly for IAA and related in doles, for which the natural fluorescence and ease of electrochemical oxida tion allow for sensitive and selective detection. However, such methods have now been largely superseded by GC-MS and immunoassays, although they may still be of considerable value.

FLUORIMETRIC DETECTION This method can be extremely sensitive, with a detection limit of 1 pg claimed for IAA when fluorimeters are used in combi nation with reverse-phase HPLC (24). The sensitivity of fluorimetry in the analysis of hormones that possess no natural fluorescence has been utilized by forming fluorescent derivatives. Esterification of the acid hormones, such as in the formation of GA methoxycoumaryl esters (25), is simple, but offers limited selectivity and is generally not suitable for most plant extracts. Some specificity has been obtained by restricting the derivatization to a narrower group of compounds. Carbonyl-containing hormones, such as ABA and jasmonic acid, have been converted to fluorescent hydrazones (1), and the cis-diol configura tions in brassinosteroids were exploited by the formation of fluorescent bisboronates (33, 34, 36). These derivatives allow detection at the low picogram level, but may not impart sufficient specificity for accurate quantification unless there is extensive prior purification of the sample.

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112 HEDDEN

ELECTROCHEMICAL DETECTION Amperometric (66) and coulometric detec

tors (149) have been described, the latter being the more sensitive with a

detection limit for IAA of about 5 pg. Wright & Doherty (149), who measured

the IAA content of a single half node of A vena !atua, used a detector with two

electrodes set at different voltages to increase selectivity, and monitored elec trochemical and fluorescence responses in series to assess peak purity. Brassinosteroid bisferroceneboronates have been prepared for electrochemical detection (35), although their selectivity is relatively low. Because electrochem ical detectors require electrically conductive liquid phases, their use is restricted

to reverse-phase and ion-exchange HPLC (112). Due to their limited applica

bility and the need for careful maintenance to sustain performance, the use of electrochemical detectors in plant hormone research has not been greatly pursued.

Quantification by HPLC-fluorimetric and electrochemical detection de pends on calibrating the detector response and assumes no change in response in the presence of the sample. Losses during purification before HPLC are corrected with the use of a radioactively-labeled internal standard, which is

recovered and counted after HPLC (66,129).

MASS SPECTROMETRIC DETECTION New developments to interfaces between HPLC columns and mass spectrometers are helping to realise the enormous potential of combined liquid chromatography-mass spectrometry (LC-MS) as an analytical tool in biochemisty. Although application of this technique to plant hormone analysis is still in its infancy, it should become important for hormone conjugates that are difficult to analyze by GC-MS because of their low volatility. Furthermore, analysis of free hormones by LC-MS could simplify current procedures by dispensing with the derivatization and GC-MS steps. However, mass spectra of sufficient quality might be difficult to obtain without extensive pre-purification of samples. The potential of LC-MS to identify underivatized

conjugates of GAs (81, 82) and IAA (16, 90, 91) was demonstrated using a

sytem fitted with a frit-fast atom bombardment interface, in which the HPLC column effluent exudes through a fine-mesh metal frit, on which it is ionized by bombardment with xenon atoms. This method cannot accommodate flow rates greater than about 5 �l per minute and was used with a capillary HPLC column. Although this restricts severely the amount of extract than can be introduced, high sensitivity provides some compensation. Other interfaces, such as atmospheric pressure chemical ionization (APCI), thermospray and particle beam, accommodate flow rates in excess of one ml per minute and are compat ible with conventional analytical columns. The application of APCI to GAs and GA-conjugates has been described (84). In this ionization method the HPLC eluant forms a plasma at atmospheric pressure by corona discharge and ionizes the samples by a chemical ionization eCI) mechanism. Ionization is aided by an

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PLANT HORMONE ANALYSIS 113 electrolyte, such as ammonium acetate, in the HPLC solvent. Thermospray LC-MS, a related technique in which the column effluent forms fine droplets by extrusion through a narrow, heated nozzle, has also been applied to GAs and their methyl (Me) esters, although conjugates were not examined (40). Because most LC-MS ionization procedures produce little fragmentation they are ideally suited to quantitative analysis. Although the sensitivity of LC-MS, with mini mum detection limits of hundreds of picograms for GAs in SIM (84), is inferior to that of GC-MS, the technology is developing rapidly and continual im provements in sensitivity are anticipated.

GC-based methods

Discounting mass spectrometric methods, there have been few recent ad vances in the use of GC detectors for plant hormones. The high sensitivity of electron capture and its selectivity for ABA has ensured its continued use for this compound (152). Formation of halogen-containing derivatives of other hormones (31, 64, 72), although enabling detection in the picogram range, provides insufficient selectivity for reliable quantification. The introduction of the flameless alkali or nitrogen-phosphorus detector as a more sensitive alter native to the alkali flame ionization detector allows detection of IAA (55) and cytokinins (126, 145) at the low picogram level with some selectivity. Whenham (145) favored the nitrogen-phosphorus detector from several GC detectors tested for methylated cytokinins. A flame photometric detector tuned for sulfur detected only S-containing cytokinins, but with poor sensitivity.

Unlike HPLC-based detectors, GC detectors are usually destructive and do not easily allow the recovery of a radioactively-labeled internal standard. Consequently, as well as depending on the efficiency of derivatization and the reproducibility of detector response, the accuracy of quantitative analyses by GC relies on precise sampling from a purified extract for GC measurement and radiocounting. The reliability of this procedure was questioned by Cohen & Schulze (21), who devised a complex isotope dilution method for IAA quantification using two internal standards, the first to correct for losses during sample preparation, the second for GC quantification. The development of mass spectrometric methods for measuring isotope dilution allows for correc tion of losses with a single internal standard without the need for precise sampling, except when adding the standard. The simplicity and accuracy of isotope dilution GC-MS have made it the method of choice for those who adhere to the physicochemical approach.

MASS SPECTROMETRIC DETECTION Analysis of plant hormones by GC-MS has been discussed in numerous review articles, including those on IAA and related compounds (3, 104, 112), ABA and its metabolites (44, 47, 79, 86, 93), GAs (6, 44-46), cytokinins (51, 52, 92), and brassinosteroids (53, 131). This

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114 HEDDEN

article is concerned with quantitative analysis, but the role of GC-MS in aiding the identification of novel hormone metabolites and in confirming the identity of target analytes is of the utmost importance. Recently, a comprehensive compilation of GA and related mass spectra, including those of the other acid

hormones, has been published (37).

There is always uncertainty about the identity of compounds that are de tected by methods that provide relatively little structural information, such as SIM (102). For example, the quantification of GA3 in barley shoots by GC

SIM was found, after full-scan GC-MS analysis, to be seriously compromised by the presence of a co-eluting compound that produced a fragment ion at

mass/charge (miz) 504 that was monitored for GA3 (23). Separation of GA3

from this unidentified C2o-GA contaminant, was achieved by changing to a

GC column of different polarity. Although it is usual in quantitative analysis

by GC-SIM to monitor multiple ions for the analyte and internal standard, this is impractical when there is little fragmentation. It is necessary to obtain confirmation of identity by full-scan mass spectral analysis of the species and tissue of interest. Some workers have been tempted by the superior sensitivity of SIM, compared with full-scan mass spectrometry, to use it for identification when tissue supply is limited. The validity of this approach, however, is seriously open to question.

Recently, tandem mass spectrometry, also known as mass spectrometry mass spectrometry (MS-MS), has been exploited in plant hormone research. In this technique, which requires two mass analyzers separated by a collision cell, a parent ion is focused by the first analyzer and allowed to dissociate with the aid of a collision gas to produce fragment (daughter) ions, which are separated in the second analyzer. Triple quadrupole instruments, in which the second quadrupole serves as the collision cell, are commonly used for this purpose. In an elegant application of MS-MS to the biosynthesis of ABA, the position and sequence of incorporation of 0 atoms from 1802 into this com

pound and its precurors and catabolites were determined (108, 153). The

method also introduces a further level of selectivity to MS analysis, as was demonstrated in the identification of brassinosteroids from Alnus glutinosa pollen after minimum purification (98). A related technique-mass-analyzed ion kinetic energy spectroscopy (MlKES)-requires double-focusing mag netic sector instruments and has been used in the structural analysis and

identification of jasmonic acid-amino acid conjugates (115).

Quantitative analysis by GC-MS Measurement of isotope dilution by mass spectrometry is well established and suitable isotopically-labeled internal stan dards have been developed for all plant hormone classes except ethylene (Table

1). For high sensitivity in quantitative analysis the mass spectrometer is nor

mally operated in the SIM mode, for which quadrupole instruments are

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PLANT HORMONE ANALYSIS Table 1 Internal standards for isotope dilution analysis

Hormone IAA ABA GAs Cytokinins Brassinosteroids Jasmonates Label [2,_2H2] [2,4,5,6,7JHsl [4,5,6,7}H4l [3a,4,5,6, 7 ,7a-13C6] [3',5',7'}H6] [6-2H3J [2,6-2H4l [13Csl [17-2H2l [1,2,3,6-2Hs] [1 7_13C,3H2]

e

Hzl [2H3]

e

Hs]

e

H6l [1,3,7,9-ISN4] [26,28-2H6l [13C,2H3]Me Compounds Reference IAA 76 IAA 76 IAA 76 IAA 18 IAA-aspartate 20 ABA 106 ABA 87

ABA glucose ester

ABA, phaseic acid 147

ABA 54 GA],GA3,GA4,GAs, Mander" GA7,GAg,GA9,GAI9, GA2o,GA29, GA53 GAI,GA20 30 GAI,GAs,GAg, 32 GA20,GA29 2-0-GA29-glucoside 116 J3-0-GA29-glucoside 130GA20(6' -2H21glucoside 130GAs[6' -2H2]glucoside Zb,(diH)Z,[9RlZ 127 [9R-5'P]Z,CdiH)[9R-5'P]Z 120 [9R]Z 43 Z,[9R]Z,[9R]CdiH)Z, 127 (OG)Z,(OG)[9R]Z, (OG)(diH)Z, (OG) [9Rl (diH)Z, [9G]Z,[7G]Z [9R]iP 43 Z,[9R]Z,[9G]Z,(OG)[9R]Z 119 brassinolide 132 castasterone typhasterol teasterone methyl jasmonate 22 115

'Professor L. N. Mander, Australian National University, Canberra has made available a number of [17-2H21GAs with very high isotopic substitution.

b Abbreviations for cytokinins as proposed by Letham & Palni, see 69.

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116 HEDDEN

ularly well suited. Several pairs of equivalent ions for the analyte and internal standard, including their molecular ions [Mtj, unless they are weak, are moni tored to allow detection of interference from contaminants. Alternatively, short scans of ion clusters that include equivalent ions for the endogenous hormone and internal standard would give sensitivity comparable with that obtained from SIM. However, because 2H-containing internal standards elute from capillary GC columns slightly earlier than the IH equivalents, it is necessary to base relative ion abundances on averaged scans over the entire GC peak. Chromato graphic separation of internal standards and analytes presents no problem in SIM because ion abundances are based on the areas or heights of the ion chromatogram peaks.

Substantial increases in MS sensitivity in recent years have resulted from improvements to the ion source and analyzer and from the appropriate choice of ionization mode and hormone derivative. For example, the lower detection limit for ABA as the Me ester, which is about 100 pg when the fragment ion at

mlz 190 is monitored in EI, is reduced to 10 pg using CI in methane with

positive ion detection (137). Moreover, due to the natural electron-capturing properties of ABA, much greater sensitivity can be obtained in CI with nega tive ion detection. A detection limit of 300 fg was obtained by monitoring the [M.] for ABA Me at m/z 278 using ammonia as reagent gas (107), and this was reduced to below 20 fg by forming the pentafluorobenzyl ester and monitoring the [M-CH2C6Hsr ion in negative ion CI with methane (88). The sensitivity of this method compares well with the most sensitive im munoassays and is adequate for all but the most demanding applications. A disadvantage of CI, despite its potential for higher sensitivity in SIM than normally obtained with EI, is that it is sometimes difficult to find more than a single suitable ion pair to monitor. Quantification of cytokinin O-glucosides by desorption CI from a heated probe has been described (70). The negative ion spectra were enhanced by fonning N-9 2-cyanoethyl and

2-chloro-2-cyanoethyl derivatives, although sensitivity was not high. Some separation

was achieved by programmed heating of the probe, but it would seem that highly-purified samples are necessary.

Quantification by isotope dilution determines the ratio of analyte and inter nal standard amounts, and is independent of their recoveries, which are as sumed to be equal. Several methods are used to calculate the relative amounts of analyte and internal standard from the measured isotope peak abundances. This relationship is complicated by the presence of naturally occurring heavy isotopes in the unlabeled analyte and by incomplete labeling of the internal standard. An empirical approach is to construct calibration curves from differ ent ratios of hormone and internal standard (45, 86,97). If there is no overlap in the ion clusters for the two compounds, which requires a mass difference of about 4 amu or greater, calibration curves are linear. Otherwise they are best

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PLANT HORMONE ANALYSIS 117 described by polynomial regressions (56). Second order regression lines give satisfactory fits in most cases. Alternatively, hormone-internal standard ratios have been calculated directly, either as the ratio of the ion pair intensities after correcting empirically for natural isotope contributions [a method used fre quently in GA analyses (8, 109, l33)], or using the isotope dilution equation (18, 76). In a different approach, a least-squares fit program has been em ployed to compare measured and assumed relative intensities for each ion in a cluster and thereby to calculate the isotopic contributions to these ions (32, 37). The method assumes that the relative intensities of the ions in the clusters for the analyte and internal standard are the same and does not require that the endogenous compound be available as a standard. The intensities of each ion in the overlapping ion clusters for these compounds need to be measured, either by SIM or by scanning over a narrow mass range. A comparison of methods commonly used in GA analyses found little difference in values obtained using calibration curves or the fit program, although estimates based on ratios of corrected ion intensities were slightly, but significantly, higher (S. J. Croker & P. Gaskin, unpublished information).

Isotopically-labeled internal standards The range of isotopically-labeled hor

mone analogs prepared chemically for isotope dilution analysis is indicated in Table 1. Deuteriated standards produced by base-catalyzed exchange, such as [2'-2H2]1AA and [2H6]ABA, are now considered unsafe because isotope ex change can occur during sample preparation (18, 87) or even during GC on capillary columns. The deuterium atom on carbon-2 of [2,4,5,6,7-2Hs]IAA is lost slowly on alkali treatment and therefore the tetradeuterio IAA standard is considered more satisfactory despite its limited availability (76).

e

3C6]1AA (9, 18) and [6-2H3]ABA (87) were introduced as stable internal standards that could be used to quantify these hormones after release from conjugates by base hydrolysis. An apparent loss of deuterium from carbon-6 of ABA was exploited in a simplified method for labeling at this position by isotope exchange in strong base (147). However,

e

H3]ABA has proved to be a satisfactory internal standard for all practical applications. The syntheses of l3C-Iabeled ABA (54) and its biosynthetic precursor xanthoxin (94) have also been reported. The need to quantify specific hormone conjugates rather than to rely on measuring the amount of hormone released after hydrolysis has prompted efforts to synthesise isotopically-substituted conjugates of most hormone classes, examples of which are given in Table 1.

Isotope effects, which are important for deuterium and tritium, can modify the chemical and chromatographic properties of molecules, the magnitude of the effect depending on the nature, number, and position of the heavy isotopes incorporated. Deuteriated hormones separate slightly from their protiated equivalents on HPLC, eluting later than the unlabeled compounds on

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118 HEDDEN

tion chromatography and earlier from reverse-phase columns (14). It is, there fore, necessary to collect broad HPLC fractions to avoid introducing errors during sample purification. Because tritium is subject to a larger isotope effect than is deuterium, the problem is more severe for tritiated standards, which are often added to extracts to aid the location of target compounds after HPLC. Incorporation of deuterium also shortens GC retention times on capillary columns of low or medium polarity, so that calculations of isotope dilution based on full-scan data require spectral averaging.

Preferably, the mass difference between the analyte and internal standard should be large enough to allow complete mass spectrometric separation of the ion clusters on which the measurement is based. This is easily achieved with small molecules such as the Me esters of IAA and ABA, but is more difficult with trimethylsilyl derivatives, which have higher molecular wei�hts and con tain silicon with its high abundance of the natural heavy isotopes 9Si and 30Si. The resulting overlap of the ion clusters, particularly severe with [17)3C]GA standards, requires that the internal standard and target compound are present in similar quantities for high precision. 14C-Labeled GA precursors, prepared biochemically from [2-14C]mevalonic acid (65), can serve as excellent inter nal standards for isotope dilution mass spectrometry (38). Because they con tain three or four 14C atoms, their mass difference from the endogenous compounds is high, and their radioactivity allows for precise application and ease of tracing during sample purification. Unfortunately, it is difficult to prepare [14C]GAs with sufficiently high specific radioactivity.

Quantification of ethylene

Because it is a gas, ethylene poses a much less severe analytical problem than do the other plant hormones. No extraction is required and there are relatively few contaminants, and these can be removed in a single gas-chromatographic step without derivatization. Quantification is normally accomplished using GC in combination with a flame ionization detector, although the low sensitivity of this technique requires that evolved gases are allowed to accumulate in a closed system or are concentrated in cooled traps. Sampling methods for ethylene have been reviewed (5, 111) and will not be considered here. Higher selectivity and sensitivity has been achieved using a photoionization detector, which, with a detection limit of 1 nl per liter, has allowed ethylene to be detected in open systems without concentration (4). However, the method has not been widely adopted and its main advantages may lie in its capacity to be used in a portable gas chromatograph run at ambient temperature with purified air as carrier gas.

PHOTOACOUSTIC LASER DETECTOR Recently the development of a highly sensitive on-line detection system based on the photoacoustic effect has enabled

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PLANT HORMONE ANALYSIS 119 ethylene to be quantified at concentrations as low as 0.06 nl per liter (41, 148). The method, which is also applicable to other atmospheric gases, involves excitation of ethylene by a C02 waveguide laser at infrared wavelengths into a higher rotational-vibrational energy state. Nonradiational relaxation leads to a redistribution of the absorbed energy into kinetic energy which, in a constant volume, results in an increase in pressure that is detected by miniature micro phones. For maximum sensitivity the laser beam is interrupted periodically, or chopped, at the resonance frequency of the photoacoustic cell, thus creating a standing wave. Experiments are performed in a flow-through system: Plant material is placed in cuvettes fed by air from which hydrocarbons have been removed over a heated catalyst. The air is then passed from the cuvettes into the cylindrical photoacoustic cell via KOH scrubbers to remove C02, which would interfere seriously with the ethylene signal. Possible interference by residual C02 and other components, such as ethanol, is corrected for by basing the quantification on the ratio of signals at two wavelengths (10.51 and 10.53 11m). In practice, ethylene levels are determined as the difference between control and treatment in different cuvettes, which are alternatively switched on-line to the monitor (148). In early reports on the use of this apparatus full equilibration between samplings required 45 min (148), but sampling intervals have now been substantially reduced. This method, which is not yet generally available, has come closer than any other to a continuous, nondestructive monitoring system for a plant hormone.

IMMUNOASSAYS

Despite their current popularity and undoubted value in plant science, the original claims for plant hormone immunoassays-universal application, a minimum of sample purification-have seldom been realized. Interference from sample components can be as serious a problem as it is in physicochemi cal assays and require substantial sample purification to remove. Using the method of successive approximation (102), in which quantification of IAA by immunoassay was compared after consecutive purification steps, HPLC was found necessary to obtain immunoassay values that were consistent and com parable with those from physicochemical assays (19, 113). The degree of purification required for immunoassay in these cases was the same as that needed for GC-MS. It is clear that some degree of purification is necessary in most plant hormone immunoassays and, as with physicochemical assays, ac curacy requires the inclusion of an internal standard to determine recoveries. The most suitable is a 3H-Iabeled hormone analog of high specific radioactiv ity. Interference in immunoassays can be specific, where contaminants com pete with the antigen leading to an overestimate of antigen concentration, or nonspecific, where impurities modify one or more processes in the assay by,

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120 HEDDEN

for example, inactivating the antibody or complexing with the antigen (57, 96, 142). Various tests for immunoassay validity have becn proposed, the most common being to assay aliquots of plant extract after spiking with different amounts of authentic analyte. In the absence of interference, plots of added versus determined analyte amounts should have the same slope regardless of aliquot size. It can, however, be difficult to detect specific interference, partic ularly when the antigen and inhibitor have similar affinities for the antibody (96). The most satisfactory validation of immunoassays is undoubtedly by verifying the values obtained using an independent, reliable technique, such as GC-MS.

Immunoassays based on antisera and McAbs have been described; the latter, although technically more difficult to prepare, provide higher specificity and an unlimited supply of antibodies. Monoclonal antibodies of high specific ity can be particularly beneficial when it is necessary to assay compounds in the presence of closely-related structures. The GAs, which are usually present in plant tissues as a family of metabolically-related compounds, pose consider able problems for immunoassays. Physiologically meaningful data will be obtained only if it is possible to quantify the active molecular species in the presence of many structurally similar compounds, often at higher concentra tion. Smith & MacMillan (123) obtained misleading results when trying to quantify Cl9-GAs in Marah macrocarpus endosperm, due to the presence of components that cross-reacted only slightly with the McAbs used, but were present at much higher concentration than the target antigens. It has not been possible to prepare McAbs that are completely specific for a single GA. Purification by HPLC is therefore a minimum prerequisite for immunoassay of particular GAs, although even with this procedure closely-related com pounds, such as GAl and GA3, are poorly separated.

Despite their limitations, properly validated immunoassays can be of con siderable value because they offer high sensitivity and convenience. Their use in plant hormone analysis, including practical aspects, has been thoroughly reviewed (58, 95, 141-43, 150). Plant hormone antibodies have also found important applications in immunolocalization, as an alternative to bioassay for hormone detection after purification and fractionation and, as discussed ear lier, in immunoaffinity purification. Furthermore, via anti-idiotypic antibod ies, they have been recruited in the quest for hormone receptors (48, 100). However, these applications are beyond the scope of this review.

Radioimmunoassays

The simplicity of radioimmunoassays (RIAs), based on competition between the antigen and a radioactively-labeled analog, encouraged their early devel opment for plant hormones. However, their requirement for radioactive tracers of high specific radioactivity and a scintillation counter is a serious disadvan tage in many laboratories. Consequently they are being supplanted by

en-Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1993.44:107-129. Downloaded from www.annualreviews.org

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PLANT HORMONE ANALYSIS 121 zyme-linked immunosorbent assays (ELISAs), which, as well as requiring less expensive equipment, have the capacity for higher sensitivity.

The sensitivity of RIAs should depend on the specific radioactivity of the tracer. In most cases 3H-Iabeled plant hormones have been used, although analogs containing 1251 have been employed in attempts to obtain higher sensitivity (60, 67, 139, 144). However, the increase in sensitivity obtained with 1251 was slight, perhaps because the relatively major structural modifica tion needed to incorporate this isotope resulted in low affinity of the tracer for the antibody.

Most workers have employed heterogeneous RIAs, in which free and anti body-bound tracer are separated, usually by ammonium sulfate precipitation of the antibody. Separation by dialysis has also been described (67), as has a solid phase system, in which the antibody was coated to the well of a microti ter plate, which was cut out and counted (136). High sensitivity was claimed for the latter method, with a measuring range of 0.03-3 pmol. Recently, an homogeneous RIA based on the use of a scintillation proximity reagent was developed for ABA (146). The reagent consisted of fluor-containing beads covalently linked to protein-A, which allowed them to be coated with the IgG McAb used in the assay. Only antibody-bound tracer ([3H]-S-ABA) is close enough to the bead to elicit scintillations and can thus be counted in the presence of unbound tracer in solution. N o separation step was necessary and the assay could be carried out in scintillation vial inserts with the minimum of manipulation.

Enzyme-linked immunosorbent assays

The availability of commercial kits, consisting of multi-well microtiter plates precoated with antihormone McAbs, has contributed to the popularity of ELISAs for plant hormone quantification. Direct ELISAs, in which sample and enzyme-coupled antigen compete for antibody binding sites, have the simplicity of RIAs and the enzymic reaction, catalyzed by alkaline phospha tase or peroxidase, affords some amplification. Detection limits of such assays are usually about 50 fmol (10 pg) with a quantification range over three orders of magnitude (140), although a detection limit of 1 fmol has been reported (2).

Monoclonal antibodies are generally favored over antisera because they offer an unlimited supply of well-characterized antibodies. Although mouse McAbs tend to bind poorly to polystyrene wells, precoating with, for example, rabbit

(42, 136, 143) or goat (154) antimouse IgG antiserum can ensure reproducibil

ity.

Numerous modifications to the standard ELISA have been developed. Coating the wells with protein-coupled antigen rather than scarce or expensive antibody would appear prudent. In this case competition between bound and sample antigen for enzyme-coupled antibodies may form the basis of the assay

(10). Alternatively, in indirect ELISAs, a second antibody, linked to enzyme,

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122 HEDDEN

is used to quantify the first, anti-hormone antibody, bound via the immobilized antigen to the well (89, 110, 138). Due to the bivalency of antibodies, the use of a second antibody offers further amplification and an increase in sensitivity of the indirect ELISA, over the direct assay, of 5-10 fold has been claimed (7). The use of biotinylated second antibody and avidin-coupled enzyme (68, 77, 125), or an enzyme substrate (4-methylumbelliferyl phosphate) that generates a fluorescent product (134) offers further opportunities for increased sensitiv ity. However, these procedures have not extended detection limits signifi cantly and it is possible that factors such as the affinity of the first antibody, or the nature of the coating antigen are more important limitations to assay sensitivity than are the final labeling and assay phases. Manning (78), in a detailed characterization of an indirect ELISA for IAA, concluded that the assay sensitivity was affected substantially by the nature of the coating anti gen. The valency of the antigen (number of IAA molecules per molecule of protein) was of particular importance, assay sensitivity decreasing with in creasing valency. At valencies above ten the amount of antibody bound in the absence of competing IAA decreased with increasing valency and the addition of low amounts of IAA increased antibody binding. This was explained as a consequence of the bivalency of antibodies, which exhibit co-operative allo steric binding. Further improvements to sensitivity were obtained by substitut ing IAA with cross-reacting haptens, such as indole-3-propionic acid, in the coating antigen to reduce antibody recognition of the bridging group.

Very high sensitivity has been obtained with an enzyme-amplified direct ELISA for ABA (42). Bound ABA-coupled alkaline phosphatase is measured from its dephosphorylation of NADP to NAD. Amplification is achieved by using the NAD in a redox cycle in which the oxidation of ethanol to acetalde h y d e by alcohol d e h y dr ogenase i s coupled to t h e reduction of iodonitrotetrazolium violet to formazan by diaphorase. The formation of formazan is measured spectrophotometric ally. The detection limit of this assay, at 0.2 fmol, is about the same as that achieved for ABA by GC-SIM

(88) and was sufficient to measure the ABA content of a guard cell pair (42).

Recently, enzyme-amplification was used in an indirect ELISA for GA4 to give a detection limit of 0.1 fmol (133a).

CONCLUSIONS

Several new analytical techniques in plant hormone analysis are emerging, some of which are destined to play prominent roles in the future. The applica tion of LC-MS to the analysis of hormone conjugates is of particular interest because it offers the first opportunity to quantify many of these compounds reliably. The rapid sampling of ethylene from single plant organs with the photoacoustic laser detector, should yield a wealth of valuable information.

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PLANT HORMONE ANALYSTS 123

Further developments in immunoaffinity chromatography are likely to sim plify plant hormone purification. The recent report of a new type of sensitive bioassay for auxins and cytokinins is the first example of the application of

recombinant DNA technology to plant hormone analysis (11). The assay uti

lizes tobacco leaf protoplasts transformed with a chimeric gene consisting of the auxin plus cytokinin-sensitive promotor from Agrobacterium tumefaciens

gene 5 coupled to the coding region of the

�

-glucuronidase gene. It is too early

to assess the potential of this assay, although the sensitivity of protoplasts to plant products may restrict it to certain tissues or highly-purified extracts.

The sensitivity of older methods, particularly of GC-MS and immunoassay, is being continually improved, allowing ever smaller quantities of tissue to be sampled. It has, for example, been possible by immunoassay to determine the ABA content of sap samples withdrawn from single cells by means of a micro-syringe (13) or in homogeneous tissue samples containing small num bers of cells (42). It is clear that much information is lost when mixtures of

tissues or cell types are pooled for extraction (122). Because extracts from

small tissue samples require relatively little purification, they can be processed rapidly and in large numbers. The resulting ability to accommodate high levels of replication adds further to the value of the data.

ACKNOWLEDGMENTS

I am grateful to those who made available preprints and unpublished informa tion, and to many colleagues at Long Ashton Research Station for their com ments on the manuscript.

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