Bioconjugate Chem. 2007, 18, 1355−1359
1355
Function and Stability of Abscisic Acid Acyl Hydrazone Conjugates by LC-MS2 of ex Vivo Samples Timothy R. Smith,† Andrew J. Clark,† Richard Napier,‡ Paul C. Taylor,† Andrew J. Thompson,‡ and Andrew Marsh*,† Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K., Warwick HRI, University of Warwick, Wellesbourne, Warwick, CV35 9EF, U.K. Received February 28, 2007; Revised Manuscript Received May 1, 2007
We prepare a biotinylated conjugate of the ubiquitous plant hormone (S)-(+)-abscisic acid via an acyl hydrazone linkage at the C4′ position and demonstrate in vivo cleavage of the otherwise stable acyl hydrazone linkage using LC-MS2. As part of a wider chemical genomic study, biological activity of the conjugate was assessed using standard epidermal peel and gravimetric transpiration assays, showing significant activity but at a level lower than the unconjugated hormone. When deuterated samples of the conjugate were fed to the plant, however, it was apparent by LC-MS2 experiments that significant levels of hydrolysis of the acyl hydrazone had taken place, contrary to in vitro stability assays in artificial sap. We conclude that abscisic acid is liberated in sufficient quantities to account for the observed physiological response and that LC-MS2 monitoring of conjugates is a simple and practical method by which such events may be assessed, whether in plants or other organisms.
INTRODUCTION The search for the biological targets of the ubiquitous plant hormone abscisic acid (ABA1) 1 (Figure 1) (1) has produced many sophisticated strategies for the preparation of analogues and conjugates (2). One favored tactic has been the use of bioconjugates such as 4′-hydrazones 2-5 (Figure 2) in order to identify anti-idiotypic monoclonal antibodies (3), to provide in ViVo fluorophore reagents (4), and as ligands in affinity chromatography (5). Each relies on the bioconjugate or derived protein exhibiting demonstrable activity in order to be relevant to the process under investigation. An example of the bioconjugate strategy working well is the use of novel aromatic annelated abscisic acid derivatives (6) in order to identify the first (7) of two recently confirmed receptors (8). Conjugates that retain some activity but at a lower level may still offer useful material, because binding may occur, but with lower affinity. Reliable assays for both biological activity and chemical composition are hence critical components in the design of successful experiments using such bioconjugates. In this Technical Note, we demonstrate the use of LC-MS2 on in Vitro and ex ViVo samples in conjunction with biological assays to determine the stability and biological activity of an acyl hydrazone conjugate of ABA. Significantly, we show that in ViVo degradation of the conjugate released low levels of ABA that were sufficient to explain the apparent activity of our conjugate.
EXPERIMENTAL PROCEDURES General Experimental. Unless otherwise noted, all materials were obtained from commercial sources and used without further purification. S-(+)-Abscisic acid (98%: purity confirmed by microanalysis) was purchased from Sichuan Lomon Bio Corporation, Chengdu Sichuan, China. Column chromatography was performed on Merck silica gel 60 H (230-400 mesh). Melting * Corresponding author e-mail:
[email protected]. † Department of Chemistry. ‡ Warwick HRI. 1 Abbreviations: ABA, abscisic acid; AS, artificial sap; MES, 2-(Nmorpholino)ethanesulfonic acid; MRM, multiple reaction monitoring; LCMS2, liquid chromatography tandem mass spectrometry.
Figure 1. (S)-(+)-Abscisic acid with formal atom numbering.
points were recorded on a Gallenkamp melting point apparatus and are uncorrected. Optical rotations (given in 10-1 deg cm2 g-1) were measured on an Optical Activity AA-1000 polarimeter. IR spectra were recorded on a Perkin-Elmer Paragon 1000 FT-IR instrument. 1H and 13C NMR spectra were recorded using a Bruker DPX300 at 300 and 75 MHz, respectively. NMR spectra were recorded in the indicated solvent, and chemical shifts (δ) are reported in parts per million (ppm) relative to residual nondeuterated solvent as an internal standard. J values are given in hertz (Hz). 2D NMR spectra were recorded using a Bruker DRX500 at 77 MHz. LSIMS mass spectrometry was performed on a Micromass Autospec mass spectrometer. ESI mass spectrometry was performed on a Bruker micrOTOF instrument. Epidermal Peel Assay. Abaxial epidermal peels from Commelina communis L. were incubated at 25 °C under continuous white light (30 µmol m-2 s-1) in Petri dishes containing aqueous buffer solution (25 mL) consisting of 8 mmol dm-3 MES‚KOH pH 6.15 and 50 mmol dm-3 KCl. A needle inserted through the lid of the Petri dish allowed CO2-free air to be bubbled through the buffer solution (100 cm3 min-1). After 2 h, the stomata were fully open. Solutions of ABA or conjugate were added and incubation continued for a further 2 h. The epidermal peels were then mounted on microscope slides, and stomatal aperture was immediately recorded digitally at 500× magnification. For each treatment, mean apertures were determined from 30 measurements made from 2 independent epidermal peels. Stomatal apertures were measured across the widest part of the opening and expressed as a percentage of an untreated control. Gravimetric Transpiration Assay. Tomato (Solanum lycoperiscum L. cv Ailsa Craig) plants were grown in a glass house in 10 dm3 pots of Levington M2 compost (Levington, Ipswich, U.K.), until they had produced approximately eight leaves. The youngest fully expanded leaf was cut from the plant, and the
10.1021/bc070069x CCC: $37.00 © 2007 American Chemical Society Published on Web 06/14/2007
1356 Bioconjugate Chem., Vol. 18, No. 4, 2007
Smith et al.
Figure 2. 4′-Acyl hydrazone conjugates of abscisic acid: biotinylated 2 (9), radio-iodinated 3 (5), tyrosyl 4 (10), and fluorescent 5 (4).
petiole was recut under degassed distilled water to avoid embolization and then immediately transferred to a 2.5 cm3 glass vial containing 2 cm3 of artificial sap (AS) consisting of 1.0 mmol dm-3 KH2PO4, 1.0 mmol dm-3 K2HPO4, 1.0 mmol dm-3 CaCl2, 0.1 mmol dm-3 MgSO4, 3.0 mmol dm-3 KNO3, and 0.1 mmol dm-3 MnSO4 (11). The sap was adjusted to pH 6.0 with aqueous HCl. Laboratory film was used to seal the petiole in the vial to reduce evaporation. Prepared leaf sets were randomized and placed on a laboratory bench under fluorescent lighting (37 µmol m-2 s-1). Each vial was weighed every 20 min for 2 h to establish an untreated transpiration rate. After 30-90 min, the rate of transpiration was seen to reach a plateau. Each leaf set was then transferred to a new vial containing the same amount of AS in addition to the specified amount of either ABA or conjugate. The rate of transpiration was determined by weighing the vial every 20 min for 2 h and expressed as a percentage of the initial transpiration rate. Up to six replicates were performed for each assay at five different concentrations of ABA and conjugate. LC-MS2 Analysis. Sample Preparation. On completion of the gravimetric transpiration assay, leaves fed with the most concentrated solution of conjugate were separated from the petiole and immediately homogenized in liquid nitrogen, lyophilized, and stored at -80 °C until required. Untreated leaves were collected and processed in a similar manner but were spiked with conjugate (75 µg) prior to homogenization as a control. Leaf material (100 mg) was extracted with 1 cm3 of water by agitation on a whirly mix for 5 min. The sample was centrifuged (2880 g, 10 min at 4 °C) and the supernatant collected and filtered through a plug of glass wool into a centrifuge filter (Vivascience Vivaspin 2, 3000 MWCO). After centrifuging (2880 g, 30 min at 4 °C), the supernatant was collected and analyzed immediately. LC-MS2 Procedure. ESI mass spectrometry was performed on a Bruker HCTplus ion trap instrument. Parameters were optimized by direct infusion of d6-abscisic acid (1 mg dm-3) in water (70%), acetonitrile (30%), and formic acid (0.01%). The optimized conditions were as follows: negative ion polarity, trap drive 34.7 (arbitrary units), octopole RF amplitude 193.4 Vpp, capillary exit -111.5 V, HV capillary 4.5 kV, drying gas (N2) temperature 320 °C, drying gas flow 9 dm3 min-1, and nebulizer 30 psi. Fragmentation parameters were handled automatically by the SmartFrag function. LC separation was performed on an Alltech Alltimer C18 column (100 mm × 2 mm, 3 µm) fitted with an Alltech C18 guard column (7.5 mm × 3.0 mm, 5 µm). For each run, 20 µL of the crude plant extract was injected onto the column. The sample was eluted using a binary solvent system comprising 0.01% formic acid in water (A) and 0.01% formic acid in acetonitrile (B). Solvents were mixed and delivered to the column by an integrated HPLC pump and autosampler (Agilent 1100 series)
at a flow rate of 0.2 cm3 min-1. The sample was separated by gradient elution starting at 74% A and 26% B and changed linearly over a 15 min period to 70% A and 30% B. Preparation of N-Succinamidylhydrazono-N′-biotinyl-3,6dioxaoctane-1,8-diamine, 7. A solution of N-succinamidyl-N′biotinyl-3,6-dioxaoctane-1,8-diamine 6 (12) (173 mg, 0.36 mmol, 1 equiv) in methanol (2 mL) was added dropwise to an ice-cooled solution of thionyl chloride (131 mg, 1.1 mmol, 3 equiv) in methanol (2 mL). The reaction was allowed to warm to room temperature and stirred for 24 h. The solvent was removed under reduced pressure before addition of a further portion of methanol (15 mL). This was removed once more under reduced pressure to yield a brown residue, which was dissolved in methanol (4 mL) and treated with hydrazine monohydrate (183 mg, 3.6 mmol, 10 equiv). The mixture was stirred at room temperature for 24 h, filtered, and then concentrated under reduced pressure. The crude product was purified by column chromatography (silica, 30% MeOH, and 3% concentrated aqueous ammonia solution in EtOAc) to yield a pale yellow solid (100 mg, 56%); Rf 0.10 (30% MeOH and 3% concentrated aqueous ammonia solution in EtOAc); mp 135-138 °C (EtOAc); [R]20D ) +12.3 (c 0.65, DMSO); Vmax(solid) 3285, 2924, 1698, 1637, 1550, 1461, 1120 cm-1; δH (300 MHz, DMSO-d6) 1.22-1.37 (m, 2H), 1.40-1.69 (m, 4H), 2.06 (t, J ) 7.4, 2H), 2.22-2.24 (m, 2H), 2.28-2.31 (m, 2H), 2.57 (d, J ) 12.2, 1H), 2.82 (dd, J ) 5.1, 12.2, 1H), 3.08-3.10 (m, 1H), 3.17-3.18 (m, 4H), 3.36-3.38 (m, 4H), 3.50 (s, 4H), 3.98 (s, 2H), 4.10-4.14 (m, 1H), 4.28-4.33 (m, 1H), 6.37 (s, 1H), 6.44 (s, 1H), 7.86 (t, J ) 5.5, 1H), 7.92 (t, J ) 5.3, 1H), 8.96 (s, 1H); δC (75 MHz, DMSO-d6) 25.6 (CH2), 28.6 (2 × CH2), 29.3 (CH2), 31.0 (CH2), 35.4 (CH2), 39.0 (2 × CH2), 40.7 (CH2), 55.7 (CH), 59.5 (CH), 61.4 (CH), 69.5 (2 × CH2), 69.9 (2 × CH2), 163.1 (C), 171.2 (C), 171.6 (C), 172.5 (C); HRMS (ESI): m/z requires (C20H37N6O6S) 489.2490, found 489.2489, 57% [M + H]+. Preparation of S-(+)-Abscisic acid 4′-(N-Succinamidyl acyl hydrazide-N′-biotinyl-3,6-dioxaoctane-1,8 diamine), 8. N-Succinamidylhydrazono-N′-biotinyl-3,6-dioxaoctane-1,8-diamine, 7, (190 mg, 0.39 mmol, 1 equiv) and S-(+)-abscisic acid (206 mg, 0.78 mmol, 2 equiv) were dissolved in methanol (10 mL). Glacial acetic acid (50 µL) and crushed 4 Å molecular sieves (100 mg) were added and the reaction stirred in darkness at room temperature under a nitrogen atmosphere for 4 days. The mixture was filtered through Celite and the solvent removed under reduced pressure. The product was purified by preparative thin-layer chromatography (silica, 15% MeOH in DCM) to yield a pale yellow solid (177 mg, 62%); Rf 0.15 (15% MeOH in DCM); [R]20D ) +267.2 (c 0.25, MeOH); Vmax(solid) 3266, 2929, 1653, 1546, 1400, 1119, 1026 cm-1; δH (300 MHz, CD3OD) 1.06 (s, 3H), 1.09 (s, 3H), 1.40-1.56 (m, 2H), 1.58-1.83 (m, 4H), 1.87 (s, 3H), 2.06 (s, 3H), 2.26 (t, J ) 7.4, 2H), 2.47-
Bioconjugate Chem., Vol. 18, No. 4, 2007 1357
Technical Notes Scheme 1. Preparation of Biotinylated Acyl Hydrazone-Linked Abscisic Acida
a
Reagents and conditions: (i) SOCl2, MeOH, then (ii) hydrazine, MeOH, 56%, (iii) (S)-(+)-abscisic acid, AcOH, MeOH, 62%.
Figure 4. A 100 µmol dm-3 solution of d6-8 in artificial sap was acidified with HCl (aq) to a specific pH. The mixture was allowed to stand for 2 h, then immediately analyzed using LC-MS2. Hydrolysis of d6-8 releases d6-abscisic acid indicated by a peak at RT ) 9.83 min. Chromatograms are offset.
Figure 3. (a) The biological activity of conjugate 8 (9) compared to (S)-(+)-abscisic acid (O) in the stomatal closure of epidermal peels. Results are displayed as a percentage of the control with fully open stomata at 100%. Error bars indicate SE of means (n ) 10). (b) The effect of feeding detached tomato leaves with AS containing conjugate d6-8 (9) or (S)-(+)-abscisic acid (O) on gravimetric transpiration. Results are displayed as a percentage of the transpiration rate of leaves that received AS alone. Error bars indicate SE of means (n ) 3).
2.70 (m, 6H), 2.74 (d, J ) 12.8, 1H), 2.96 (dd, J ) 4.9, 12.8, 1H), 3.21-3.28 (m, 1H), 3.39-3.42 (m, 4H), 3.55-3.63 (m, 4H), 3.66 (s, 4H), 4.33-4.37 (m, 1H), 4.52-4.56 (m, 1H), 5.75 (s, 1H), 6.19 (s, 1H), 6.19 (d, J ) 16.0, 1H), 7.73 (d, J ) 16.0, 1H); δC (75 MHz, CD3OD) 19.7 (CH3), 23.6 (CH3), 24.4 (CH3), 25.3 (CH3), 27.3 (CH2), 29.9 (CH2), 30.2 (CH2), 31.0 (CH2), 32.3 (CH2), 37.2 (CH2), 38.7 (C), 40.7 (CH2), 40.8 (CH2), 41.5 (CH2), 50.3 (CH2), 57.4 (CH), 62.0 (CH), 63.8 (CH), 71.1 (2 × CH2), 71.7 (2 × CH2), 80.8 (C), 125.4 (CH), 125.7 (CH), 130.0 (CH), 136.4 (CH), 144.5 (C), 151.9 (C), 156.0 (C), 166.5 (C), 167.9 (C), 172.3 (C), 175.2 (C), 176.6 (C); HRMS (LSIMS): m/z requires (C35H54N6NaO9S) 757.3571, found 757.3564, 100% [M + Na]+. Preparation of S-(+)-d6-Abscisic acid 4′-(N-Succinamidyl acyl hydrazide-N′-biotinyl-3,6-dioxaoctane-1,8 diamine), d68. S-(+)-d6-Abscisic acid, prepared from two successive rounds of deuterium exchange (13), was reacted with N-succinamidyl-
hydrazono-N′-biotinyl-3,6-dioxaoctane-1,8-diamine 7, in an identical manner to the preparation of 8, yielding the title product (154 mg, 53%) as a pale yellow solid; [R]20D ) +272.0 (c 0.30, MeOH); Vmax(solid) 3282, 2933, 1655, 1534, 1458, 1127, 1026 cm-1; δH (300 MHz, CD3OD) 1.01 (s, 3H), 1.05 (s, 3H), 1.421.47 (m, 2H), 1.54-1.76 (m, 4H), 1.97 (s, 3H), 2.22 (t, J ) 7.6, 2H), 2.58 (d, J ) 5.4, 2H), 2.63 (d, J ) 5.4, 2H), 2.70 (d, J ) 12.8, 1H), 2.88 (dd, J ) 5.0, 12.8, 1H), 3.17-3.23 (m, 1H), 3.36-3.38 (m, 4H), 3.54-3.56 (m, 4H), 3.63 (s, 4H), 4.29-4.32 (m, 1H), 4.48-4.51 (m, 1H), 5.75 (s, 1H), 6.05 (d, J ) 16.0, 1H), 7.60 (d, J ) 16.0, 1H); δD (77 MHz, CH3OH) 0.88 (s, br, 3D), 1.63 (s, br, 2D), 7.71 (s, 1D); δC (125 MHz, CD3OD) 19.4 (CH3), 22.9 (CH3), 23.6 (CH3), 26.5 (CH2), 28.5 (CH2), 28.8 (CH2), 29.7 (CH2), 30.9 (CH2), 36.0 (CH2), 38.9 (C), 39.1 (CH2), 39.3 (CH2), 40.1 (CH2), 56.0 (CH), 60.6 (CH), 62.4 (CH), 69.60 (CH2), 69.63 (CH2), 70.31 (CH2), 70.34 (CH2), 79.3 (C), 122.0 (CH), 128.4 (CH), 136.4 (CH), 146.4 (C), 150.0 (C), 154.4 (C), 165.1 (C), 170.7 (C), 170.8 (C), 173.7 (C), 175.2 (C); HRMS (ESI): m/z requires (C35H48N6O9SD6Na) 763.3942, found 763.3957, 100% [M + Na]+.
RESULTS AND DISCUSSION Acyl hydrazones including 2-5 have been previously reported (4, 5, 9, 10) and used in bioassays; hence, as part of a chemical genomic study on the action of abscisic acid requiring well-defined conjugates, related acyl hydrazone 8 was prepared (Scheme 1). A flexible and hydrophilic linker derived from 2-(2(2-aminoethoxy)ethoxy)ethanamine was used for conjugating D-biotin to S-(+)-abscisic acid. Biotin hydrazide 7 was prepared from previously reported acid 6 (12) and then coupled to commercial S-(+)-abscisic acid, yielding 8 through a modifica-
1358 Bioconjugate Chem., Vol. 18, No. 4, 2007
Smith et al.
Figure 5. Deuterated ABA conjugate d6-8.
tion of the method reported by Kohler et al. (5). Biological activity of this conjugate was observed in a stomatal aperture assay (Figure 3) and shows similar levels of activity to the biotinylated abscisic acid conjugate 2 reported by Yamazaki et al. (9). Conflicting reports concerning the stability of acyl hydrazones to hydrolysis led us to assay the purity of our new hydrazone by LC-MS before and after in Vitro storage under a variety of conditions including in artificial sap (AS) (11) at a range of pH values (Figure 4), in addition to the usual methods used to confirm identity (see Experimental Section). This data clearly showed that the acyl hydrazone was stable in acidic medium at pH g 4.0, thus validating its use in the relatively less acidic in ViVo plant environment but in line with a general acid-catalyzed hydrolysis mechanism, decomposed at pH e 3.0. It has been proposed that acyl hydrazone conjugates of abscisic acid may hydrolyze in ViVo (14), but this hypothesis has not been fully tested, so we extended this work to the study of their stabilities in ViVo using a modification of the LC-MS2 technique described by Go´mez-Cadenas and co-workers (15). The detection of ABA in plant extracts by LC-MS2 has been rigorously studied (16-18) and provides a rapid means of assessing whether in ViVo hydrolysis has taken place. The labeled conjugate d6-8 (Figure 5) was prepared analogously to 8 using d6-ABA, which allows the conjugated ABA to be distinguished from endogenous ABA in the plant. We were able to enrich the total deuterium content of our d6-ABA to about 94% (determined by 1H NMR) by submitting S-(+)-ABA to two successive rounds of deuterium exchange as described by Hirai and co-workers (13). The same authors demonstrated that the deuterium labels in this d6-ABA were sufficiently stable for use as an internal standard for quantitative analysis. The deuterium content of conjugate d6-8 was similarly assessed and estimated at 92%. If in ViVo hydrolysis of the deuterated biotinylated conjugate d6-8 occurs, then free abscisic acid would be released, indicating an apparent biological activity erroneously attributed to the conjugate. In the absence of in ViVo hydrolysis, it is reasonable to assume that the intact conjugate is active. ABA and the deuterated conjugate d6-8 was fed to detached tomato leaves in a solution of AS. The rate of uptake of the AS was determined gravimetrically and compared to the rate of untreated leaves. Since ABA and our nondeuterated conjugate reduce transpiration by effecting stomatal closure, the rate of AS uptake was, as expected, reduced (Figure 3). The tomato leaves treated with d6-8 were homogenized, lyophilized, extracted with water, and immediately submitted to LC-MS2 analysis. Although extraction of abscisic acid using water has been validated in previous work (15), it should be noted that these conditions may not be optimal for isolation of conjugate d6-8. Spectrometric analysis of the extract was performed in negative-mode ESI to detect the well-characterized (15, 18) fragmentation pattern of abscisic acid (263-153 Da)
Figure 6.
and our d6-abscisic acid (269-159 Da). Multiple reaction monitoring (MRM) of the fragmentation to secondary ion peaks together with comparison of retention times of known samples provides unequivocal confirmation of the presence of these compounds. The ESI mass spectrometry parameters were optimized for d6-abscisic acid by direct infusion of pure samples; however, a low level of d6-8 could also be detected. A pure sample of d6-8 gave fragments from 739 Da, [M]- to 455 and 409 Da. The exact nature of the latter fragment is unknown, but the 455 Da species is consistent with the elemental composition C20H31N4O6S, which we postulate to be the deprotonated ion of the structure shown in Figure 6. This fragment was the most intense, so we chose to monitor the 739455 Da fragmentation as part of our MRM settings. The results (Figure 7) show the presence of a large peak representing endogenous abscisic acid and a small peak for the conjugate d6-8. Crucially, d6-abscisic acid was also detected with a peak area of about 15% of the endogenous abscisic acid (we found that d6-abscisic acid and its fragments have similar ion intensity to those of nondeuterated abscisic acid, allowing comparison of these two peak areas). This result was observed in two biological replicates. A control experiment was performed to ensure that hydrolysis of d6-8 had not occurred in sample preparation or during the LC-MS2 run. Plant cuttings were fed untreated AS, then prior to homogenization were spiked with d6-8. After processing these cuttings in the manner described above, the plant extract was analyzed by LC-MS2. Only endogenous abscisic acid and d6-8 were detected (data not shown), showing that decomposition of d6-8 had indeed occurred inside the living plant. We conclude that the hydrolysis of acyl hydrazone conjugates takes place in living plant tissue at a rate sufficient to account for observed biological activity, although the mechanism and site of decomposition are presently unknown. The data support earlier reports of the inactivity of 4′-carbonyl conjugates (14, 19). The exogenous ABA from the hydrolysis of d6-8 will be rapidly metabolized, making an accurate quantitative analysis
Figure 7. LC-MS2 chromatograms of a plant extract from tomato leaves fed with conjugate d6-8 (100 µmol dm-3) showing endogenous ABA (10.55 min), d6-abscisic acid (10.52 min), and d6-8 (5.59 min). Chromatograms are offset.
Bioconjugate Chem., Vol. 18, No. 4, 2007 1359
Technical Notes
of the extent of hydrolysis problematic. Quantification of deuterated ABA catabolites phaseic acid, dihydrophaseic acid, and abscisic acid glucose ester would need to be incorporated into any final quantitative analysis. Exogenous ABA is also likely to trigger increased biosynthesis and metabolism of endogenous ABA (20). In summary, knowledge of the in ViVo stability of bioconjugates is important to the assessment of bioactivity: LC-MS2 is widely available and ideally suited to such evaluations.
ACKNOWLEDGMENT We thank the BBSRC for a BMS Committee Studentship (T.R.S.), and thank Dr. A. J. Clarke, Mr. J. C. Bickerton for technical assistance with NMR, mass spectrometry, respectively, and Dr. C. Corre for assistance with LC-MS experiments. We thank the reviewers for helpful comments.
LITERATURE CITED (1) Schroeder, J. I., and Kuhn, J. M. (2006) Plant biology-abscisic acid in bloom. Nature (London) 439, 277-278. (2) Zaharia, L. I., Walker-Simmon, M. K., Rodriguez, C. N., and Abrams, S. R. (2005) Chemistry of abscisic acid, abscisic acid catabolites and analogs. J. Plant Growth Regul. 24, 274-284. (3) Quarrie, S. A., and Galfre, G. (1985) Use of different hapten-protein conjugates immobilised on nitrocellulose to screen monoclonal antibodies to abscisic acid. Anal. Biochem. 151, 389-399. (4) Asami, T., Tao, L., Yamamoto, S., Robertson, M., Min, Y-K., Murofushi, N., and Yoshida, S. (1997) Fluorescence-labeled abscisic acid possessing abscisic acid-like activity in barley aleurone protoplasts. Biosci. Biotechnol. Biochem. 61, 1198-1199. (5) Kohler, A. D., Beale, M. H., Rollason, R., Barratt, D. H. P., Lewis, M. J., Van der Meulen, R. M., and Wang, M. (1997) A radioiodinated abscisic acid photoaffinity probe. J. Chem. Soc., Perkin Trans. 1 (10), 1543-1547. (6) Nyangulu, J. M., Galka, M. M., Jadhav, A., Gai, Y., Graham, C. M., Nelson, K. M., Cutler, A. J., Taylor, D. C., Banowetz, G. M., and Abrams, S. R. (2005) An affinity probe for isolation of abscisic acid-binding proteins. J. Am. Chem. Soc. 127, 1662-1664. Nyangulu, J. M., Nelson, K. M., Rose, P. A., Gai, Y., Loewen, M., Lougheed, B., Quail, J. W., Cutler, A. J., and Abrams, S. R. (2006) Synthesis and biological activity of tetralone abscisic acid analogues. Org. Biomol. Chem. 7, 1400-1412. (7) Razem, F. A., El-Kereamy, A., Abrams, S. R., and Hill, R. D. (2006) The RNA-binding protein FCA is an abscisic acid receptor. Nature (London) 439, 290-294. (8) Shen, Y. Y., Wang, X. F., Wu, F. Q., Du, S. Y., Cao, Z., Shang, Y., Wang, X. L., Peng, C. C., Yu, X. C., Zhu, S. Y., Fan, R. C., Xu, Y. H., and Zhang, D. P. (2006) The Mg-chelatase H subunit is an abscisic acid receptor. Nature (London) 443, 823-826.
(9) Yamazaki, D., Yoshida, S., Asami, T., and Kuchitsu, K. (2003) Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells. Plant J. 35, 129-139. (10) Hornberg, C., and Weiler, E. W. (1984) High-affinity binding sites for abscisic acid on the plasmalemma of Vicia faba guard cells. Nature (London) 310, 321-324. (11) Wilkinson, S., and Davies, W. J. (1997) Xylem sap pH increase: a drought signal received at the apoplastic face of the guard cell that involves the suppression of saturable abscisic acid uptake by the epidermal symplast. Plant Physiol. 113, 559-573. (12) Trester-Zedlitz, M., Kamada, K., Burley, S. K., Fenyo¨, D., Chait, B. T., and Muir, T. W. (2003) A modular cross-linking approach for exploring protein interactions. J. Am. Chem. Soc. 125, 24162425. (13) Hirai, N., Kondo, S., and Ohigashi, H. (2003) Deuterium-labeled phaseic acid and dihydrophaseic acids for internal standards. Biosci. Biotechnol. Biochem. 67, 2408-2415. (14) Hite, D. R. C., Outlaw, W. H., and Seavy, M. A. (1994) Substitution of hydrazones for the 4′ carbonyl on abscisic acid rendered it ineffective in a rapid stomatal-opening-inhibition bioassay. Physiol. Plant. 92, 79-84. (15) Go´mez-Cadenas, A., Pozo, O. J., Garcı´a-Augustı´n, P., and Sancho, J. V. (2002) Direct analysis of abscisic acid in crude plant extracts by liquid chromatography-electrospray/tandem mass spectroscopy. Phytochem. Anal. 13, 228-234. (16) Lo´pez-Carbonell, M., and Ja´uregui, O. (2005) A rapid method for analysis of abscisic acid (ABA) in crude extracts of water stressed Arabidopsis thaliana plants by liquid chromatography-mass spectrometry in tandem mode. Plant Physiol. Biochem. 43, 407-411. (17) Durgbanshi, A., Arbona, V., Pozo, O., Miersch, O., Sancho, J. V., and Go´mez-Cadenas, A. (2005) Simultaneous determination of multiple phytohormones in plant extracts by liquid chromatographyelectrospray tandem mass spectroscopy. J. Agric. Food Chem. 53, 8437-8442. (18) Ross, A. R. S., Ambrose, S. J., Cutler, A. J., Feurtado, J. A., Kermode, A. R., Nelson, K., Zhou, R., and Abrams, S. R. (2004) Determination of endogenous and supplied deuterated abscisic acid in plant tissue by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring. Anal. Biochem. 329, 324-333. (19) Van der Meulen, R. M., Heidekamp, F., Jastorff, B., Horgan, R., and Wang, M. (1993) Effects of abscisic acid analogues on abscisic acid-induced gene expression in barley aleurone protoplasts: relationship between structure and function of the abscisic acid molecule. J. Plant Growth Regul. 12, 13-19. (20) Lin, B., Wang, H., Wang, J., Zaharia, L. I., and Abrams, S. R. (2005) Abscisic acid regulation of heterophylly in Marsilea quadrifolia L.: effects of R-(-) and S-(+) isomers. J. Exp. Bot. 56, 2935-2948. BC070069X
