Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [¹³C₆]-IAA or [²H₆]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.     

Detection of endogenous gibberellins and their relationship to hypocotyl elongation in soybean seedlings

Four gibberellins, GA₅₃, GA₁₉, GA₂₀, and GA₁, were detected by bioassay, chromatography in two HPLC systems, and combined gas chromatography-mass spectroscopy-selected ion monitoring (GC-MS-SIM) in etiolated soybean (Glycine max [L.] Merr.) hypocotyls. GC-MS-SIM employed [²H₂]-labeled standards for each endogenous gibberellin detected, and quantities estimated from bioassays and GC-MS-SIM were similar. This result plus the tentative detection of GA₄₄ and GA₈ (standards not available) indicates that the early-C-13-hydroxylation pathway for gibberellin biosynthesis predominates in soybean hypocotyls. Other gibberellins were not detected. Growth rates decreased after transfer to low water potential (ψ_w) vermiculite and were completely arrested 24 hours after transfer. The GA₁ content in the elongating region of hypocotyls had declined to 38% of the 0 time value at 24 hours after transfer to low ψ_w vermiculite, a level which was only 13% of the GA₁ content in control seedlings at the same time (24 hours posttransfer). Rewatering seedlings following 24 hours growth in low ψ_w vermiculite resulted in a complete recovery in elongation rate, an increase in GA₁ (20% at 2 hours, two-fold at 8 hours, eightfold at 24 hours), and a decrease in ABA levels (tenfold at 2 hours). Treatment of well-watered seedlings with the GA-synthesis inhibitor tetcyclacis (TCY) resulted in lowered GA₁ levels and increased ABA levels. When seedlings grown 24 hours in low ψ_w vermiculite were rewatered with TCY, recovery of the elongation rate was delayed and reduced, and the decline in ABA levels was slowed. Addition of GA₃ restored the elongation rate inhibited by TCY. Seedlings were growth responsive to exogenous GA₃, and this GA₃-promoted growth was inhibited by exogenous ABA. The data are consistent with the hypothesis that changes in GA₁ and ABA levels play a role in adjusting hypocotyl elongation rates. However, the changes observed are not of sufficient magnitude nor do they occur rapidly enough to suggest they are the primary regulators of elongation rate responses to rapidly changing plant water status.     

Abscisic Acid Biosynthesis in Isolated Embryos of Zea mays L

Previous labeling experiments with ¹⁸O₂ have supported the hypothesis that stress-induced abscisic acid (ABA) is synthesized through an indirect pathway involving an oxygenated carotenoid (xanthophyll) as a precursor. To investigate ABA formation under nonstress conditions, an ¹⁸O₂ labeling experiment was conducted with isolated embryos from in vitro grown maize (Zea mays L.) kernels. Of the ABA produced during the incubation in ¹⁸O₂, three-fourths contained a single ¹⁸O atom located in the carboxyl group. Approximately one-fourth of the ABA synthesized during the experiment contained two ¹⁸O atoms. These results suggest that ABA synthesized in maize embryos under nonstress conditions also proceeds via the indirect pathway, requiring a xanthophyll precursor. It was also found that the newly synthesized ABA was preferentially released into the surrounding medium.     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable ¹³C₆-isotope of pABA (p- amino[aromatic-¹³C₆]benzoic acid ([¹³C₆]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[¹³C₆]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [¹³C₆]pABA or [¹³C₆]4HB generate both ¹³C₆-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product ¹³C₆-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ₆. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ₆ quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.     

Characterization and use in ELISA of a new monoclonal antibody for quantitation of abscisic acid in senescing rice leaves

A new monoclonal antibody (mAb) was generated against abscisic acid (ABA), and an indirect enzyme-linked immunosorbent assay (ELISA) using this mAb was developed for convenient quantitative analysis of ABA levels in rice leaf extracts. The mAb, raised against (+-)-ABA conjugated to bovine serum albumin (BSA) through its carboxyl group (C₁), reacted preferentially with the (+)-ABA enantiomer, and equally well with both free and methyl-ester (+-)-ABA. Cross-reactivity with several ABA-related compounds was negligible. Linearity was obtained between 3 and 1000 pmo1 of (+)-ABA. The ABA-mAb was further used to quantitate pmol quantities of (+)-ABA in attached and detached rice leaves. Results obtained with such ELISA quantitation showed an increase in the free ABA content of detached rice leaves at progressive stages of senescence, which was regarded as a senescence-related response. This quantitation compared favorably with other presently used techniques for ABA determination, with regard to their detection limits, cost and assay time. The results suggest that the combination of a specific mAb with a sensitive ELISA technique is quite promising for quantitation of ABA.     

Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

Effect of drought and ABA on growth, photosynthesis and antioxidant system of Cotinus coggygria seedlings under two different light conditions

We exposed seedlings of Cotinus coggygria var. cinerea to drought and exogenous abscisic acid (ABA) under two different light conditions. Two watering regimes (well-watered and drought), two exogenous ABA applications (no ABA and with ABA) and two light regimes (full sunlight and shade) were employed. Compared with well-watered treatment, drought treatment significantly reduced the relative growth rate, relative water content (RWC), net photosynthesis rate (A) and transpiration (E), but increased chlorophyll a (chla), carbon isotope (δ¹³C), endogenous ABA, malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents, and guaiacol peroxidase (POD) and catalase (CAT) activities. There was an apparent alleviation of drought effects by shade, as indicated by the lower relative growth rate, and chlorophyll, MDA and H₂O₂ contents, and increases in indoleacetic acid (IAA) and reduced glutathione (GSH) contents. On the other hand, the exogenous ABA application under shade induced protective effects on drought-stressed seedlings, as visible in RWC, MDA, A, stomatal conductance (g_s), E, δ¹³C, ABA and IAA values. In all, our results suggest that seedlings of C. coggygria are more sensitive to drought under full-light than under shade.     

Xanthophylls and abscisic Acid biosynthesis in water-stressed bean leaves

Experiments were designed to obtain evidence about the possible role of xanthophylls as abscisic acid (ABA) precursors in water-stressed leaves of Phaseolus vularis L. Leaves were exposed to ¹⁴CO₂ and the specific activities of several major leaf xanthophylls and stress-induced ABA were determined after a chase in ¹²CO₂ for varying periods of time. The ABA specific radioactivities were about 30 to 70% of that of lutein and violaxanthin regardless of the chase period. The specific activity of neoxanthin, however, was only about 15% of that of ABA. The effects of fluridone on xanthophyll and ABA levels and the extent of labeling of both from ¹⁴CO₂ were determined. Fluridone did not inhibit the accumulation of ABA when leaves were stressed once, although subsequent stresses in the presence of fluridone did lead to a reduced ABA accumulation. The incorporation of ¹⁴C from ¹⁴CO₂ into ABA and the xanthophylls was inhibited by fluridone and to about the same extent. The incorporation of ¹⁸O into ABA from violaxanthin which had been labeled in situ by means of the violaxanthin cycle was measured. The results indicated that a portion of the ABA accumulated during stress was formed from violaxanthin which had been labeled with ¹⁸O. The results of these experiments are consistent with a preformed xanthophyll(s) as the major ABA precursor in water-stressed bean leaves.     

Changes in the levels of abscisic acid and its metabolites resulting from chilling of tomato fruits

The 6,6,6-[²H]-analogues of abscisic acid (ABA), phaseic (PA) and dihydrophaseic (DPA) acids were used in GC-MS-SIM determination of free and total alkali hydrolyzable ABA, PA and DPA in the pericarp of tomato (Lycopersicon esculentum L. cv. Pik Red) fruit. Determinations were made on breaker-stage fruit stored 1, 2, 3 or 4 weeks at 2.5°C or at 10°C, and after subsequent ripening for 1 week in darkness at 20°C. Two-fold increases in levels of ABA occurred after storage at low temperatures with a slightly but significantly greater increase in ABA level occurring with 2.5°C storage. These increases in ABA levels were not associated with the appearance of damage symptoms that occurred with storage at the chilling temperature (2.5°C). Differences in ABA metabolism were found resulting from storage at the two temperatures, 2.5 or 10°C. Significantly greater DPA levels were found after 10°C storage than after 2.5°C storage (2 weeks). Levels of ABA ester-conjugates increased with 20°C ripening only after 10°C storage while free ABA levels decreased after both storage temperature conditions. Levels of DPA conjugates also increased only after 20°C ripening following 10°C storage. A longer period of storage resulted in decreases of free DPA levels after 10°C storage but increased DPA levels were found after 2.5°C storage.Abbreviations ABA abscisic acid - PA phaseic acid - DPA dihydrophaseic acid - GC-MS-SIM gas chromatography-mass spectrometry-selected ion monitoring - HPLC high pressure liquid chromatography - fw. fresh weight author for correspondence     

The stereochemistry of cyclization in abscisic acid

The hydroxylation of the pro-6′-(R)-methyl of (+)-abscisic acid, which then cyclises to phaseic acid, was used to define the origin in mevalonate of the 6′-methyl groups. Abscisic acid (ABA), biosynthesised from [2-¹⁴C, 2-³H₂]-mevalonate, was metabolized to phaseic acid by tomato shoots. The slight loss of [³H] from the phaseate, and to a lesser extent from the ABA, suggested that the unlabelled 6′-methyl was hydroxylated. This was confirmed by Kuhn-Roth oxidation of methyl phaseate to give [¹⁴C, ³H]-acetate. The data also suggest that ABA is converted to dihydrophaseate via free phaseate, the conjugates being formed from each free acid.     

Implication of Gibberellins in Head Smut (Sporisorium reilianum) of Sorghum bicolor

The head smut fungus, Sporisorium reilianum ([Kuhn] Landon and Fullerton), was shown to reduce plant height in infected Sorghum bicolor ([L.] Moench) plants. The major reductions occurred in the internodes nearest the panicle and were more severe in naturally infected than in inoculated plants. Less affected plants developed reproductively sterile panicles, and eventually smutted panicles developed phyllodied growths which progressed into leafy shoots. Extracts of smutted, sterile, and healthy (control) panicles of field-grown plants exhibited gibberellin (GA)-like activity in the dwarf rice bioassay. When extracts were purified and assayed with deuterium-labeled GA standards by gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM), GA₁, GA₃, GA₁₉, GA₂₀, and GA₅₃ were detected based on coelution with the standards, identical Kovats retention index values, and matching ion masses and relative abundances for three major ions. In addition, based on published Kovats retention index values, ion masses, and relative abundance values, GA₄, GA₇, GA₈, GA₁₄, GA₂₉, and GA₄₄ were tentatively identified. Quantitative analysis revealed that panicles of healthy control plants contained from 60 to 100% higher total concentrations of GAs than panicles of smutted plants. These comparisons were most striking for the early 13-hydroxylation pathway precursors GA₅₃, GA₄₄, and GA₁₉ but not for GA₂₀. Extracts of S. reilianum sporidia and culture medium exhibited GA-like bioactivity, and GA₁ and GA₃ were detected based on GC-MS-SIM assay with ²H-labeled internal standards. Quantitative analysis of these GAs showed increasing concentrations from 4 to 7 to 10 days of culture and a decline at 20 days. This is the first GC-MS-SIM detection of GAs in a non-Ascomycete fungus, and the disease symptoms and quantitative data suggested that fungal infection may interfere with biosynthesis of GAs by the host plant.     

Chemical and hydraulic signals regulate stomatal behavior and photosynthetic activity in maize during progressive drought

The objectives of this study were to investigate stomatal regulation in maize seedlings during progressive soil drying and to determine the impact of stomatal movement on photosynthetic activity. In well-watered and drought-stressed plants, leaf water potential (Ψ _leaf), relative water content (RWC), stomatal conductance (g _s), photosynthesis, chlorophyll fluorescence, leaf instantaneous water use efficiency (iWUE_leaf), and abscisic acid (ABA) and zeatin-riboside (ZR) accumulation were measured. Results showed that g _s decreased significantly with progressive drought and stomatal limitations were responsible for inhibiting photosynthesis in the initial stages of short-term drought. However, after 5 days of withholding water, non-stomatal limitations, such as damage to the PSII reaction center, became the main limiting factor. Stomatal behavior was correlated with changes in both hydraulic and chemical signals; however, changes in ABA and ZR occurred prior to any change in leaf water status. ABA in leaf and root tissue increased progressively during soil drying, and further analysis found that leaf ABA was negatively correlated with g _s (R ² = 0.907, p < 0.05). In contrast, leaf and root ZR decreased gradually. ZR in leaf tissue was positively correlated with g _s (R ² = 0.859, p < 0.05). These results indicate that ABA could induce stomatal closure, and ZR works antagonistically against ABA in stomatal behavior. In addition, the ABA/ZR ratio also had a strong correlation with g _s, suggesting that the combined chemical signal (the interaction between ABA and cytokinin) plays a role in coordinating stomatal behavior. In addition, Ψ _leaf and RWC decreased significantly after only 3 days of drought stress, also affecting stomatal behavior.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.     

Quantitative models characterizing seed germination responses to abscisic Acid and osmoticum

Mathematical models were developed to characterize the physiological bases of the responses of tomato (Lycopersicon esculentum Mill. cv T5) seed germination to water potential (ψ) and abscisic acid (ABA). Using probit analysis, three parameters were derived that can describe the germination time courses of a seed population at different ψ or ABA levels. For the response of seed germination to reduced ψ, these parameters are the mean base water potential (¯ψ_b, MPa), the standard deviation of the base water potential among seeds in the population (σ_ψb, MPa), and the “hydrotime constant” (θ_H, MPa·h). For the response to ABA, they are the log of the mean base ABA concentration ([unk]ABA_b, m), the standard deviation of the base ABA concentration among seeds in the population (σ_ABAb, log[m]), and the “ABA-time constant” (θ_ABA, log[m]·h). The values of ¯ψ_b and [unk]ABA_b provide quantitative estimates of the mean sensitivity of germination rate to ψ or ABA, whereas σ^ψ_b and σ_ABAb account for the variation in sensitivity among seeds in the population. The time constants, θ_H and θ_ABA, indicate the extent to which germination rate will be affected by a given change in ψ or ABA. Using only these parameters, germination time courses can be predicted with reasonable accuracy at any medium ψ according to the equation probit(g) = [ψ - (θ_H/t_g) - ¯ψ_b]/σ_ψb, or at any ABA concentration according to the equation probit(g) = [log[ABA] - (θ_ABA/t_g) - log[[unk]ABA_b]]/σ_ABAb, where t_g is the time to radicle emergence of percentage g, and ABA is the ABA concentration (m) in the incubation solution. In the presence of both ABA and reduced ψ, the same parameters can be used to predict seed germination time courses based upon strictly additive effects of ψ and ABA in delaying the time of radicle emergence. Further analysis indicates that ABA and ψ can act both independently and interactively to influence physiological processes preparatory for radicle growth, such as the accumulation of osmotic solutes in the embryo. The models provide quantitative values for the sensitivity of germination to ABA or ψ, allow evaluation of independent and interactive effects of the two factors, and have implications for understanding how ABA and ψ may regulate growth and development.     

Gas Exchange, Stomatal Behavior, and deltaC Values of the flacca Tomato Mutant in Relation to Abscisic Acid

The relationship between stomatal conductance and capacity for assimilation was investigated in flacca, a mutant of tomato (Lycopersicon esculentum Mill.) that has abnormal stomatal behavior and low abscisic acid (ABA) content. The assimilation capacity, determined by measuring assimilation rate as a function of intercellular CO₂ pressure, did not differ in leaves of flacca and its parent variety, Rheinlands Ruhm (RR). On the other hand, stomatal conductance of flacca leaves was greater than that of RR, and could be phenotypically reverted by spraying with 30 micromolar ABA. Stomatal conductance of flacca leaves was also reduced by increasing CO₂ pressure, increasing leaf to air vapor pressure difference, and decreasing quantum flux, irrespective of ABA treatment.

The high conductance of flacca leaves resulted in a high intercellular CO₂ pressure. This allowed greater discrimination against ¹³CO₂, as evidenced by more negative δ ¹³C values for flacca as compared to RR. The δ ¹³C values of both flacca and RR plants as influenced by ABA treatment were consistent with predictions based on gas exchange measurements, using a recent model of discrimination.

     

Rapid separation and quantification of abscisic Acid from plant tissues using high performance liquid chromatography

Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-¹⁴C]ABA, of which 98.9% was recovered.     

Abscisic alcohol is an intermediate in abscisic Acid biosynthesis in a shunt pathway from abscisic aldehyde

It has previously been shown that the abscisic acid (ABA)-deficient flacca and sitiens mutants of tomato are impaired in ABA-aldehyde oxidation and accumulate trans-ABA-alcohol as a result of the biosynthetic block (IB Taylor, RST Linforth, RJ Al-Naieb, WR Bowman, BA Marples [1988] Plant Cell Environ 11: 739-745). Here we report that the flacca and sitiens mutants accumulate trans-ABA and trans-ABA glucose ester and that this accumulation is due to trans-ABA biosynthesis. ¹⁸O labeling of water-stressed wild-type and mutant tomato leaves and analysis of [¹⁸O]ABA by tandem mass spectrometry show that the tomato mutants synthesize a significant percentage of their ABA and trans-ABA as [¹⁸O]ABA with two ¹⁸O atoms in the carboxyl group. We further show, by feeding experiments with [²H₆]ABA-alcohol and ¹⁸O₂, that this doubly-carboxyl-labeled ABA is synthesized from [¹⁸O]ABA-alcohol with incorporation of molecular oxygen. In vivo inhibition of [²H₆]ABA-alcohol oxidation by carbon monoxide establishes the involvement of a P-450 monooxygenase. Likewise, carbon monoxide inhibits the synthesis of doubly-carboxyl-labeled ABA in ¹⁸O-labeling experiments. This minor shunt pathway from ABA-aldehyde to ABA-alcohol to ABA operates in all plants examined. For the ABA-deficient mutants impaired in ABA-aldehyde oxidation, this shunt pathway is an important source of ABA and is physiologically significant.     

Abscisic Acid accelerates adaptation of cultured tobacco cells to salt

Adaptation of tobacco (Nicotiana tabacum L. var Wisconsin 38) cells to NaCl was accelerated by (±) abscisic acid (ABA). In medium with 10 grams per liter NaCl, ABA stimulated the growth of cells not grown in medium with NaCl (unadapted, S-0) with an increasing response from 10⁻⁸ to 10⁻⁴ molar. ABA (10⁻⁵ molar) enhanced the growth of unadapted cells in medium with 6 to 22 grams per liter NaCl but did not increase the growth of cells previously adapted to either 10 (S-10) or 25 (S-25) grams per liter NaCl unless the cells were inoculated into medium with a level of NaCl higher than the level to which the cells were adapted. The growth of unadapted cells in medium with Na₂SO₄ (85.5 millimolar), KCl (85.5 or 171 millimolar), K₂SO₄ (85.5 millimolar) was also stimulated by ABA. ABA (10⁻⁸-10⁻⁴ molar) did not accelerate the growth of unadapted cells exposed to water deficits induced by polyethylene glycol (molecular weight 8000) (5-20 grams per 100 milliliters), sorbitol (342 millimolar), mannitol (342 millimolar) or sucrose (342 millimolar). These results suggest that ABA is involved in adaptation of cells to salts, and is not effective in promoting adaptation to water deficits elicited by nonionic osmotic solutes.