Planta 9 by Springer-Verlag 1979
Planta 144, 255-263 (1979)
Radioimmunoassay for the Determination of Free and Conjugated Abscisic Acid* Elmar W. Weiler Lehrstuhl ftir Pflanzenphysiologie,Ruhr-UniversitfitBochum, Postfach 102148, D-4630 Bochum, Federal Republic of Germany
Abstract. The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum a l b u m i n - ( i ) - A B A conjugate have a high affinity for ABA (Ka= 1.3 x 109 1 11101-1). Trans, t r a n s - A B A and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25 x 10 12 tool ABA, the measuring range extends to 20• 10 12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi. Key words: Abscisic acid noassay
Persea -
Radioimmu-
Introduction
The plant hormone abscisic acid (ABA) is known to be involved in many physiological processes such as: dormancy; abscission; growth inhibition; stomatal closure; and stress defense although many functional aspects still remain and are subject for controversial discussion (for reviews, see D6rffling, 1972, 1976; Milborrow, 1974; Jones, 1978). * Part 7 in the Series: "Use of Immunoassayin Plant Science" Abbreviations." ABA= abscisic acid; BHT=2.6-di-t-butyl-4-methyl phenol; TLC=thin-layer chromatography; HSA=human serum albumin.
The quantitative measurement of the endogeneous levels of abscisic acid is extremely difficult because of its instability and the low concentration of the hormone in plants (in the ng/g fresh weight range). For the determination and quantitation of abscisic acid, several methods including bioassays and chromatographic procedures are currently employed. At present, it is possible to detect about 0.5 I~g mlof ABA by optical rotatory dispersion (Milborrow, 1967). UV-spectroscopy permits the detection of 1 3 ~tg (D6rffling, 1970) whereas chromatography, alone or in combination with mass spectrometry, is being used by several groups. ABA is measured either as its methyl ester (Gaskin and Macmillan, 1968; Seeley and Powell, t970) or is converted to a volatile trimethyl silyl derivative (Davis and Addicott, 1972) and detected with a flame ionization detector (Lenton et al., 1971) or by electron capture (Seeley and Powell, 1970). Whereas 10-100 ng of ABA may be detected by flame ionization, the electron-capture detector is sensitive down to 10-100 pg. More recently, highpressure liquid chromatography has been used to quantitate ABA (Diiring and Bachmann, 1975; Durley et al., 1977; Quebedaux et al., 1976). This method has a detection limit of 1-2 ng. All of these analytical techniques require prior preparation of highly purified extracts which are achieved by one or more differential solvent extractions in addition to at least one chromatographic step, and often a derivative synthesis (Milborrow, 1967; Milborrow and Mallaby, 1975; Zevaart, 1974). Because of their lack of specificity, the same degree of purification is required for all known ABAbioassays (Milborrow, 1974; Tillberg, 1975). The sensitivity of biotests is between 10-6 to 10-11 M and about 100-200 ng ml- 1 of ABA may be detected (Milborrow, 1974; Sivori etal., 1971; Tucker and Mansfield, 1974; Tanada, 1967). The most sensitive
0032-0935/78/0144/0255/$01.80
256
(down to 10-11 M), although not specific, biotest today is the Lemna growth bioassay, as described by Tillberg (1975). 5 10 ng of ABA produces about 50% abscission in the cotton explant bioassay. The wheat coleoptile assay requires about 200-300 ng ml- 1 of ABA for 50% growth inhibition (Milborrow, 1967, 1974). In order to overcome some of the limitations of the currently available methods the possibilities of producing antibodies specific for ABA and of measuring this hormone by radioimmunoassay, in unpurified or only partly purified plant extracts, was investigated. Radioimmunoassay combines the unique specificity of the antigen-antibody reaction with the sensitivity of a trace determination of radioactivity. For many low and high molecular weight compounds it provides the most sensitive analytical method available (Eckert, 1976; Landon and Moffat, 1976), with detection limits between 1 0 - 1 2 - 1 0 - 15 mol of antigen. An immunological assay, involving precipitating measurements of antibody complexes, has been reported for ABA (Fuchs et al., 1972) but has proven to be insensitive. Therefore, no data on its specificity were given. A radioimmunological assay has been reported in preliminary from (Walton and Galson, 1977) but no details on the procedure or its use have yet been given. This paper describes the production and characterization of anti-ABA antibodies with high specificity and the development of a radioimmunoassay of sufficient sensitivity to allow the detection and quantitation of ABA in unpurified plant extracts. The assay described here proves to be one of the most sensitive and accurate known for ABA. Since no purification steps are involved, large numbers of samples can be assayed per day. In addition, ABA levels have been measured in different plants and plant organs, and a brief taxonomic survey on the occurrence of immunoassayable material has been done.
Materials and Methods
E.W. Weiler : Determination of Free and Conjugated Abscisic Acid cocktail was PCS (Amersham-Searle). All other solvents used were of highest purity available. Automated radioimmunoassays were performed as described previously (Weiler and Zenk, 1976). Radioactivity determinations were made in a p-scintillation counter (Berthold) with punched tape output. The calculations were done on a programmable desktop computer (HP 9825, Hewlett-Packard), using the spline-approximation method (Marschner et al., 1974) for genera!ion of standard curves.
Preparations of ABA Derivatives and Plant Pigments Methyl-abscisate was prepared by combining ( + ) - A B A with etheral diazomethane and was purified with TLC (solvent system: toluene/ethyl-acetate/HAc= 75:22:3 (v/v), Rf = 0.39, ABA: Rf = 0.25, chromatographic purity: > 99%). ( _+)-Abscisyl-(2,3,4,6-0-tetraacetyl)-/LD-glucose ester was synthesized according to Lehmann and Schfitte (1977) and purified with TLC of the ethylacetate fraction after ABA had been removed by extraction with aqueous NaHCO3 (solvent system: toluene/ethylacetate/HAc=25:15:2 (v/v), P~.=0.38, ABA: Rr=0.44 ). The compound was further characterized by m.p. (70 75 ~ C, lit. 75 ~ C) and the mass spectrum was in accordance with that given by Lehmann and Schiitte (1977). The cis-diol of methyl-abscisate was prepared by a sodium borohydride treatment and purified and identified with TLC according to the protocol given by Cornforth et al. (1967). Dihydrophaseic acid was prepared by the sodium-borohydride reduction of phaseic acid. Violaxanthin and other plant pigments were prepared from leaf extracts of Spinacea oleracea, as described by Hager and Meyer-Bertenrath (1966), and identified by Rf-values and UV-data (Hager and Meyer-Bertenrath, 1967).
Extraction of Plant Material All extractions were carried out in dim, indirect light. Extracts were stored at 4 ~ in the dark and analyzed within 2d.
Extraction procedure a) : Tissues were frozen in liquid nitrogen, powdered, and then 50 ml of 90% MeOH, containing 10 rag/1 2,6di-t-butyl-4-methyl phenol (BHT) (Milborrow and Mallaby, 1975), were added per 5 g fresh weight. After extraction at 4 ~ C for 48 h and then filtration, the methanol was evaporated under vacuum (30 ~ C) to one-tenth volume. The aqueous phase was then cleared by centrifugation and diluted with water to give a 25 ml per 200 ml original-extraction solvent. Suitable dilutions were immunoassayed. Extracts were done in duplicate. For recovery analysis, 2 gg of (_+)-ABA per 5 g plant material was added to one sample at the brei stage, or to extracts prior to dilution.
Plant Material
Extraction procedure b): 100 300 mg of finely-shredded, fresh
Ripe avocado fruits (Persea gratissima Gaertn.) were purchased from commercial sources. All other plant material was collected from the greenhouses of the University Botanical Garden and extracted immediately. Except when otherwise stated, leaf samples were assayed. Fungal material was obtained from liquid cultures. Spirulina ptatensis, a gift from Dr. Soeder, Dortmund, was stored frozen.
plant material was extracted with 5 ml of the MeOH/BHT solution for 48 h at 4 ~ C, with intermittant stirring. After decanting and adjusting the volume to 5 ml, an aliquot of the extracts was diluted (five times) with water and then immunoassayed. 50 ng of (_+)ABA was added at the beginning of the extraction to duplicate tubes as an internal standard.
Thin Layer Chromatography Chemicals and Equipment (+_)-Abscisic acid (ABA) and the mixed isomers of ABA and farnesol were obtained from Sigma. [SH]( +_)-ABA was purchased from The Radiochemical Center, Amersham (TRQ 1263, Spec. Act.22.5 Ci/mmol). Human serum albumin (HSA) was obtained from Serva, and bovine serum was supplied by Mediapharm. The scintillation
To test for the distribution of immunoreactive material on TLC plates, the ether-soluble fraction of leaf material was prepared from an acidified (pH 2.5) aqueous extract. The extracts were applied as a narrow band of 10 cm length on silica gel plates (0.25 mm), developed, and immediately after drying, cut into 0.5 cm strips which were eluted overnight in 1 ml of MeOH. After
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid being diluted ten times with the assay buffer, 0.6 ml aliquots were immunoassayed. In order to minimize background, it proved necessary to use plates which had been developed at least twice with the M e O H / B H T solvent. A B A marker spots were included on the plates, but a 3 4 mln band of silica gel was removed between A B A and the extracts, because it was found that considerable a m o u n t s of marker A B A diffused into the extract regions, if this precaution was not taken.
Immunoassay Reagents For all radioimmunoassay work, an 0.2 M acetate/NaOH buffer pH 4.0 was used and the bovine serum and ammonium-sulfate solutions were prepared as previously described (Weiler and Zenk, 1976). Just prior to use, [3H](_+)-ABA was diluted stepwise with water from a stock solution of 10 gCi/ml. Standards were stored under N2 at 4 ~ in the dark. Although prepared freshly each day, it was found that these solutions were stable for at least 14 days under the conditions employed.
257
Results
Assay Conditions and Properties of Antiserum All the immunized animals produced antibodies which strongly bound [3H](+)-ABA, but in this study, pooled serum from only one animal was used. The titre of the antiserum (that dilution which bound 30% of 0.75 pmol [3H](_+)-ABA under normal assay conditions) was 1:1,350. From a Scatchard-analysis (Scatchard, 1949) of standard curve data (cf. Fig. 1), a maximum affinity constant of K a = 1.3 x 109 I/tool was calculated.
Reaction Parameters
Coupling of ( +_)-ABA to Human Serum Albumine (HSA) To a solution of 250 mg H S A in water (pH 8.5), 132 mg ( + ) - A B A , dissolved in 3 ml of H20/dimethyl formamide (1:2) was added dropwise and the p H adjusted to 8 with 1M NaOH. 210 m g of N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride was added in 4 portions over a period of 90 min and the solution was stirred in the dark for 19 h at 4 ~ C. The solution was then dialyzed against tap water for 4 days and aliquots of the conjugate were stored frozen.
Preparation of Anti-ABA Antisera The conjugate which was emulsified in a 1 : 1 mixture of phosphate buffer p H 7.4 and Freund's complete adjuvant (Difco), was injected into 12 14 week old rabbits. After an intradermal preimmunization, intramuscular booster injections were given monthly and blood was collected both one and two weeks later. Antisera were stored frozen at - 1 8 ~ C.
Radioimmunoassay All incubation steps were carried out in the dark and pipetting was done in dim light. Standards were assayed in tripIicate and samples in either duplicate or triplicate. 0.1 ml of the sample or standard solution was pipetted into 12 • 63 m m glass tubes and 0 . 5 m l buffer added. 0.1 ml bovine serum (l:10diluted) was added as the carrier protein and 0.1 ml dilute [3H](_+)-ABA as the tracer (0.75 pmol). After mixing, 0.1 mI of diIuted antiserum was added and the tubes mixed again. For the determination of unspecific binding, water was used instead of antiserum. The tubes were incubated for 90 min at 4 ~ C and then 1.2 ml of a m m o n i u m sulfate solution were added. Precipitation was allowed to occur for 30 rain at r o o m temperature (23 ~ C) and, after centrifuging and decanting, the pellets were washed once with 1 ml of halfsaturated a m m o n i u m sulfate and then recentrifuged. The washed pellets were dissolved in 0.15 ml of water. 1 ml of scintillation cocktail was added. After mixing, the tubes were inserted into modified scintillation vials and counted for 2 min.
Calculations All data were corrected for unspecific binding and the antigen response expressed as B/B 0 values (B = antibody radioactivity in the presence of unlabelled antigen, either standard or u n k n o w n ; B o = a n t i b o d y radioactivity in the absence of unlabelled antigen), or as the logit B/B o = I n [B/Bo/100- B/B0]. C p m values were used t h r o u g h o u t and counting efficiencies were 19.6% + 1.8% for standards and 1 9 . 1 % • 1.1% for samples.
Maximum binding of [3H](+)-ABA to the antibodies was observed between pH 3.5 and 4.5. When the pH was increased to 6.5 binding decreased and remained constant up to pH 10. Thus, the protonated form of the carboxyl group of ABA was required for optimal reactivity. At 4 ~ C, binding was markedly higher (16%) than at room temperature (23 ~ C), and from the association kinetics it was found that reaction equilibrium was achieved after 90 min. Ammonium sulfate precipitation curves of the antibody complexes showed a broad optimum from 50-60% saturation and 52% was used for subsequent assays. [3H](• did not precipitate at any saturation tested (1 70%) and unspecific binding values were in the range of 1-2%. The ammonium sulfate technique is not time dependent (Farr, 1958) and therefore introduces no variation into the assay, even if large numbers of samples have to be processed. Solvent effects on antibodybinding ability did not occur with up to 20% MeOH and/or 20/ag of BHT in the sample volume.
Assay Sensitivity Figure 1 shows the standard curve for (_)-ABA in the (sigmoid) B/B o versus log ABA and the (linearized) logit B/Bo versus log ABA plots. The linearity of the latter also shows that reaction equilibrium has been reached within the incubation time. The detection limit, defined as the smallest amount of ABA which can be distinguished at the 99.5% confidence limit, is 0 . 2 5 • (66pg). 1 . 9 x 1 0 - 1 2 m o l (0.49 ng) of ABA gives 50% inhibition of binding. The measuring range (range of linearity of logit-plot) is from 0.4 x 10 -12 to 20x 10- 12 mol (0.1-5 ng). This radioimmunoassay can be included among the most sensitive assay methods yet developed for ABA. The standard curve is highly reproducible (cf. Fig. 1). Intra-assay variabilities of standard B/B o
258
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid i
80
~_
,--,1-
enD. -g
.-'.60
/,0
-.
20 ~
(• 1'o
acid[pmol]
'
1'0
16o
,600
('1- abscisic acid [pmol]
Assay Specificity
Table 1. The specificity of antiserum to abscisic acid Compound
pmol required to yield B/Bo = 50%
Cross reactivity (%)
I
(_+)-abscisic acid
1.75
100
II
(_+)-abscisyt-(2, 3, 4, 60-tetraacetyl)B-D-glucose ester
1.70
103
III
methyl-( + )-abscisate
0.96
182
I+IV
1:1 mixture of cis-transand trans-trans-abscisic acid
3.45
47
V
methyl-( _+)-abscisatecis-diol
VI VII VIII
vomifoliol
IX X
268
0.60
phaseic acid
1,180
0.16
dihydrophaseic acid
1,525
0.12
> 10,000"
0
xanthoxin
> 2,500"
0
2-cis-epoxyfi-ionylideneacetic acid
>> 4,000"
0
XI
~-ionylideneacetic acid
> 4,000 a
XII
lunularic acid
>> 4,000"
XIII
violaxanthin
>> 3,300 a
XIV
farnesoI, mixed isomers
> 10,000"
"
Fig. 1. Standard curve for abscisic acid in a sigmoid and a linear plot. = mean _+ S.D. of n = 16 consecutive assays
Highest concentration assayed
values, expressed as coefficients of variation, were 0.9% to 4% throughout the measuring range. Interassay variabilities (n = 16) ranged from 4-9% between 0.4-4 pmol ABA and 13-20% from 4 ~ 0 pmol. The analytical recovery was tested by including triplicates of the same standard solution in 16 consecutive assays (added: 0.7 ng, found: 0.73 ng_+0.17 rig).
Cross reactivities of structurally or physiologically related compounds were determined by comparing the concentration required to yield B / B o = 5 0 % to the value obtained for (+)-ABA. The data given in Table 1 (cf. Fig. 2) show the specific binding of abscisic acid in its free or esterified forms. The acetylated glucose ester exhibits the same reactivity as free ABA, and methyl-( +_)-abscisate cross reacts at 182%. Thus the formation of methyl-abscisate, which may occur as an extraction artifact, can be avoided by including acid in the extraction mixture or by using acetone (Milborrow and Mallaby, 1975). Slight changes in any part of the ABA molecule other than the carboxyl group result in a nearly complete loss of reactivity. Since a 1:1 mixture of the cis,trans- and trans,trans-isomers of ABA cross reacts only 47%, it is apparent that trans,trans-ABA does not react with the antibody. The importance of the correct side chain for antibody binding is further stressed by the absence of reactivity with vomifoliol (VIII), a molecule differing from ABA only in the length of the side chain. Xanthoxin (IX), phaseic acid (VI) and dihydrophaseic acid (VII) did not cross react significantly, nor did the synthetic derivatives (X) and (XI). Reduction of the keto group also leads to a severely reduced binding affinity (0.6% for the cis-diol ofABA). Violaxanthin did not inhibit binding of [3H](_+)-ABA at any concentration tested nor did neoxanthin, antheraxanthin, lutein, zeaxanthin, the carotenoids or the chlorophylls. To test further antibody specificity, the distribution of immunoreactive material on chromatograms of plant extracts was tested. When raw methanolic extracts were applied, the plates had to be heavily loaded to detect the ABA-related compounds, however, a clear separation could not be obtained. In
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid
02H
~ Ill R=H (II) R=[3-D-GLc(Ar )/. [Ill) R=CH3
~C02Mr HO
259
(tl-ABAHHtrans'ABA Nicotiana tabacum extract
(lVJ
100.
< 50-
0~C02 H (Vl)
(vj
i §
(t)-ABA~. Htrans-ABA
OH Ho~CO2H
0~ O H
o
H
HO~-'~CHO
o.,~
0.6
6.8
1'.o
Rf Fig. 3. Distribution of immunoreactive material on a thin-layer c h r o m a t o g r a m of an ether-soluble fraction of an acidified Nicotiana tabaeum leaf extract. Plate: silica gel 0.25 ram; Solvent system: toluene/ethyl acetate/HAc = 25 : 15: 3 (v/v)
(VIII)
(VIII
o,2
~ ' ~ C 02H
(IXl
(X)
~COzH (Xl)
CO~zH OH (XII)
one immunoreactive band was found in this crude fraction and it corresponded to marker ABA (Rf= 0.6). From this chromatogram, the amount of ABA in the leaves was calculated as being 190 ng/g fresh weight. This value compared favorably with the 210 ng/g fresh weight value, determined by directly immunoassaying the crude methanolic extract.
OH
X X X cH'~
Assay Variability and Recovery (XIVl
{XIIIJ
Fig. 2. C o m p o u n d s assayed for cross reactivity with anti-ABA antiserum
some extracts, a second small peak appeared at the origin and most likely represented ABA-glucose ester. Figure 3 shows the distribution of immunoreactive material on a chromatogram of the ether-soluble acid fraction of a Nicotiana tabacum leaf extract. Only
The average coefficient of variation of double determinations was 9% in the standard curve range from 0-0.5 ng and 10% in the range from 0.5-5 ng ABA. If desired, the variability could be further reduced by correcting each determination for counting efficiency. Recovery of (+)-ABA added to methanolic plant extracts prior to dilution was 104% when added prior
Table 2. Distribution of A B A in the fruit of Persea gratissima (avocado). Extraction procedure (a) was used. fr.wt. = f r e s h weight, dr.wt. = dry weight Part of fruit
Total fr.wt. (g)
A m o u n t of A B A present
A B A level
(nmol)
(pmol g z fr.wt.)
(% of totat)
Literature value" (rag kg ~ fr.wt.) (mg kg - I fr.wt.) ORD
peel green pulp yellow pulp
testa embryo
a
Milborrow, 1967
23 90 56
0.57 23
18.7 16.0 12.2
0.19 4.2
36.5 31.3 23.7
0.36 8.1
820 178 216
0.216 0.047 0.057
pericarpmean:
0.073
342 178
0.090 0.047
seed mean:
0.049
racemate dilution
0.76
0.034
0.11
260
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid
Table 3. Distribution of abscisic acid in some plant families. Leaves were assayed except for some cases, where leaves and shoots (1) or whole plants (2) were analyzed. (3)=aquatic plant, growing submerged; (4)= aquatic plant, emerged leaves analyzed; (5)= aquatic plant, submerged leaves analyzed; (6)=succulent; n.d.= not detected. Extraction procedure (b) was used. fr.wt.=fresh weight; dr.wt.=dry weight Family
Species
ABA level -
Cyanophyceae: Oscillatoriaceae
Remarks
n.d.
Gibberella fujikuroi
n.d.
n.d.
Hyphomycetes
Curvularia lunata
n.d.
n.d.
Hepaticae : Marchantiaceae
Marchantia polymorpha n.d.
n.d.
Lycopodium squarrosum 1.16 Selaginella uncinnata n.d. Isogtes matogrossense 0.25
7.4 n.d. 3.7
(1) (1) (4)
Equisetum arvense
0.36
3.2
(1)
Asplenium dimorphum Asplenium adiantum nigrum Polypodium heimionitis Woodwardia radicans Bolbitis heudelotii Azolla mexicana Salvinia auriculata Marsilea hirsuta Pilularia globulifera Ceratopteris malictroides Ceratopteris malictroides
n.d. 0.25
n.d. 1.0
0.58
3.2
0.65 0.73 0.27 0.26 0.82 0.48 0.25
3.0 6.3 4.5 4.8 2.5 3.6 4.2
0.50
6.4
Ascomycetes : Fungi imperfecti."
Equisetaceae:
Equisetaceae Filicatae: Polypodiaceae
Salviniaceae Marsileaceae Parkeriaceae
Gymnospermae : Pinaceae
Cupressaceae
Podocarpaceae
Picea sitchensis Pinus halepensis Pinus pinea Pinus radiata Cupressus sempervirens Juniperus bermudiana Podocarpus macrophyllus
Angiospermae, Dicotyledonae : Lauraceae Persea americana Apollonias barbujana Cinnamonum ceylanicum Laurus nobilis Nymphaeaceae Cabomba sp. Nymphaea caerulea Nymphaea lutea Nymphaea lutea
0.78 1.02 0.52 0.80 1.10
4.5 2.7 1.5 2.1 2.6
0.95 0.99
2.6 2.2
0.87 1.70 1.10
2.8 3.7 2.6
1.00 n.d. 0.58 0.71 0.59
2.1 n.d. 4.0 3.8 5.7
ABA level 7
n.d.
Lycopodiatae : Lycopodiaceae Selaginellaceae Iso~taceae
Species
7
Spirulina platensis
Hypocreaceae
Family
(3) (2), (4) (4) (4) (1), (3) (1), (4)
(3) (4) (4) (5)
Ceratophyllaceae Ceratophyllum sp. Papaver somniferum Papaveraceae Fagaceae Castanea sativa Cyclobalanopsis glauca Quercus ilex Dorstenia urceolata Moraceae Crassulaceae Echeveria pringlei Kalanchoe velutina Sedum palmeri Prunus ilicifolia Rosaceae Mimosa pudica Mimosaceae Caesalpina spinosa Caesalpinaceae Drosophyllum Droseraceae lusitanicum Myriophyllum Haloragaceae brasiliense Myrtus communis Myrtaceae Rotala rotundifolia Lythraceae Oxalis articulata Oxalidaceae Pelargonium articulata Geraniaceae Aquifoliaceae Ilex aquifolium Euphorbia pteroneura Euphorbiaceae Euphorbia millii Euphorbia tirucalli Phyllanthus fluitans Hydrocotyle Umbellifereae bonariensis Begonia glabra Begoniaceae Carica quercifolia Caricaceae Arbutus unedo Ericaceae Dracophyllum Epacridaceae secundum Manilkara zapota Sapotaceae Cyclamen Primulaceae mederifolium Samolus floribundus Menyanthaceae Nymphoides humboldtianum Catharanthus roseus Apocynanceae Nerium oleander Rauwolfia vomitoria Pachypodium lamerei Plumbaginaceae Limonium fruticans Viburnum tinus Caprifoliaceae Symphoricarpus foetidus Pterocephalus Dipsacaceae dementorum Phillyrea latifolia Oleaceae Jasminium simplicifolium Datara arborea Solanaceae Datura sanguinea Solanum laciniatum Digitalis lanata ScrophulariIsoptexis canariensis aceae Phygelius capensis
Remarks
7
n.d. 0.60 0.35 0.91 0.69 0.57 0.11 0.14 0.15 0.80 1.20 1.10 0.92
n.d. 5.5 0.8 1.7 1.4 2.0 2.5 4.1 3.4 2.2 4.2 3.9 7.3
(1), (3)
0.92
9.1
(l), (3)
0.83 0.66 0.80 0.17 0.82 0.28 0.45 0.37 0.39 0.25
1.9 6.2 6.4 1.8 1.7 2.7 2.8 3.0 3.2 1.9
0.23 0.39 0.51 0.91
3.6 2.6 1.5 1.7
0.86 0.29
2.0 3.2
0.15 0.39
2.0 6.3
0.56 0.60 0.30 0.60 0.08 1.30 0.60
3.8 1.5 1.9 5.1 0.4 3.0 7.2
0.38
2.5
1.30 1.00
2.5 3.0
1.00 1.10 1.80 0.90 0.85 0.60
7.8 7.8 10.2 4.3 3.0 3.3
(6) (6) (6)
(1), (3)
(1), (6) (l) (1), (6) (4)
(4)
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid Table 3 (continued) Family
Species
Determination of ABA in Plant Extracts ABA level I
Lentibulariaceae Labiatae Verbenaceae
Aeschynanthus speciosus Utricularia vulgaris
Teucrium flavum Avicennia marina Durantha repens Campanulaceae Campanula rupestris Lobeliaceae Lobelia cardinalis Asteraceae Artemisia arborescens Centaurea canariensis Senecio cruentus
Remarks
I hi)
Gesneriaceae
0.20
2.4
0.10
1.9
1.00 3.4 0.87 2.9 0.85 3.8 1.20 9.3 0.67 10.2 0.85 3.7 0.96 6.1 0.37 3.6
.
(1), (3)
(3)
Angiospermae, Monocotyledonae: Alismataceae
Echinodorus cordifolius Sagittaria latifolia HydrocharitElodeacanadensis aceae Vallisneria gigantea Lil~aceae Aloe statuides Dracaena indivisa Sanseveria trifasciata Smilax aspera Nolinaceae Beaucarneastricta Agavaceae Yucca arizonica Orchidaceae Cymbidiumhybrida Phalaenopsis hybrida Bromeliaceae Guzmaniaminor Cyperaceae Cyperushaspan Cyperus textilis Commelinaceae Cochliostema odoratissimum Restioniaceae Leptocarpussimilis Poaceae Oryza sativa Arecaceae Chamaerops humilis Elaeis guineensis Araceae Anubias sp. Cryptocoryne aponogetifolia Cryptocoryne wendtii Lemnaceae Lemna minor
261
0.70
3.2
(4)
n.d. 0.16 0.34 0.17 0.91 0.23 0.60 0.49 0.32 1.20 0.70 0.52 1.00 0.32 0.24
n.d. 2.7 5.7 2.7 2.3 2.6 1.8 1.9 1.0 4.8 4.7 2.1 6.3 1.4 3.2
(3) (1), (3) (4) (3)
0.59 0.78 0.60 1.70 1.10 0.78
1.6 4.4 1.3 4.9 7.8 7.9
(3) (3)
0.51 0.14
8.5 2.0
(3) (2), (4)
(1) (1)
The distribution of ABA within the avocado fruit, which is frequently chosen for biosynthetic work on ABA (Noddle and Robinson, 1969; Milborrow and Robinson, 1973; Milborrow, I976), is shown in Table 2. The data obtained from the seed show a strong correlation to published values (Milborrow, 1967), however, the level of ABA in the pulp was much lower in our material. This might be due to differences in the ripeness or in the variety used. Of the total amount of ABA present in the fruit, about 90% is located in the pericarp, and 70% of this total is found in the green tissues. This is in close agreement with reports that ABA synthesis seems to be associated with functional chloroplasts (Railton etal., 1974; Milborrow, 1974). In another experiment, the distribution of immunoreactive material in more than 100 species of plants was investigated (Table 3). ABA was found in all higher plant families, but could not be detected in the 9 blue-green alga, Spirulinaplatensis, the liverwort Marchantia polymorpha, or the two fungi tested. In most plants, ABA levels were between 2 ~ nmol g-1 dry weight (0.5 1 mg k g - t ) , although as much as 1 0 n m o l g -1 ( 2 . 5 m g k g -1) were found in some species, namely the Solanaceae. There is, however, a much larger variation in ABA levels when expressed on a fresh weight basis. ABA was very low in succulent plants (0.1-0.3 nmol g- 1=0.025-0.075 mg k g - 1), whereas a number of non-succulent plants yielded values higher than 1 nmol g-1. A wide variation of ABA content was observed in aquatic plants which were grown submerged. In some species (e.g. Sagitta latifolia) ABA could not be detected. Milborrow and Robinson (1972) also report very low levels of ABA in fresh samples of Ceratophyllum demersum and Callitriche stagnalis (submerged parts).
Discussion
to extraction of the plant material. Recovery was 103% with a standard deviation of 28% between different plant materials. Therefore, all plant extracts were corrected for recovery. This correction also compensated for any isomerization to trans,trans-ABA which might have occured during extraction and assay. Extract dilution curves did not exactly parallel the standard curves. However, since parallelism could be restored by adding increasing amounts of (_+)ABA to the extracts, it is likely that the naturally occuring ( + ) - A B A differs in reactivity from the commercial ABA used for standardization.
The experimental data presented in this paper demonstrate that antibodies with high specificity for ABA and its esters have been obtained. These can be utilized in a radioimmunoassay which permits the direct quantitative estimation of this hormone between 0.4x 10-12 mol to 20x 10-12 tool in crude plant extracts. Xanthoxin, phaseic acid and vomifoliol may occur naturally in amounts comparable to ABA. Dihydrophaseic acid may occur in hundredfold higher amounts (Zeevaart, 1974; B6ttger, 1978; Tinelli et al., 1973). However, the assay is not affected by these structurally-related compounds at any concentration within the physiological range. In addition, neither
262
carotenoids, xanthophylls, nor physiologically related compounds (i.e. farnesol or lunularic acid) show any interference. Because of the identical reactivities of (+)-ABA and its tetraacetyl glucose ester, the sum of free and esterified ABA is calculated from standard curves. Since the acetylated glucose ester was the only derivative available for cross-reactivity studies, we assume that for the purpose of total ABA determination, the naturally occurring glucose ester does not differ significantly from this compound in its reactivity. However, if it is necessary to differentiate between free and esterified abscisic acid, this can be achieved by differential solvent extraction (Zeevaart, 1974; D6rffling, 1974). The antiserum used in this study was raised and standardized against (_+)-ABA, however, plant material contains only (+)-ABA. The fact that it was possible to inhibit completely the binding of [3H](+_)ABA by adding increasing amounts of plant extracts, and the linearity of these extract dilution curves in the logit/log plot showed that (+)-ABA also competes very well with antibody-bound [3H](-)-ABA. Thus, differences in reactivities of the ( + ) and ( - ) forms must be small. Since the glucose and methyl esters react equal to or better than the free acid, and since structural changes in any other part of the molecule result in almost total loss of immunoreactivity, it would appear that the entire ABA molecule, except for the carboxyl group, binds to the antibody. The taxonomic distribution of ABA and its levels, determined by this method, are in agreement with the data obtained by other analytical techniques (Pryce, 1970, 1971; Gorham, 1977). A wide range of species, selected from as many families as possible and grown under conditions prevailing in the greenhouse, were used for this experiment. The results demonstrate that this radioimmunoassay can be used to quantitate ABA in the most diverse plant material. Furthermore, the data presented in Table 3 strongly support the hypothesis (Pryce, 1971) that ABA may generally occur in higher plants, although at very low levels in some species. In addition, the measuring range of this assay covers the whole range of ABA levels found in plant material thus far examined. A broad survey, such as that presented in Tabel 3, has never been possible before using other analytical techniques. This demonstrates the potential of the RIA, which, in its semi-automated performance (Weiler and Zenk, 1976) permits the processing of several hundred samples per day. Thus a number of problems can now be approached efficiently, such as the monitoring of ABA levels and its precise distribution in plants or plant organs in relation to physiological
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid
state, stress response, and other factors. Other physiological studies using this assay method are currently in progress. Phaseic acid was a gift from Prof. Raschke, East Lansing. The xanthoxin was from Dr. B6ttger, Hamburg, and the vomifoliol was a gift from Dr. Kapil, Lucknow. Prof. Kindl, Marburg, provided lunularic acid, and Dr. Okita, Sendal, provided compounds (X) and (XI). The excellent technical assistance of Mrs. P. Westekemper is also acknowledged, as well as the proof reading of the manuscript by Prof.R. Mansell, Tampa.
References B6ttger, M.: The occurrence of cis, trans- and trans, trans-xanthoxin in pea roots. Z.Pflanzenphysiol. 86, 265-268 (1978) Cornforth, J.W., Draber, W., Milborrow, B.V., Ryback, G. : Absolute stereochemistry of (+)-abscisin II. Chem. Commun. 114-116 (1967) Davis, L.A., Addicott, F.T. : Abscisic acid: correlations with abscission and with development in the cotton fruit. Plant Physiol. 49, 644.648 (1972) D6rffling, K.: Quantitative Verfinderungen des Abscisins~inregehaltes w~ihrend der Fruchtentwicklung yon Solanum lycopersicure L. Planta 93, 233-242 (1970) D6rffiing, K.: Abscisinsfiure und Xanthoxin. Fortschr.Bot. 34, 190-192 (1972) D6rffling, K.: Abscisic acid and related compounds. Progr.Bot. 38, 153-156 (1976) D6rffling, K., Sonka, B., Tietz, D.: Variation and metabolism of abscisic acid in pea seedlings during and after water stress. Planta 121, 57~66 (1974) Dfiring, H., Bachmann, O. : Abscisic acid analysis in Vitis vinifera in the period of endogeneous bud dormancy by high pressure liquid chromatography. Physiol. Plant. 34, 201-203 (1975) Durley, R.C., Kannangara, T., Simpson, G.M. : Analysis of abscisins and 3-indolylacetic acid in leaves of Sorghum bicolor by high-performance liquid chromatography. Can.J.Bot. 56, 157 161 (1978) Eckert, H.G.: Die Technik des Radioimmunoassays. Angew. Chem. 88, 565 574 (1976) Farr, R.S. : A quantitative immunochemical measure of the primary interaction of I*BSA and antibody. J. Infect. Dis. 103, 239 262
(1958) Fuchs, Y., Mayak, S., Fuchs, S.: Detection and quantitative determination of abscisic acid by immunological assay. Planta 103, 117-125 (1972) Gaskin, P., Macmillan, J. : Plant hormones. VII. Identification and estimation of abscisic acid in a crude plant extract by combined gas chromatography - mass spectrometry. Phytochemistry 7, 1699 1701 (1968) Gorham, J. : Lunularic acid and related compounds in liverworts, algae and Hydrangea. Phytochemistry 16, 249-253 (1977) Hager, A., Meyer-Bertenrath, T. : Die Isolierung und quantitative Bestimmung der Carotinoide und Chlorophytle yon Blfittern, Algen und isolierten Chloroplasten mit Hilfe dfinnschichtchromatographischer Methoden. Planta 69, 198 217 (1966) Hager, A., Meyer-Bertenrath, T.: Die Identifizierung der an Dfinnschichten getrennten Carotinoide grfiner Blfitter und A1gen. Planta 76, 149-168 (1967) Jones, H.G. : How plants respond to stress. Nature 271, 610 (1978) Landon, J., Moffat, A.C. : The radioimmunoassay of drugs. Analyst 101, 225 243 (1976)
E.W. Weiler : Determination of Free and Conjugated Abscisic Acid Lehmann, H., Sch/itte, H.R.: Zur Darstellung O-substituierter Zuckerester. J. Prakt. Chem. 319, 117-122 (1977) Lenton, J.R., Perry, V.M., Saunders, P.F. : The identification and quantitative analysis of abscisic acid in plant extracts by gasliquid chromatography. Planta 96, 271-280 (1971) Marschner, I., Dobry, H., Erhardt, F., Landersdorfer, T., Popp, B., Ringel, C., Scriba, P.C.: Berechnung radioimmunologischer Mel3werte mittels Spline-Funktionen. Arztl. Lab. 20, 184-191 (1974) Milborrow, B.V.: The identification of (+)-abscisin II ((+)-dormin) in plants and measurement of its concentrations. Planta 76, 93 113 (1967) Milborrow, B.V. : The chemistry and physiology of abscisic acid. Ann.Rev. Plant Physiol. 25, 259 307 (1974) Milborrow, B.V., Mallaby, R. :Occurrence of methyl-( +)-abscisate as an artefact of extraction. J.Exp.Bot. 26, 741-748 (1975) Milborrow, B.V., Robinson, D.R.: Factors affecting the biosynthesis of abscisic acid. J.Exp.Bot. 24, 537 548 (1973) Noddle, R.C., Robinson, D.R.: Biosynthesis of abscisic acid: Incorporation of radioactivity from [2-14C]mevalonic acid by intact fruit. Biochem. J. 112, 547-548 (1969) Pryce, R.J. : Lunularic acid, a common endogenous growth inhibitor of liverworts. Planta 97, 354-357 (1971) Pryce, R.J. : The occurrence of lunularic and abscisic acid in plants. Phytochemistry 11, 1759-1761 (1972) Quebedaux, B., Sweetser, P.B., Rowell, J.C.: Abscisic acid in soybean reproductive structures during development. Plant Physiol. 58, 363 366 (1976) Railton, I.D., Reid, D.M., Gaskin, P., Macmillan, J. : Characterization of abscisic acid in chloroplasts of Pisum sativum L. cv.
263 Alaska by combined gas-chromatography-mass spectrometry. Planta 117, 179-182 (1974) Seeley, S.D., Powell, L.E.: Electron capture-gas chromatography for sensitive assay of abscisic acid. Anal.Biochem. 35, 530-533 (1970) Sivory, E.M., Sonvico, V., Fernandez, N.O.: Determination of abscisic acid following Paleg's method. Plant Cell Physiol. 12, 993-996 (1971) Tanada, T. : A rapid photoreversible response of barley root tips in the presence of 3-indoleacetic acid. Proc.Nat. Acad. Sci.USA 59, 376-380 (1968) Tillberg, E.: An abscisic acid-like substance in dry and soaked Phaseolus vulgaris seeds determined by the Lemna growth bioassay. Physiol. Plant. 34, 192-195 (1975) Tinelli, E.T., Sondheimer, E., Walton, D.C.: Metabolites of [2-14C]abscisic acid. Tetrahedron Lett. 2, 139-140 (1973) Tucker, D.J., Mansfield, T.A.: A simple bioassay for detecting "antitranspirant" activity of naturally occurring compounds such as abscisic acid. Planta 98, 157-163 (1971) Walton, D., Galson, E.: A radioimmunoassay for abscisic acid. Plant Physiol. 59, Suppl. 77 (1977) Weiler, E.W., Zenk, M.H. : Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. Phytochemistry 15, i537-1545 (1976) Zeevaart, J.A.D. : Levels of (+)-abscisic acid and xanthoxin in spinach under different environmental conditions. Plant Physiol. 53, 644-648 (1974)
Received 21 July; accepted 1 October 1978
Planta 144, 255-263 (1979)
Radioimmunoassay for the Determination of Free and Conjugated Abscisic Acid* Elmar W. Weiler Lehrstuhl ftir Pflanzenphysiologie,Ruhr-UniversitfitBochum, Postfach 102148, D-4630 Bochum, Federal Republic of Germany
Abstract. The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum a l b u m i n - ( i ) - A B A conjugate have a high affinity for ABA (Ka= 1.3 x 109 1 11101-1). Trans, t r a n s - A B A and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25 x 10 12 tool ABA, the measuring range extends to 20• 10 12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi. Key words: Abscisic acid noassay
Persea -
Radioimmu-
Introduction
The plant hormone abscisic acid (ABA) is known to be involved in many physiological processes such as: dormancy; abscission; growth inhibition; stomatal closure; and stress defense although many functional aspects still remain and are subject for controversial discussion (for reviews, see D6rffling, 1972, 1976; Milborrow, 1974; Jones, 1978). * Part 7 in the Series: "Use of Immunoassayin Plant Science" Abbreviations." ABA= abscisic acid; BHT=2.6-di-t-butyl-4-methyl phenol; TLC=thin-layer chromatography; HSA=human serum albumin.
The quantitative measurement of the endogeneous levels of abscisic acid is extremely difficult because of its instability and the low concentration of the hormone in plants (in the ng/g fresh weight range). For the determination and quantitation of abscisic acid, several methods including bioassays and chromatographic procedures are currently employed. At present, it is possible to detect about 0.5 I~g mlof ABA by optical rotatory dispersion (Milborrow, 1967). UV-spectroscopy permits the detection of 1 3 ~tg (D6rffling, 1970) whereas chromatography, alone or in combination with mass spectrometry, is being used by several groups. ABA is measured either as its methyl ester (Gaskin and Macmillan, 1968; Seeley and Powell, t970) or is converted to a volatile trimethyl silyl derivative (Davis and Addicott, 1972) and detected with a flame ionization detector (Lenton et al., 1971) or by electron capture (Seeley and Powell, 1970). Whereas 10-100 ng of ABA may be detected by flame ionization, the electron-capture detector is sensitive down to 10-100 pg. More recently, highpressure liquid chromatography has been used to quantitate ABA (Diiring and Bachmann, 1975; Durley et al., 1977; Quebedaux et al., 1976). This method has a detection limit of 1-2 ng. All of these analytical techniques require prior preparation of highly purified extracts which are achieved by one or more differential solvent extractions in addition to at least one chromatographic step, and often a derivative synthesis (Milborrow, 1967; Milborrow and Mallaby, 1975; Zevaart, 1974). Because of their lack of specificity, the same degree of purification is required for all known ABAbioassays (Milborrow, 1974; Tillberg, 1975). The sensitivity of biotests is between 10-6 to 10-11 M and about 100-200 ng ml- 1 of ABA may be detected (Milborrow, 1974; Sivori etal., 1971; Tucker and Mansfield, 1974; Tanada, 1967). The most sensitive
0032-0935/78/0144/0255/$01.80
256
(down to 10-11 M), although not specific, biotest today is the Lemna growth bioassay, as described by Tillberg (1975). 5 10 ng of ABA produces about 50% abscission in the cotton explant bioassay. The wheat coleoptile assay requires about 200-300 ng ml- 1 of ABA for 50% growth inhibition (Milborrow, 1967, 1974). In order to overcome some of the limitations of the currently available methods the possibilities of producing antibodies specific for ABA and of measuring this hormone by radioimmunoassay, in unpurified or only partly purified plant extracts, was investigated. Radioimmunoassay combines the unique specificity of the antigen-antibody reaction with the sensitivity of a trace determination of radioactivity. For many low and high molecular weight compounds it provides the most sensitive analytical method available (Eckert, 1976; Landon and Moffat, 1976), with detection limits between 1 0 - 1 2 - 1 0 - 15 mol of antigen. An immunological assay, involving precipitating measurements of antibody complexes, has been reported for ABA (Fuchs et al., 1972) but has proven to be insensitive. Therefore, no data on its specificity were given. A radioimmunological assay has been reported in preliminary from (Walton and Galson, 1977) but no details on the procedure or its use have yet been given. This paper describes the production and characterization of anti-ABA antibodies with high specificity and the development of a radioimmunoassay of sufficient sensitivity to allow the detection and quantitation of ABA in unpurified plant extracts. The assay described here proves to be one of the most sensitive and accurate known for ABA. Since no purification steps are involved, large numbers of samples can be assayed per day. In addition, ABA levels have been measured in different plants and plant organs, and a brief taxonomic survey on the occurrence of immunoassayable material has been done.
Materials and Methods
E.W. Weiler : Determination of Free and Conjugated Abscisic Acid cocktail was PCS (Amersham-Searle). All other solvents used were of highest purity available. Automated radioimmunoassays were performed as described previously (Weiler and Zenk, 1976). Radioactivity determinations were made in a p-scintillation counter (Berthold) with punched tape output. The calculations were done on a programmable desktop computer (HP 9825, Hewlett-Packard), using the spline-approximation method (Marschner et al., 1974) for genera!ion of standard curves.
Preparations of ABA Derivatives and Plant Pigments Methyl-abscisate was prepared by combining ( + ) - A B A with etheral diazomethane and was purified with TLC (solvent system: toluene/ethyl-acetate/HAc= 75:22:3 (v/v), Rf = 0.39, ABA: Rf = 0.25, chromatographic purity: > 99%). ( _+)-Abscisyl-(2,3,4,6-0-tetraacetyl)-/LD-glucose ester was synthesized according to Lehmann and Schfitte (1977) and purified with TLC of the ethylacetate fraction after ABA had been removed by extraction with aqueous NaHCO3 (solvent system: toluene/ethylacetate/HAc=25:15:2 (v/v), P~.=0.38, ABA: Rr=0.44 ). The compound was further characterized by m.p. (70 75 ~ C, lit. 75 ~ C) and the mass spectrum was in accordance with that given by Lehmann and Schiitte (1977). The cis-diol of methyl-abscisate was prepared by a sodium borohydride treatment and purified and identified with TLC according to the protocol given by Cornforth et al. (1967). Dihydrophaseic acid was prepared by the sodium-borohydride reduction of phaseic acid. Violaxanthin and other plant pigments were prepared from leaf extracts of Spinacea oleracea, as described by Hager and Meyer-Bertenrath (1966), and identified by Rf-values and UV-data (Hager and Meyer-Bertenrath, 1967).
Extraction of Plant Material All extractions were carried out in dim, indirect light. Extracts were stored at 4 ~ in the dark and analyzed within 2d.
Extraction procedure a) : Tissues were frozen in liquid nitrogen, powdered, and then 50 ml of 90% MeOH, containing 10 rag/1 2,6di-t-butyl-4-methyl phenol (BHT) (Milborrow and Mallaby, 1975), were added per 5 g fresh weight. After extraction at 4 ~ C for 48 h and then filtration, the methanol was evaporated under vacuum (30 ~ C) to one-tenth volume. The aqueous phase was then cleared by centrifugation and diluted with water to give a 25 ml per 200 ml original-extraction solvent. Suitable dilutions were immunoassayed. Extracts were done in duplicate. For recovery analysis, 2 gg of (_+)-ABA per 5 g plant material was added to one sample at the brei stage, or to extracts prior to dilution.
Plant Material
Extraction procedure b): 100 300 mg of finely-shredded, fresh
Ripe avocado fruits (Persea gratissima Gaertn.) were purchased from commercial sources. All other plant material was collected from the greenhouses of the University Botanical Garden and extracted immediately. Except when otherwise stated, leaf samples were assayed. Fungal material was obtained from liquid cultures. Spirulina ptatensis, a gift from Dr. Soeder, Dortmund, was stored frozen.
plant material was extracted with 5 ml of the MeOH/BHT solution for 48 h at 4 ~ C, with intermittant stirring. After decanting and adjusting the volume to 5 ml, an aliquot of the extracts was diluted (five times) with water and then immunoassayed. 50 ng of (_+)ABA was added at the beginning of the extraction to duplicate tubes as an internal standard.
Thin Layer Chromatography Chemicals and Equipment (+_)-Abscisic acid (ABA) and the mixed isomers of ABA and farnesol were obtained from Sigma. [SH]( +_)-ABA was purchased from The Radiochemical Center, Amersham (TRQ 1263, Spec. Act.22.5 Ci/mmol). Human serum albumin (HSA) was obtained from Serva, and bovine serum was supplied by Mediapharm. The scintillation
To test for the distribution of immunoreactive material on TLC plates, the ether-soluble fraction of leaf material was prepared from an acidified (pH 2.5) aqueous extract. The extracts were applied as a narrow band of 10 cm length on silica gel plates (0.25 mm), developed, and immediately after drying, cut into 0.5 cm strips which were eluted overnight in 1 ml of MeOH. After
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid being diluted ten times with the assay buffer, 0.6 ml aliquots were immunoassayed. In order to minimize background, it proved necessary to use plates which had been developed at least twice with the M e O H / B H T solvent. A B A marker spots were included on the plates, but a 3 4 mln band of silica gel was removed between A B A and the extracts, because it was found that considerable a m o u n t s of marker A B A diffused into the extract regions, if this precaution was not taken.
Immunoassay Reagents For all radioimmunoassay work, an 0.2 M acetate/NaOH buffer pH 4.0 was used and the bovine serum and ammonium-sulfate solutions were prepared as previously described (Weiler and Zenk, 1976). Just prior to use, [3H](_+)-ABA was diluted stepwise with water from a stock solution of 10 gCi/ml. Standards were stored under N2 at 4 ~ in the dark. Although prepared freshly each day, it was found that these solutions were stable for at least 14 days under the conditions employed.
257
Results
Assay Conditions and Properties of Antiserum All the immunized animals produced antibodies which strongly bound [3H](+)-ABA, but in this study, pooled serum from only one animal was used. The titre of the antiserum (that dilution which bound 30% of 0.75 pmol [3H](_+)-ABA under normal assay conditions) was 1:1,350. From a Scatchard-analysis (Scatchard, 1949) of standard curve data (cf. Fig. 1), a maximum affinity constant of K a = 1.3 x 109 I/tool was calculated.
Reaction Parameters
Coupling of ( +_)-ABA to Human Serum Albumine (HSA) To a solution of 250 mg H S A in water (pH 8.5), 132 mg ( + ) - A B A , dissolved in 3 ml of H20/dimethyl formamide (1:2) was added dropwise and the p H adjusted to 8 with 1M NaOH. 210 m g of N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride was added in 4 portions over a period of 90 min and the solution was stirred in the dark for 19 h at 4 ~ C. The solution was then dialyzed against tap water for 4 days and aliquots of the conjugate were stored frozen.
Preparation of Anti-ABA Antisera The conjugate which was emulsified in a 1 : 1 mixture of phosphate buffer p H 7.4 and Freund's complete adjuvant (Difco), was injected into 12 14 week old rabbits. After an intradermal preimmunization, intramuscular booster injections were given monthly and blood was collected both one and two weeks later. Antisera were stored frozen at - 1 8 ~ C.
Radioimmunoassay All incubation steps were carried out in the dark and pipetting was done in dim light. Standards were assayed in tripIicate and samples in either duplicate or triplicate. 0.1 ml of the sample or standard solution was pipetted into 12 • 63 m m glass tubes and 0 . 5 m l buffer added. 0.1 ml bovine serum (l:10diluted) was added as the carrier protein and 0.1 ml dilute [3H](_+)-ABA as the tracer (0.75 pmol). After mixing, 0.1 mI of diIuted antiserum was added and the tubes mixed again. For the determination of unspecific binding, water was used instead of antiserum. The tubes were incubated for 90 min at 4 ~ C and then 1.2 ml of a m m o n i u m sulfate solution were added. Precipitation was allowed to occur for 30 rain at r o o m temperature (23 ~ C) and, after centrifuging and decanting, the pellets were washed once with 1 ml of halfsaturated a m m o n i u m sulfate and then recentrifuged. The washed pellets were dissolved in 0.15 ml of water. 1 ml of scintillation cocktail was added. After mixing, the tubes were inserted into modified scintillation vials and counted for 2 min.
Calculations All data were corrected for unspecific binding and the antigen response expressed as B/B 0 values (B = antibody radioactivity in the presence of unlabelled antigen, either standard or u n k n o w n ; B o = a n t i b o d y radioactivity in the absence of unlabelled antigen), or as the logit B/B o = I n [B/Bo/100- B/B0]. C p m values were used t h r o u g h o u t and counting efficiencies were 19.6% + 1.8% for standards and 1 9 . 1 % • 1.1% for samples.
Maximum binding of [3H](+)-ABA to the antibodies was observed between pH 3.5 and 4.5. When the pH was increased to 6.5 binding decreased and remained constant up to pH 10. Thus, the protonated form of the carboxyl group of ABA was required for optimal reactivity. At 4 ~ C, binding was markedly higher (16%) than at room temperature (23 ~ C), and from the association kinetics it was found that reaction equilibrium was achieved after 90 min. Ammonium sulfate precipitation curves of the antibody complexes showed a broad optimum from 50-60% saturation and 52% was used for subsequent assays. [3H](• did not precipitate at any saturation tested (1 70%) and unspecific binding values were in the range of 1-2%. The ammonium sulfate technique is not time dependent (Farr, 1958) and therefore introduces no variation into the assay, even if large numbers of samples have to be processed. Solvent effects on antibodybinding ability did not occur with up to 20% MeOH and/or 20/ag of BHT in the sample volume.
Assay Sensitivity Figure 1 shows the standard curve for (_)-ABA in the (sigmoid) B/B o versus log ABA and the (linearized) logit B/Bo versus log ABA plots. The linearity of the latter also shows that reaction equilibrium has been reached within the incubation time. The detection limit, defined as the smallest amount of ABA which can be distinguished at the 99.5% confidence limit, is 0 . 2 5 • (66pg). 1 . 9 x 1 0 - 1 2 m o l (0.49 ng) of ABA gives 50% inhibition of binding. The measuring range (range of linearity of logit-plot) is from 0.4 x 10 -12 to 20x 10- 12 mol (0.1-5 ng). This radioimmunoassay can be included among the most sensitive assay methods yet developed for ABA. The standard curve is highly reproducible (cf. Fig. 1). Intra-assay variabilities of standard B/B o
258
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid i
80
~_
,--,1-
enD. -g
.-'.60
/,0
-.
20 ~
(• 1'o
acid[pmol]
'
1'0
16o
,600
('1- abscisic acid [pmol]
Assay Specificity
Table 1. The specificity of antiserum to abscisic acid Compound
pmol required to yield B/Bo = 50%
Cross reactivity (%)
I
(_+)-abscisic acid
1.75
100
II
(_+)-abscisyt-(2, 3, 4, 60-tetraacetyl)B-D-glucose ester
1.70
103
III
methyl-( + )-abscisate
0.96
182
I+IV
1:1 mixture of cis-transand trans-trans-abscisic acid
3.45
47
V
methyl-( _+)-abscisatecis-diol
VI VII VIII
vomifoliol
IX X
268
0.60
phaseic acid
1,180
0.16
dihydrophaseic acid
1,525
0.12
> 10,000"
0
xanthoxin
> 2,500"
0
2-cis-epoxyfi-ionylideneacetic acid
>> 4,000"
0
XI
~-ionylideneacetic acid
> 4,000 a
XII
lunularic acid
>> 4,000"
XIII
violaxanthin
>> 3,300 a
XIV
farnesoI, mixed isomers
> 10,000"
"
Fig. 1. Standard curve for abscisic acid in a sigmoid and a linear plot. = mean _+ S.D. of n = 16 consecutive assays
Highest concentration assayed
values, expressed as coefficients of variation, were 0.9% to 4% throughout the measuring range. Interassay variabilities (n = 16) ranged from 4-9% between 0.4-4 pmol ABA and 13-20% from 4 ~ 0 pmol. The analytical recovery was tested by including triplicates of the same standard solution in 16 consecutive assays (added: 0.7 ng, found: 0.73 ng_+0.17 rig).
Cross reactivities of structurally or physiologically related compounds were determined by comparing the concentration required to yield B / B o = 5 0 % to the value obtained for (+)-ABA. The data given in Table 1 (cf. Fig. 2) show the specific binding of abscisic acid in its free or esterified forms. The acetylated glucose ester exhibits the same reactivity as free ABA, and methyl-( +_)-abscisate cross reacts at 182%. Thus the formation of methyl-abscisate, which may occur as an extraction artifact, can be avoided by including acid in the extraction mixture or by using acetone (Milborrow and Mallaby, 1975). Slight changes in any part of the ABA molecule other than the carboxyl group result in a nearly complete loss of reactivity. Since a 1:1 mixture of the cis,trans- and trans,trans-isomers of ABA cross reacts only 47%, it is apparent that trans,trans-ABA does not react with the antibody. The importance of the correct side chain for antibody binding is further stressed by the absence of reactivity with vomifoliol (VIII), a molecule differing from ABA only in the length of the side chain. Xanthoxin (IX), phaseic acid (VI) and dihydrophaseic acid (VII) did not cross react significantly, nor did the synthetic derivatives (X) and (XI). Reduction of the keto group also leads to a severely reduced binding affinity (0.6% for the cis-diol ofABA). Violaxanthin did not inhibit binding of [3H](_+)-ABA at any concentration tested nor did neoxanthin, antheraxanthin, lutein, zeaxanthin, the carotenoids or the chlorophylls. To test further antibody specificity, the distribution of immunoreactive material on chromatograms of plant extracts was tested. When raw methanolic extracts were applied, the plates had to be heavily loaded to detect the ABA-related compounds, however, a clear separation could not be obtained. In
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid
02H
~ Ill R=H (II) R=[3-D-GLc(Ar )/. [Ill) R=CH3
~C02Mr HO
259
(tl-ABAHHtrans'ABA Nicotiana tabacum extract
(lVJ
100.
< 50-
0~C02 H (Vl)
(vj
i §
(t)-ABA~. Htrans-ABA
OH Ho~CO2H
0~ O H
o
H
HO~-'~CHO
o.,~
0.6
6.8
1'.o
Rf Fig. 3. Distribution of immunoreactive material on a thin-layer c h r o m a t o g r a m of an ether-soluble fraction of an acidified Nicotiana tabaeum leaf extract. Plate: silica gel 0.25 ram; Solvent system: toluene/ethyl acetate/HAc = 25 : 15: 3 (v/v)
(VIII)
(VIII
o,2
~ ' ~ C 02H
(IXl
(X)
~COzH (Xl)
CO~zH OH (XII)
one immunoreactive band was found in this crude fraction and it corresponded to marker ABA (Rf= 0.6). From this chromatogram, the amount of ABA in the leaves was calculated as being 190 ng/g fresh weight. This value compared favorably with the 210 ng/g fresh weight value, determined by directly immunoassaying the crude methanolic extract.
OH
X X X cH'~
Assay Variability and Recovery (XIVl
{XIIIJ
Fig. 2. C o m p o u n d s assayed for cross reactivity with anti-ABA antiserum
some extracts, a second small peak appeared at the origin and most likely represented ABA-glucose ester. Figure 3 shows the distribution of immunoreactive material on a chromatogram of the ether-soluble acid fraction of a Nicotiana tabacum leaf extract. Only
The average coefficient of variation of double determinations was 9% in the standard curve range from 0-0.5 ng and 10% in the range from 0.5-5 ng ABA. If desired, the variability could be further reduced by correcting each determination for counting efficiency. Recovery of (+)-ABA added to methanolic plant extracts prior to dilution was 104% when added prior
Table 2. Distribution of A B A in the fruit of Persea gratissima (avocado). Extraction procedure (a) was used. fr.wt. = f r e s h weight, dr.wt. = dry weight Part of fruit
Total fr.wt. (g)
A m o u n t of A B A present
A B A level
(nmol)
(pmol g z fr.wt.)
(% of totat)
Literature value" (rag kg ~ fr.wt.) (mg kg - I fr.wt.) ORD
peel green pulp yellow pulp
testa embryo
a
Milborrow, 1967
23 90 56
0.57 23
18.7 16.0 12.2
0.19 4.2
36.5 31.3 23.7
0.36 8.1
820 178 216
0.216 0.047 0.057
pericarpmean:
0.073
342 178
0.090 0.047
seed mean:
0.049
racemate dilution
0.76
0.034
0.11
260
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid
Table 3. Distribution of abscisic acid in some plant families. Leaves were assayed except for some cases, where leaves and shoots (1) or whole plants (2) were analyzed. (3)=aquatic plant, growing submerged; (4)= aquatic plant, emerged leaves analyzed; (5)= aquatic plant, submerged leaves analyzed; (6)=succulent; n.d.= not detected. Extraction procedure (b) was used. fr.wt.=fresh weight; dr.wt.=dry weight Family
Species
ABA level -
Cyanophyceae: Oscillatoriaceae
Remarks
n.d.
Gibberella fujikuroi
n.d.
n.d.
Hyphomycetes
Curvularia lunata
n.d.
n.d.
Hepaticae : Marchantiaceae
Marchantia polymorpha n.d.
n.d.
Lycopodium squarrosum 1.16 Selaginella uncinnata n.d. Isogtes matogrossense 0.25
7.4 n.d. 3.7
(1) (1) (4)
Equisetum arvense
0.36
3.2
(1)
Asplenium dimorphum Asplenium adiantum nigrum Polypodium heimionitis Woodwardia radicans Bolbitis heudelotii Azolla mexicana Salvinia auriculata Marsilea hirsuta Pilularia globulifera Ceratopteris malictroides Ceratopteris malictroides
n.d. 0.25
n.d. 1.0
0.58
3.2
0.65 0.73 0.27 0.26 0.82 0.48 0.25
3.0 6.3 4.5 4.8 2.5 3.6 4.2
0.50
6.4
Ascomycetes : Fungi imperfecti."
Equisetaceae:
Equisetaceae Filicatae: Polypodiaceae
Salviniaceae Marsileaceae Parkeriaceae
Gymnospermae : Pinaceae
Cupressaceae
Podocarpaceae
Picea sitchensis Pinus halepensis Pinus pinea Pinus radiata Cupressus sempervirens Juniperus bermudiana Podocarpus macrophyllus
Angiospermae, Dicotyledonae : Lauraceae Persea americana Apollonias barbujana Cinnamonum ceylanicum Laurus nobilis Nymphaeaceae Cabomba sp. Nymphaea caerulea Nymphaea lutea Nymphaea lutea
0.78 1.02 0.52 0.80 1.10
4.5 2.7 1.5 2.1 2.6
0.95 0.99
2.6 2.2
0.87 1.70 1.10
2.8 3.7 2.6
1.00 n.d. 0.58 0.71 0.59
2.1 n.d. 4.0 3.8 5.7
ABA level 7
n.d.
Lycopodiatae : Lycopodiaceae Selaginellaceae Iso~taceae
Species
7
Spirulina platensis
Hypocreaceae
Family
(3) (2), (4) (4) (4) (1), (3) (1), (4)
(3) (4) (4) (5)
Ceratophyllaceae Ceratophyllum sp. Papaver somniferum Papaveraceae Fagaceae Castanea sativa Cyclobalanopsis glauca Quercus ilex Dorstenia urceolata Moraceae Crassulaceae Echeveria pringlei Kalanchoe velutina Sedum palmeri Prunus ilicifolia Rosaceae Mimosa pudica Mimosaceae Caesalpina spinosa Caesalpinaceae Drosophyllum Droseraceae lusitanicum Myriophyllum Haloragaceae brasiliense Myrtus communis Myrtaceae Rotala rotundifolia Lythraceae Oxalis articulata Oxalidaceae Pelargonium articulata Geraniaceae Aquifoliaceae Ilex aquifolium Euphorbia pteroneura Euphorbiaceae Euphorbia millii Euphorbia tirucalli Phyllanthus fluitans Hydrocotyle Umbellifereae bonariensis Begonia glabra Begoniaceae Carica quercifolia Caricaceae Arbutus unedo Ericaceae Dracophyllum Epacridaceae secundum Manilkara zapota Sapotaceae Cyclamen Primulaceae mederifolium Samolus floribundus Menyanthaceae Nymphoides humboldtianum Catharanthus roseus Apocynanceae Nerium oleander Rauwolfia vomitoria Pachypodium lamerei Plumbaginaceae Limonium fruticans Viburnum tinus Caprifoliaceae Symphoricarpus foetidus Pterocephalus Dipsacaceae dementorum Phillyrea latifolia Oleaceae Jasminium simplicifolium Datara arborea Solanaceae Datura sanguinea Solanum laciniatum Digitalis lanata ScrophulariIsoptexis canariensis aceae Phygelius capensis
Remarks
7
n.d. 0.60 0.35 0.91 0.69 0.57 0.11 0.14 0.15 0.80 1.20 1.10 0.92
n.d. 5.5 0.8 1.7 1.4 2.0 2.5 4.1 3.4 2.2 4.2 3.9 7.3
(1), (3)
0.92
9.1
(l), (3)
0.83 0.66 0.80 0.17 0.82 0.28 0.45 0.37 0.39 0.25
1.9 6.2 6.4 1.8 1.7 2.7 2.8 3.0 3.2 1.9
0.23 0.39 0.51 0.91
3.6 2.6 1.5 1.7
0.86 0.29
2.0 3.2
0.15 0.39
2.0 6.3
0.56 0.60 0.30 0.60 0.08 1.30 0.60
3.8 1.5 1.9 5.1 0.4 3.0 7.2
0.38
2.5
1.30 1.00
2.5 3.0
1.00 1.10 1.80 0.90 0.85 0.60
7.8 7.8 10.2 4.3 3.0 3.3
(6) (6) (6)
(1), (3)
(1), (6) (l) (1), (6) (4)
(4)
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid Table 3 (continued) Family
Species
Determination of ABA in Plant Extracts ABA level I
Lentibulariaceae Labiatae Verbenaceae
Aeschynanthus speciosus Utricularia vulgaris
Teucrium flavum Avicennia marina Durantha repens Campanulaceae Campanula rupestris Lobeliaceae Lobelia cardinalis Asteraceae Artemisia arborescens Centaurea canariensis Senecio cruentus
Remarks
I hi)
Gesneriaceae
0.20
2.4
0.10
1.9
1.00 3.4 0.87 2.9 0.85 3.8 1.20 9.3 0.67 10.2 0.85 3.7 0.96 6.1 0.37 3.6
.
(1), (3)
(3)
Angiospermae, Monocotyledonae: Alismataceae
Echinodorus cordifolius Sagittaria latifolia HydrocharitElodeacanadensis aceae Vallisneria gigantea Lil~aceae Aloe statuides Dracaena indivisa Sanseveria trifasciata Smilax aspera Nolinaceae Beaucarneastricta Agavaceae Yucca arizonica Orchidaceae Cymbidiumhybrida Phalaenopsis hybrida Bromeliaceae Guzmaniaminor Cyperaceae Cyperushaspan Cyperus textilis Commelinaceae Cochliostema odoratissimum Restioniaceae Leptocarpussimilis Poaceae Oryza sativa Arecaceae Chamaerops humilis Elaeis guineensis Araceae Anubias sp. Cryptocoryne aponogetifolia Cryptocoryne wendtii Lemnaceae Lemna minor
261
0.70
3.2
(4)
n.d. 0.16 0.34 0.17 0.91 0.23 0.60 0.49 0.32 1.20 0.70 0.52 1.00 0.32 0.24
n.d. 2.7 5.7 2.7 2.3 2.6 1.8 1.9 1.0 4.8 4.7 2.1 6.3 1.4 3.2
(3) (1), (3) (4) (3)
0.59 0.78 0.60 1.70 1.10 0.78
1.6 4.4 1.3 4.9 7.8 7.9
(3) (3)
0.51 0.14
8.5 2.0
(3) (2), (4)
(1) (1)
The distribution of ABA within the avocado fruit, which is frequently chosen for biosynthetic work on ABA (Noddle and Robinson, 1969; Milborrow and Robinson, 1973; Milborrow, I976), is shown in Table 2. The data obtained from the seed show a strong correlation to published values (Milborrow, 1967), however, the level of ABA in the pulp was much lower in our material. This might be due to differences in the ripeness or in the variety used. Of the total amount of ABA present in the fruit, about 90% is located in the pericarp, and 70% of this total is found in the green tissues. This is in close agreement with reports that ABA synthesis seems to be associated with functional chloroplasts (Railton etal., 1974; Milborrow, 1974). In another experiment, the distribution of immunoreactive material in more than 100 species of plants was investigated (Table 3). ABA was found in all higher plant families, but could not be detected in the 9 blue-green alga, Spirulinaplatensis, the liverwort Marchantia polymorpha, or the two fungi tested. In most plants, ABA levels were between 2 ~ nmol g-1 dry weight (0.5 1 mg k g - t ) , although as much as 1 0 n m o l g -1 ( 2 . 5 m g k g -1) were found in some species, namely the Solanaceae. There is, however, a much larger variation in ABA levels when expressed on a fresh weight basis. ABA was very low in succulent plants (0.1-0.3 nmol g- 1=0.025-0.075 mg k g - 1), whereas a number of non-succulent plants yielded values higher than 1 nmol g-1. A wide variation of ABA content was observed in aquatic plants which were grown submerged. In some species (e.g. Sagitta latifolia) ABA could not be detected. Milborrow and Robinson (1972) also report very low levels of ABA in fresh samples of Ceratophyllum demersum and Callitriche stagnalis (submerged parts).
Discussion
to extraction of the plant material. Recovery was 103% with a standard deviation of 28% between different plant materials. Therefore, all plant extracts were corrected for recovery. This correction also compensated for any isomerization to trans,trans-ABA which might have occured during extraction and assay. Extract dilution curves did not exactly parallel the standard curves. However, since parallelism could be restored by adding increasing amounts of (_+)ABA to the extracts, it is likely that the naturally occuring ( + ) - A B A differs in reactivity from the commercial ABA used for standardization.
The experimental data presented in this paper demonstrate that antibodies with high specificity for ABA and its esters have been obtained. These can be utilized in a radioimmunoassay which permits the direct quantitative estimation of this hormone between 0.4x 10-12 mol to 20x 10-12 tool in crude plant extracts. Xanthoxin, phaseic acid and vomifoliol may occur naturally in amounts comparable to ABA. Dihydrophaseic acid may occur in hundredfold higher amounts (Zeevaart, 1974; B6ttger, 1978; Tinelli et al., 1973). However, the assay is not affected by these structurally-related compounds at any concentration within the physiological range. In addition, neither
262
carotenoids, xanthophylls, nor physiologically related compounds (i.e. farnesol or lunularic acid) show any interference. Because of the identical reactivities of (+)-ABA and its tetraacetyl glucose ester, the sum of free and esterified ABA is calculated from standard curves. Since the acetylated glucose ester was the only derivative available for cross-reactivity studies, we assume that for the purpose of total ABA determination, the naturally occurring glucose ester does not differ significantly from this compound in its reactivity. However, if it is necessary to differentiate between free and esterified abscisic acid, this can be achieved by differential solvent extraction (Zeevaart, 1974; D6rffling, 1974). The antiserum used in this study was raised and standardized against (_+)-ABA, however, plant material contains only (+)-ABA. The fact that it was possible to inhibit completely the binding of [3H](+_)ABA by adding increasing amounts of plant extracts, and the linearity of these extract dilution curves in the logit/log plot showed that (+)-ABA also competes very well with antibody-bound [3H](-)-ABA. Thus, differences in reactivities of the ( + ) and ( - ) forms must be small. Since the glucose and methyl esters react equal to or better than the free acid, and since structural changes in any other part of the molecule result in almost total loss of immunoreactivity, it would appear that the entire ABA molecule, except for the carboxyl group, binds to the antibody. The taxonomic distribution of ABA and its levels, determined by this method, are in agreement with the data obtained by other analytical techniques (Pryce, 1970, 1971; Gorham, 1977). A wide range of species, selected from as many families as possible and grown under conditions prevailing in the greenhouse, were used for this experiment. The results demonstrate that this radioimmunoassay can be used to quantitate ABA in the most diverse plant material. Furthermore, the data presented in Table 3 strongly support the hypothesis (Pryce, 1971) that ABA may generally occur in higher plants, although at very low levels in some species. In addition, the measuring range of this assay covers the whole range of ABA levels found in plant material thus far examined. A broad survey, such as that presented in Tabel 3, has never been possible before using other analytical techniques. This demonstrates the potential of the RIA, which, in its semi-automated performance (Weiler and Zenk, 1976) permits the processing of several hundred samples per day. Thus a number of problems can now be approached efficiently, such as the monitoring of ABA levels and its precise distribution in plants or plant organs in relation to physiological
E.W. Weiler: Determination of Free and Conjugated Abscisic Acid
state, stress response, and other factors. Other physiological studies using this assay method are currently in progress. Phaseic acid was a gift from Prof. Raschke, East Lansing. The xanthoxin was from Dr. B6ttger, Hamburg, and the vomifoliol was a gift from Dr. Kapil, Lucknow. Prof. Kindl, Marburg, provided lunularic acid, and Dr. Okita, Sendal, provided compounds (X) and (XI). The excellent technical assistance of Mrs. P. Westekemper is also acknowledged, as well as the proof reading of the manuscript by Prof.R. Mansell, Tampa.
References B6ttger, M.: The occurrence of cis, trans- and trans, trans-xanthoxin in pea roots. Z.Pflanzenphysiol. 86, 265-268 (1978) Cornforth, J.W., Draber, W., Milborrow, B.V., Ryback, G. : Absolute stereochemistry of (+)-abscisin II. Chem. Commun. 114-116 (1967) Davis, L.A., Addicott, F.T. : Abscisic acid: correlations with abscission and with development in the cotton fruit. Plant Physiol. 49, 644.648 (1972) D6rffling, K.: Quantitative Verfinderungen des Abscisins~inregehaltes w~ihrend der Fruchtentwicklung yon Solanum lycopersicure L. Planta 93, 233-242 (1970) D6rffiing, K.: Abscisinsfiure und Xanthoxin. Fortschr.Bot. 34, 190-192 (1972) D6rffling, K.: Abscisic acid and related compounds. Progr.Bot. 38, 153-156 (1976) D6rffling, K., Sonka, B., Tietz, D.: Variation and metabolism of abscisic acid in pea seedlings during and after water stress. Planta 121, 57~66 (1974) Dfiring, H., Bachmann, O. : Abscisic acid analysis in Vitis vinifera in the period of endogeneous bud dormancy by high pressure liquid chromatography. Physiol. Plant. 34, 201-203 (1975) Durley, R.C., Kannangara, T., Simpson, G.M. : Analysis of abscisins and 3-indolylacetic acid in leaves of Sorghum bicolor by high-performance liquid chromatography. Can.J.Bot. 56, 157 161 (1978) Eckert, H.G.: Die Technik des Radioimmunoassays. Angew. Chem. 88, 565 574 (1976) Farr, R.S. : A quantitative immunochemical measure of the primary interaction of I*BSA and antibody. J. Infect. Dis. 103, 239 262
(1958) Fuchs, Y., Mayak, S., Fuchs, S.: Detection and quantitative determination of abscisic acid by immunological assay. Planta 103, 117-125 (1972) Gaskin, P., Macmillan, J. : Plant hormones. VII. Identification and estimation of abscisic acid in a crude plant extract by combined gas chromatography - mass spectrometry. Phytochemistry 7, 1699 1701 (1968) Gorham, J. : Lunularic acid and related compounds in liverworts, algae and Hydrangea. Phytochemistry 16, 249-253 (1977) Hager, A., Meyer-Bertenrath, T. : Die Isolierung und quantitative Bestimmung der Carotinoide und Chlorophytle yon Blfittern, Algen und isolierten Chloroplasten mit Hilfe dfinnschichtchromatographischer Methoden. Planta 69, 198 217 (1966) Hager, A., Meyer-Bertenrath, T.: Die Identifizierung der an Dfinnschichten getrennten Carotinoide grfiner Blfitter und A1gen. Planta 76, 149-168 (1967) Jones, H.G. : How plants respond to stress. Nature 271, 610 (1978) Landon, J., Moffat, A.C. : The radioimmunoassay of drugs. Analyst 101, 225 243 (1976)
E.W. Weiler : Determination of Free and Conjugated Abscisic Acid Lehmann, H., Sch/itte, H.R.: Zur Darstellung O-substituierter Zuckerester. J. Prakt. Chem. 319, 117-122 (1977) Lenton, J.R., Perry, V.M., Saunders, P.F. : The identification and quantitative analysis of abscisic acid in plant extracts by gasliquid chromatography. Planta 96, 271-280 (1971) Marschner, I., Dobry, H., Erhardt, F., Landersdorfer, T., Popp, B., Ringel, C., Scriba, P.C.: Berechnung radioimmunologischer Mel3werte mittels Spline-Funktionen. Arztl. Lab. 20, 184-191 (1974) Milborrow, B.V.: The identification of (+)-abscisin II ((+)-dormin) in plants and measurement of its concentrations. Planta 76, 93 113 (1967) Milborrow, B.V. : The chemistry and physiology of abscisic acid. Ann.Rev. Plant Physiol. 25, 259 307 (1974) Milborrow, B.V., Mallaby, R. :Occurrence of methyl-( +)-abscisate as an artefact of extraction. J.Exp.Bot. 26, 741-748 (1975) Milborrow, B.V., Robinson, D.R.: Factors affecting the biosynthesis of abscisic acid. J.Exp.Bot. 24, 537 548 (1973) Noddle, R.C., Robinson, D.R.: Biosynthesis of abscisic acid: Incorporation of radioactivity from [2-14C]mevalonic acid by intact fruit. Biochem. J. 112, 547-548 (1969) Pryce, R.J. : Lunularic acid, a common endogenous growth inhibitor of liverworts. Planta 97, 354-357 (1971) Pryce, R.J. : The occurrence of lunularic and abscisic acid in plants. Phytochemistry 11, 1759-1761 (1972) Quebedaux, B., Sweetser, P.B., Rowell, J.C.: Abscisic acid in soybean reproductive structures during development. Plant Physiol. 58, 363 366 (1976) Railton, I.D., Reid, D.M., Gaskin, P., Macmillan, J. : Characterization of abscisic acid in chloroplasts of Pisum sativum L. cv.
263 Alaska by combined gas-chromatography-mass spectrometry. Planta 117, 179-182 (1974) Seeley, S.D., Powell, L.E.: Electron capture-gas chromatography for sensitive assay of abscisic acid. Anal.Biochem. 35, 530-533 (1970) Sivory, E.M., Sonvico, V., Fernandez, N.O.: Determination of abscisic acid following Paleg's method. Plant Cell Physiol. 12, 993-996 (1971) Tanada, T. : A rapid photoreversible response of barley root tips in the presence of 3-indoleacetic acid. Proc.Nat. Acad. Sci.USA 59, 376-380 (1968) Tillberg, E.: An abscisic acid-like substance in dry and soaked Phaseolus vulgaris seeds determined by the Lemna growth bioassay. Physiol. Plant. 34, 192-195 (1975) Tinelli, E.T., Sondheimer, E., Walton, D.C.: Metabolites of [2-14C]abscisic acid. Tetrahedron Lett. 2, 139-140 (1973) Tucker, D.J., Mansfield, T.A.: A simple bioassay for detecting "antitranspirant" activity of naturally occurring compounds such as abscisic acid. Planta 98, 157-163 (1971) Walton, D., Galson, E.: A radioimmunoassay for abscisic acid. Plant Physiol. 59, Suppl. 77 (1977) Weiler, E.W., Zenk, M.H. : Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. Phytochemistry 15, i537-1545 (1976) Zeevaart, J.A.D. : Levels of (+)-abscisic acid and xanthoxin in spinach under different environmental conditions. Plant Physiol. 53, 644-648 (1974)
Received 21 July; accepted 1 October 1978
