9e(tiea for the termination 01
I
1
weredescribed
has paralleled the his chemical methods of phytohormones h analyses of these immunological methods. number of compounds in a single sample, and
marriage of current me analysis with immunosor assays promises to exte our understanding of ph plant growth and develo
638A
ANALYTICAL CHEMISTRY. VOL. 57
ction limits and mones’ roles in
NI
i 4 Y 1985
Molecules involved in plant growth regulation and development have received increasing attention in the past decade. These molecules are referred to collectively as plant hormones, phytohormones, plant growth substances, or plant growth regulators. Plant hormones are secondary metabolites that are generally segregated into five groups (see Table I). They are small organic molecules that are often synthesized in one part of a plant and transported to another where they act. Phytohormones may also be effective in regulating events within the cells in which their synthesis takes place. As indicated in Table I each hormone group is identified with certain developmental or reproductive phenomena. It is also known that hormone groups can act in concert with one another. An example of this is the expression of apical dominance in shoot tissue regulated by the balance of cytokinin to auxin. A low cytokinin to auxin ratio suppresses lateral bud development, producing a plant that is usually elongated with a single axis and little branching (e.g., pea, sunflower). A high cytokinin to auxin ratio results in a highly branched or bushy plant (e.g., tomato, potato). Identification of hormonally regulated control points that could be manipulated through chemical applications or genetic engineering could well be a key to improving yield, disease resistance, and tolerance to environmental stress. As such, plant hormone research is a critical element of many plant biology programs. The mechanisms of phytohormone action in regulating plant growth and development are not well understood. Advances in our understanding of plant hormone physiolosy have been hampered by a lack of analytical methodology that is able to isolate these compounds and measure them a t the levels a t which they are found. In the past, multiple extractions and multicolumn cleanup steps were often necessary before quantitation was possible. As a result, sample recoveries 0003-2700185/0357-638A$Ol ,5010 @ 1985 American Chemical Society
Gregory C. Davis Mich B. Hein Brian C. Neely C. Ray Sharp Michael G. Carnes Monsanto Agricultural Products Company 700 Chesterfield Village Parkway Chesterfield,Mo. 63198
were usually low. To try to overcome the detectability problems, large amounts (kilograms) of plant tissue were reonired to nrovide sufficient compo&d to be measured. Although this approach proved successful in quantitating pooled hormone levels, important information regarding individual tissue levels was lost. Modern methods of analysis are helping to bring about a fundamental understanding of phytohormone action hy allowing sensitive and selective detection of these molecules in small plant samples.
In this REPORT we will descrihe various methods for purifying and analyzing plant hormones. The presentation reflects the historical develomlent of analyses for plant hormones; beginning with bioassays and ending with the novel immunochemical assays. Those wishing a more comprehensive and detailed review of plant hormone methods will want to consult References 1 6 . isoiatlon and purification Generally, phytohormones are analyzed in organic extracts of plant tis-
sues. Analyses are typically performed after a variety of chemical partitioning and classical or modern chromatographic purifications. Fractions separated by these methods are then analyzed for specific plant hormones by biological assays, physicochemical methods, or immunological assays. The earliest analyses of plant hormones focused on a single hormone that was isolated by solvent partitioning. Hormones were separated on the basis of differential solubility. This was achieved by using aqueous buffers a t varying pH and organic solvents
Table I. Plant hormone groups TYp*.l lewis (Unplo
Honrmm 0”P
Auxins
haw*
rn
CHi-
n m w c acid
Abscisins
1-io3
phaseic acid
StaMtal ciosure; embryo dormancy: inhibit many
10-2 X 10‘ Adsorption: isomerization of pentadiene to trans,
Gibberellins
Adsorption; chemical
oxidation: photwxidation
responses of gibberellins, cytokinins. and auxins
.m
Nb CN
AMW* P-mm
Maintain apical dominance; cell expansion and elongation
isopentenyl adenine; doH Czeatin riboside
Cytokinins
lbslm hnh w e W )
Phenylacetic acid 4-Chlorc-indoleacetic acid
OOH
0 ,+,*DcU*
EunWsd-1 Msrrm and .ah
othn .=MIPI.
mS*m
trans configuration
Cell division; break of bud and seed dormancy; inhibit apical dominance of main stem axis
l-lOs
Severe adsorption to glassware and silica
Stem elongation: fruit growth: induce enzymes for seed gemination
1-103
Oxidation; mwe than 60 similar molecules; no unique chromophore
-
Volatile: contamination from many plastics and polymers
*z..lin
J$xp
HO
*llh
A>
>60 gibberellins varying in position and degree of -OH and -CWH substitution
Senescence; epinasty: climacteric ripening of fruit
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
639A
rigure 1. Reversed-phase separation 01 cytoKinins Beckman Ultrasphere ODS; solvent A-40 mM triethylammOniumacetate IpH 3.4). solvent &methanol/ acetonlblle (13): gradient-lnltial Conditions 10% B fw 1 mi”, followed by a linear increase to 18% B WB( 19 min, and finally a linear increase to 70% B over 23 min: flow rate 2.0 mL/mln. 2-90, zeatin-$glucoside: 2-00. zeatln-Oglumide; Z,zeatin: DKZ. dihydromtln:c-2. cis-zeatin: ZR. zeatin ribside: DKZR, dihydroreallnriboride: KIN, kinetin; iPA. isapentenyl&nosine: iP, isopentenylBdeniw. Samples 10 ng except 2-90 and Z-OG (20 ng). Chromalogram coutlesy of R. Dvley. Oregon Slate University
such as chloroform, methylene chloride, diethyl ether, and ethyl acetate. The solvent partitioning schemes yielded fractions that could be tested for hormone activity in biological assays. Initially the “active” fractions contained numerous compounds from the original extract, one or several of which possessed biological activity. To deduce the chemical nature of the active component(s), careful attention was paid to the solubility characteristics of an active fraction. Paper and thin-layer chromatography were also used to purify phytohormones. As other separation methods including liquid-liquid partition, adsorption, and ion exchange chromatography were developed they were applied to purification of hormones. UV absorbance
flow monitors or stand-alone spectrophotometers were used to monitor for elution of compounds to he tested for biological activity. These absorbance detectors also began to be used as “detectors” of plant hormones, although no proof of biological activity could be implied by UV absorbance. The resolution of gravity flow or low-pressure columns was limited so that the extraction protocol and sample manipulation prior to these columns were critical. Later, use of Sephadex LH-20 provided good resolution of some hormone groups by reversed-phase separation (6),but this method was soon replaced hy more powerful and versatile preparative HPLC methods. Since 1975 reversedphase LC has played an increasingly
important role in the separation of plant growth substances. Today it is an integral part of the vast majority of published analytical methods, either in the isolation steps or in the final analysis. One of the reasons for the popularity of reversed-phase LC is that it can be used with aqueous mobile phases. As a result it is ideally suited for the examination of plant samples, which are by nature predominantly aqueous. Flexibility in the choice of mobile phases allows one to find the separation conditions to isolate hormones that differ very little in structure. Figure 1demonstrates a separation of a number of cytokinins by reversed-phase HPLC. All of these methods, from initial tissue extraction through Partitioning and chromatographic separation, are time-consuming and often require large volumes of solvents and many separate manipulations of a single sample. This can result in losses of phytohormones that are present a t trace levels. The use of isotopically laheled internal standards is necessary to estimate recovery and to quantify these molecules with acceptable accuracy ( 2 , n . Biological assays
Biological assay is the oldest and most fundamental method of identifying and quantifying plant hormones. Isolated plant tissues, organs, or perhaps whole seedlings are grown under rigidly controlled environmental conditions to maximize tissue sensitivity and reduce variability. These tissues are then exposed to phytohormones or to purified fractions of plant extracts, and the response is quantitated. The measured responses include stem elongation, stem curvature, pigment biosynthesis, and increase in biomass.
. . I
Figure 2. Avena coleoptile cury_._.
.
I
Ths Avena coleoptile curvatwe bloassay was developed by Fritz Went (4.Subapical coleoptile segments are incubated with a small agar block in contact wilh one side of Uw cul apical surface. Cvvature results horn differential elongationof cells induced by auxin hn one side of the coleoptile 840,.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
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An example of a biological assay for auxin is illustrated in Figure 2. (A Listing of many of the well-described biological assays for plant hormones appears in Reference 4.) Bioassays have many inherent problems. Precision is hampered by the variability of the plant tissues that are the basis of the assays. Time of analysis is prohibitive, minimally hours and often days to weeks. Potentially active substances must be applied exogenously to the assay tissues. This can result in low sensitivity because of problems with uptake and metabolism. These problems, coupled with the requirement for large numbers of replicates to achieve precision of analysis, mean that large amounts of a plant hormone must be used in a single analysis. Because of the inherently low concentrations of phytohormones in plant tissues it is often impractical or impossible to acquire sufficient hormone for a bioassay. Most bioassays have limited or undefined selectivity, which places a strict requirement on fractionation procedures to provide homogeneous samples for analysis. Any impurities in a biological extract, including inhibitors (a common problem) or other active molecules, compromise the interpretation of the bioassay. These problems have been the impetus for development of modern Chromatographic,spectrometric, and immunologicalanalyses. Bioassays do provide a valuable function despite numerous drawbacks. Some plant hormones are defined by their activity in uniquely selective assays (e.g., the Avena coleoptile curvature assay for auxins, Figure 2). Although hormones are ubiquitous in higher plants, the relative and specific activities differ among plant species. Therefore, rudimentary bioassays are often necessary in a particular species to determine the significance of a phytohormone. Ultimately all putative plant hormones must be demonstrated to have activity in a pertinent biological assay.
Modern methods of chemical analysis Modern methods of analysis have been applied to all of the plant hormone groups. These methods have provided sensitivity, precision, and speed of analysis unattainable with biological assays. The more recently developed assays for plant hormones rely on chromatographic separations for some degree of qualitative analysis. Reliance on retention characteristics for compound identification is more easily justified for simpler samples such as serum, urine, and defined chemical systems than for complex plant extracts. Most of the presently used analyses for phytohormones de642A
Flgure 3. Indole acetic acid (IAA) from corn kernel extract with tandem fluorescent (F) and electrochemical (EC) detectors E I O PR ~ P-~ ~soivem-0.05 . H phosphate (PH 3.0) wim 18% memano1 (WJ: flow rate 1.0 m V mm: Kialos FS950,sxcltatlon a1 280 nm and ern1sIon a1 340 nm: BAS LC-3. glassy carbon elenrode a1 0 85 V y6. A g - A S I reference
pend on HPLC or GC separations. Judicious choice of a detection method, coupled with one of these chromatographic techniques. optimizes the qualitative and quantitative information from the assays. HPLC can be used to quantify all of the plant hormones except ethylene. After HPLC separation, detection by UV absorbance is commonly used, but it is not a method of choice because of its lack of selectivity. The cytokinins, abscisic acid, and indole-3-acetic acid
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, MAY 1985
are readily detected a t 254 nm and 280 nm, but the gibberellins only absorb UV radiation a t wavelengths below 230 nm. Derivatization of phytohormones to facilitate HPLC detection has been used to enhance sensitivity but rarely improves selectivity because many components of the heterogeneous sample are derivatized. Detectors relying on more selective phenomena than UV absorbance, such as fluorescence or electroactivity, greatly enhance the power of HPLC analyses. Routine determination of indole-3-acetic acid by HPLC with fluorescence or amperometric detection is a method of choice (Figure 3). Coupling these detectors can provide valuable qualitative as well as quantitative information if detector response ratios are observed (2,9). The analysis of plant growth substances by GC requires (with the obvious exception of ethylene) derivatization. However, all of the hormones are amenable to common derivatization schemes such as silylation, alkylation, and acetylation. Initially, GC separations of plant hormones were achieved with packed columns. Since 1980 most GC hormone separations have used high-resolution capillary columns. Detedors for GC, such as the flame ionization detector, suffer from a lack of selectivity, placing them under the same rigorous requirements for sample cleanup as the LC methods. The application of specific detectors has helped dramatically in this regard (2). One exception to this is the determination of ethylene. Ethylene is sampled from the gaseous atmosphere around plants and readily determined by packed-column GC with flame ionization detection. Since abcisic acid is naturally electronegative, metbylation renders this compound detectable by GC with electron capture (EC) detection. Other compounds such as the gibberellins (GA) require derivatization to volatile EC-active conjugates such as GA-perfluorobenzyl esters. The nitrogen atoms in the adenine ring of the cytokinins have been used to advantage in analyses of these hormones by GC coupled to either nitrogen-phosphorus or flame ionization detection. Agiant step forward in hormone research came in the late 19608 and early 19108 with the application of CC/ MS to the analysis and identification of plant hormones. This moved the field away from reliance on column retention times and bioactivity. Jake MacMillan and his colleagues a t the University of Bristol were pioneers in this approach and were instrumental in the interfacing of the computer to the mass spectrometer to facilitate structural analysis. Although their main area of emphasis was gibberel-
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643A
l i s , other researchers have explored the remaining hormone groups (3). More recently, GC/FT-IR has been used to detect and identify plant growth substances. Application of this technique can be very helpful in cases where mass spectral data leave some ambiguity in identification. This is commonly the case for the gibberellins and related comDounds that occur as many structural isomers (Figure 4). Both GCIMS and GCIFT-IR provide fundamental information for ;ompound identification that is not available from other detection methods. However, these methods are too expensive and time-consuming for routine multiple-sample analyses. Microbore HPLC has yet to make a significant impact on plant hormone research. As the importance of microsampling increases tn satisfy the need to understand hormonal regulation at the tissue level, microbore LC will be required for HPLC methods. In addition, LC/MS of phytohormones will be facilitated by microbore LC application. Other innovations in modern separation and detection technology will certainly continue to improve phytohormone methodology.
lmmunochemlcal methods The application of immunochemical methods to plant hormone analysis is
the most promising development to occur since the application of GC/MS to structure elucidation more than a decade ago. Radioimmunoassay (RIA) and enzyme immunoassay (EIA), which have long been valuable analytical tools for clinical research, can provide selectivity and sensitivity equal to or better than the best physicochemical methods. In addition, immunochemical methods permit the processing of large numbers of samples in a short period of time. The analysis of several hundred samples per day via RIA or EIA is well within the ability of these methods. The first step in the production of reagent antibodies is the preparation of a suitable antigen. Plant hormones are too small to elicit an immune response when introduced into an animal. They must first he conjugated to a large carrier molecule such as bovine serum albumin (BSA), lipopolysaccharide (LPS), or keyhole limpet hemocyanin (KLH). When this hormone carrier complex is injected into an animal, a fraction of the antihodies produced are against the hapten (hormone) located on the surface of the carrier. Figure 5 illustrates the sites of attachment that have been used for hormone carrier conjugation. The coupling of the hormone and the way it is presented to the immune system can
have a dramatic effect on the selectivity of the resultant antibody. Mertens et al. (IO)have shown that when ahscisic acid is conjugated through the C-1 carboxyl group, antibodies are produced that will recognize hoth the free and C-1 conjugate forms of the hormone. However, when abscisic acid is linked through the 4’ketone, antibodies are produced that will recognize only the free acid. The final orientation of hapten on the carrier surface is a factor to consider when designing the immunoassay selectivity. With the exception of ethylene, reagent antibodies have been successfully produced against all of the phytohormones. The vast majority of antihodies to date have been polyclonal (heterogeneous antihody population of varying selectivity and binding ahility). A few monoclonal8 (unique antibodies produced in culture from individual cell lines) are now being reported in the literature and a t meetings, some with amazing selectivity (10). Monoclonal antibody production is more difficult, laborious, and timeconsuming than polyclonal antibody production. The effort is worthwhile because of the ability to obtain a constant supply of well-defined antibody. In Table 11, several of the published ’ RIA and EIA methods are compared
Figure 4. Vapor phase IR spectrum of GA,methyl ester H e w l a Packard 5880/\: &lec+x-lW 1W98 FT-IR. 20-m gold-xaled lbht pipe at 250 OC: columnJBW D E I , 15 m X 0.25 mm i.d. (0.25-firn wating): iniecOC; own m m r e - ( g a d l e n l ) 50 “C to 280 OC at 20 OCImin: splless injwtlar: carrier g a d e at a hear velocihl of 25 CmlS. Specbun m u M y of Joan E. Nerner, lowa State Ulivaslty
tor temperahue-250
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ANALYTICAL CKMISTRY. VOL. 57, NO. 6, MAY 1985
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to physicochemicalapproaches. As can be seen, the immunoassays are comparable to, if not better than, the physieoehemical detection limits. In application the immunoassay techniques outperform the other approaches because of their ability to better handle sample volume and delivery. For example, an entire 100-pL extract can be examined by immunoassay, but only a few microliters can
be used for GC or GCMS analysis. This ultimately affects the original sample size requirementa for the analysis. Although with HPLC one can "trace enrich" plant hormones therehy making it possible to handle larger sample volumes, this approach is usually impractical without extensive sample cleanup. One must remember that plant hormones are found in 1ooO-fold less concentration compared
-
Table 11. Comparison of typical detection limits for physicochemical assays and immunoassays Halmcfm
Indole acetic acid
u*hoo
LCIF LClEC EIA
Abscisic acid
GClEC RIA
Cytokinins
GClMS
RIA
Gibberellins
GClMS
EIA
D.trtlon H I H ( m w
0.6 x
3 x 10-13 2 x 10-1.
4 9 11
4 x 10-19 2 x 10-1'
4 10
3x 3x
4 12
10-14
lo-'? 10-14
3 x 10-12 I x 10-15
4
13
LCIF-liquid chromat&~aphywith f l u a e s a m n t deteotion:LCIEC-liquid chromatography wltl etaclrtrochemical detection;EIA-enzyme immunassay;QClECgaS chomamgraphywlth el= W o n Capture detection: RIA--rBdioimunmssay: GCIMS-gas ChMnamgaPhy wlth m858 spec tmmeby.Adapted from Reference 14
646A
ANALYTICAL CHEMISTRY, VOL. 57. NO. 6. MAY I985
(min)
lure 6. Reversed-phase separation of soybean testa extract (a) Separation of onehan of soybean testa exbact. Blophaps RP-8: solvenl A-water (pH 3.0): solvent &aoemnilrile; &lent-0 to 50% B in 30 min; flow rata 1.0 mlmin. (b) separatiar d second half of s o w n testa axtract followinglmmuroatflnnypu rificatIon.lmmunoafllnny column prepared w h mizeatin ribosidemIi88Ta. Condllions BS in (a)
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647 A
to other secondary metabolites present in the plant extract. As mentioned earlier, the selectivity of the antibody translates directly into speed of analysis (Le., the more selective the antibody, the less sample manipulation prior to analysis). Although some of the antibodies that have been reported are selective enough to discriminate among compounds of similar structure in a crude extract, for many hormones this may not he practical. Consider the case of gibberellins: More than 60 gibberellins have been identified, all of which differ only in varying degrees of oxidation of the gihbane skeleton. To produce antibodies unique to each gibberellin would be a herculean task. A more realistic approach may be to raise antibodies of defined cross-reactivity that will he applicable to the majority of gibberellins present in plants. These antibodies would he used in conjunction with a simple separation step (HPLC). The rigorous cleanup requirements previously needed would he reduced because the marriage of the antibody selectivity with column separation would be quite powerful. Although not as fast as crude extract analysis, this would still represent a significant increase in sample processing compared to current procedures. Morris and his colleagues have used this approach in an HPLC/RIA procedure for cytokinins (1.5). Using antibodies that recognized zeatin- and isopentenyl-type cytokinins, they were able to rapidly measure multiple cytokinins from plant and bacterial sources in the picogram range. Alternatively one could use immunoaffinity columns of immohilized cross-reactive antisera prior to a physicochemical method. These columns would act as selective chemical filters passing all of the unwanted chemical “noise” while retaining the compounds of interest. Immunoaffinity columns have been used for a number of years for large molecule isolation, but they are still somewhat novel for small-molecule adsorption. Figure ti illustrates the cleanup possible using immunoaffinity columns. The crude extract injected directly onto the HPLC is impossihle to interpret or quantitate. The cytokinin in the sample is easily measured after cleanup on the column. lmmunoaffinity columns can also he used to separate optical isomers. Future applications of immunological methods to hormone physiology include cytolocalization (16) and identification of hormone receptor sites (17),
(2) Brenner, M. L. Ann. Re”. Plant Physio/. 198l.32,511. (3) Hillman, J. R., Ed. “Isolation of Plant Growth Substances”; Society for Experimental Riology, Seminar Series 4; Camhridge University Press: Cambridge, England, 1978. (4) Reeve. D. R.; Crozier, A. ‘‘Molecular and Sub-cellular Aspects of Hormonal Regulation in Plants”;Springer-Verlag: Berlin. 1980; Vol. I, p. 1. (5) Weiler, E. W. Riochem. SOC.Trans. l9H3, 11, 485. (6) Carnes, M. G.; Brenner, M. L.;Andersen, C. R. “Plant Growth Substances”; Hirokawa Publishing Company: Tokyo, 1974; p. 99. (7) Randurski, R. S.; Schultze, A. Plant Physiol. 1974,54, 257.
(8) Went, F. R e d . Tmu. Rot. N e e d 1928, 25,l. (9) Sweetser, P.B.; Swartzfager, D. G. Plant Physiol. 1978,61.254. (10) Mertens. A.; Deus-Neumann, R.; Weiler, E. W. FEHSLett. 1983,160, 269. (11) Weiler. E. W.; Jourdan, P. S.; Conrad, W. Plonto 1981, 153, 561. (12)Weiler, E. W. Planto 1980. 149.155. (13) Atzom. H.;Weiler, E. W. I’lonta 19113,
Greg Davis received a RS i n chemist r y from Southeast Missouri State University in 1975 and a PhD i n analytical chemistry f r o m Purdue University i n 1980. U p o n completinn of his doctoral work, h e joined Monsanto i n S t . Louis and since 1984 has been a research group leader i n the Plant Sciences Division. His research interests include plant hormone physiology, immunological methods, and bioseparations.
Immunoassay Division. I n 1983, he joined Monsanto’s Plant Sciences Division where he has been applying immunological techniques to plant hormone analysis.
Mich Hein received a RS i n botany from Ohio University and a n M S and PhD i n plant physiology from t h e University of Minnesota. He has worked i n the Plant Sciences Division of t h e Monsanto Agricultural Products Company since 1.982and is presently a project leader i n the Cellular and Molecular Biology Group. His research interests include separation science and plant reproductive biology. Rrian Neely received his R A i n biology from t h e University of Missouri i n 1975. He worked for four years i n
References (1) Rrenner, M. L. “Plant Growth Suhstances”; ACS Washington, D.C., 1979 Vol. 111, p. 212. 648A
ANALYTICAL CHEMISTRY, VOL. 57. NO. 6 . MAY 1985
159.7.
Physiol. Plant. 19H2.54, 230. (15) MacDanald, E. M. S.;Akiyoshi, D. E.; Morris,R.O. J.Chmmotugr.1981.2/4,101. (16) Zavala. M. E.; Rrandon, D. I,.J . Cell Rid. 1983,97, 1235. (17) .Jacobs, M.; Gilhert, S.F. Science 1983,22fI, 1297. (14) Weiler, E. W.
Ray Sharp received his RS i n chemist r y from the Uniuersity of MissnuriS t . Louis i n 1970. He worked as a research chemist for Sigma Chemical Company for two years and for Mallinckrodt’s Immunoassay Division for 10 years, Since joining the Plant Sciences Division i n 1983, his research has been directed toward deuelopment of immunological techniques for plant hormone analysis. Michael Carnes jnined Monsanto’s Agricultural Research Division i n 1974 after receiving his PhD i n plant physiologyfrom the University of Minnesota. He also holds a RS i n bota n y from Oklahoma University (1966) and a n M S in botany and plant pathology from Oklahoma State University (1969).Since 1982, when he was appointed Science Fellow, his research interests have centered on
