Abscisic acid biosynthesis in roots

The pathway of water-stress-induced abscisic acid (ABA) biosynthesis in etiolated and light-grown leaves has been elucidated (see A.D. Parry and R. Horgan, 1991, Physiol. Plant. 82, 320–326). Roots also have the ability to synthesise ABA in response to stress and it was therefore of interest to examine root extracts for the presence of carotenoids, including those known to be ABA precursors in leaves. All-trans- and 9-cis-neoxanthin, all-trans- and 9-cis-violaxanthin, antheraxanthin (all potential ABA precursors), lutein and -carotene were identified on the basis of absorbance spectra, reactions with dilute acid, retention times upon high-performance liquid chromatography and by comparison with leaf carotenoids that had been analysed by mass spectrometry. The source of the extracted carotenoids was proved to be root tissue, and not contaminating compost or leaf material. The levels of total carotenoids in roots varied between 0.03–0.07% of the levels in light-grown leaves (Arabidopsis thaliana (L.) Heynh, Nicotiana plumbaginifolia Viv., Phaseolus vulgaris L. and Pisum sativum L.) up to 0.27% (Lycopersicon esculentum Mill.). The relative carotenoid composition was very different from that found in leaves, and varied much more between species. All-trans-neoxanthin and violaxanthin were the major carotenoids present (64–91 % of the total), but while Lycopersicon contained 67–80% all trans-neoxanthin, Phaseolus, Pisum and Zea mays L. contained 61–79% all-trans-violaxanthin. Carotenoid metabolism also varied between species, with most of the carotenoids in older roots of Phaseolus being esterified. Roots and leaves of the ABA-deficient aba mutant of Arabidopsis had reduced epoxy-xanthophyll levels compared to the wild-type.Abbreviations ABA abscisic acid - r.p.HPLC reversed-phase high performance liquid chromatography The authors would like to thank Dr. B.H. Davies for helpful discussions and Mrs. A.F. Rees for her excellent technical assistance. A.D.P. was supported by a grant from the Agricultural and Food Research Council, from whom funds were also obtained to purchase the HPLC-photodiode-array detector.     

Carotenoids and abscisic acid (ABA) biosynthesis in higher plants

Recent research has revealed that abscisic acid (ABA), synthesised in response to water stress, is an apo-carotenoid. Two potential carotenoid precursors, 9'- cis -neoxanthin and 9- cis -violaxanthin, have been identified in light-grown and etiolated leaves, and in roots of a variety of species. Experiments utilizing etiolated Phaseolus vulgaris leaves and deuterium oxide strongly suggest that 9'- cis -neoxanthin, synthesised from all- trans -violaxanthin, is the immediate pre-cleavage precursor of ABA. The cleavage of 9'- cis -neoxanthin, performed by an inducible and specific dioxygenase, is likely to be the rate-limiting step in ABA biosynthesis. Any apocarotenoids formed as by-products of cleavage are probably rapidly degraded by lipoxygenase or related enzymes. After cleavage xanthoxin is converted via ABA-aldehyde to ABA by constitutive enzymes in the cytosol.     

Abscisic acid metabolism —vacuolar/extravacuolar distribution of metabolites

The biotransformation of abscisic acid (ABA) was studied in cell suspension cultures of Lycopersicon esculentum. The ABA was converted by the cells to phaseic acid, nigellic acid, dihydrophaseic acid, abscisic acid--D-glucopyranosyl ester (ABA-Glc) and other ABA and phaseic acid conjugates. Investigation of their cellular distribution showed that the conjugated forms were located only in the vacuoles whereas ABA and its acidic metabolites were found mainly in the extravacuolar fractions. Our results, together with a number of studies on the increase of ABA-Glc as a response to stress, allow us to propose that ABA-Glc is irreversibly compartmented in the vacuoles of plant cells.Abbreviations ABA abscisic acid - ABA-Glc -D-glucopyranosyl ester of ABA - DPA 4-dihydrophaseic acid; nigellic acid=3-methyl-5-(1-hydroxy-2-hydroxymethyl-6-dimethyl-4-oxo-cyclohex-2-enyl)-penta-2Z, 4E-dienoic acid - PA phaseic acid     

The effects of 2,2′-bipyridine and other metal chelators on the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in rice

Effects of metal chelators, 2,2-bipyridine, 8-hydroxyquinoline and 1,10-phenenthroline, on the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in detached leaves of light-grown rice (Oryza sativa) seedlings and detached shoots of etiolated rice seedlings were investigated. Metal chelators strongly inhibited the in vivo ACC oxidase activity in detached leaves and detached etiolated shoots. This inhibition could be partially recovered by Fe²⁺. Our results support the notion that Fe²⁺ is an essential cofactor for the conversion of ACC to ethylene in vivo.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - BP 2,2-bypyridine - HQ 8-hydroxylquinoline - MJ methyl jasmonate - PA 1,10-phenanthroline - Put putrescine     

Dormancy termination of western white pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy ABA (7OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC–tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7OH ABA and ABA-GE also accumulated during moist chilling; however, 7OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.Abbreviations ABA abscisic acid - ABA-GE abscisic acid glucose ester - DPA dihydrophaseic acid - 7OH ABA 7-hydroxy abscisic acid - 8OH ABA 8-hydroxy abscisic acid - MRM multiple reaction monitoring - PA phaseic acid     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase     

Xanthoxin levels and metabolism in the wild-type and wilty mutants of tomato

Using ¹³C-labelled internal standards and gas chromatography-mass spectrometry/multiple-ion monitoring the levels of xanthoxin (Xan) and 2-trans-xanthoxin (t-Xan) have been determined in stressed and non-stressed leaves of wildtype tomato (Lycopersicon esculentum Mill cv. Ailsa Craig), and the wilty mutants, notabilis (not), flacca (flc) and sitiens (sit). Levels of Xan were very low in all tissues. Ratios of t-Xan: Xan ranged from 10:1 to <500:1. In the wild-type and flc, t-Xan levels increased following stress. The results from feeding experiments using [¹³C]Xan and t-Xan demonstrated that whilst wild-type and not plants readily converted Xan into abscisic acid (ABA), flc and sit plants converted only a small amount of applied Xan into ABA. In all plants t-Xan was not converted into ABA. These results indicate that the flc and sit mutants are impaired in ABA biosynthesis because they are unable to convert Xan into ABA, whereas the not mutant is blocked at a metabolic step prior to Xan. Another possible ABA precursor, ABA-1,4-trans-diol (ABA-t-diol) was found to occur in wild-type and mutant tissue. All four tissues could convert [²H]ABA-t-diol to ABA. Incubation of stressed leaves in the presence of ¹⁸O₂ provided evidence consistent with Xan and ABA originating via oxidative cleavage of a xanthophyll such as violaxanthin.Abbreviations ABA abscisic acid - ABA-t-diol abscisic acid-1,4-trans-diol - DDC sodium diethyldithiocarbamate - FW fresh weight - GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - MIM multiple-ion monitoring - PA phaseic acid - Xan xanthoxin - flc flacca - not notabilis - sit sitiens     

Identification by combined gas chromatography-mass spectrometry of phaseic acid and dihydrophaseic acid and characterization of further abscisic acid metabolites in pea seedlings

Seven day old seedlings of Pisum sativum L., cv. Kleine Rheinländerin, were wilted for 3 days. After partially removing the roots, they were rewatered and at the same time radioactive abscisic acid([1-¹⁴C]ABA, spec. activity 1.7·10⁸d s^-1mmol^-1) was applied for 1 h via the xylem of the roots. After 24 h, 4 days, and 12 days the seedlings were extracted and the metabolites of ABA were analyzed by means of thin-layer and gas chromatography in combination with mass spectrometry, autoradiography, and scintillation counting. Phaseic acid (PA) and dihydrophaseic acid (DPA) were identified as metabolites of ABA. The presence of another ABA-metabolite was also demonstrated. From its mass spectrum it has been postulated that this metabolite is 4-desoxy-ABA. In addition to these substances, several other metabolites, which are more polar than ABA and its known degradation products, were present in the seedlings. The quantity and number of these unknown metabolites increased with time.Abbreviations ABA abscisic acid - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen bis(5-phenyloxazole)     

Water stress and the catabolism of (±)-abscisic acid by excised leaves of Hordeum vulgare L. cv. Dyan

When excised, light-grown leaves of Hordeum vulgare were fed with (±)-[2-¹⁴C]-abscisic acid and stressed until they had lost 12% of their original fresh weight, marked changes in the distribution of radioactivity between abscisic acid and its catabolites were observed. Wilted leaves were less able than their turgid counterparts to transform (±)-[2-¹⁴C]-abscisic acid into 2-hydroxymethyl abscisic acid, dihydrophaseic acid and water-soluble conjugates of abscisic acid. Water stress had little effect on the production of phaseic acid although refeeding studies with [¹⁴C]-phaseic acid showed that the step from phaseic acid to dihydrophaseic acid was inhibited in wilted leaves. Evidence was obtained which suggested that these changes did not result from dilution of applied, radiolabelled substrate by endogenous abscisic acid. The catabolites of (±)-abscisic acid were identified by capillary gas chromatography-mass spectrometry.     

Induction of 3'-O-beta-D-ribofuranosyl adenosine during compatible, but not during incompatible, interactions of Arabidopsis thaliana or Lycopersicon esculentum with Pseudomonas syringae pathovar tomato

All hitherto identified aromatic compounds accumulating in leaves of Arabidopsis thaliana (L.) Heynh. upon infection with virulent or avirulent strains of Pseudomonas syringae pathovar tomato (Pst) were indolic metabolites. We now report the strong accumulation of a novel type of natural product, 3-O--d-ribofuranosyl adenosine (3RA), exclusively during compatible interactions. In contrast to the various indolic metabolites, 3RA was undetectable in incompatible interactions of A. thaliana leaves with an avirulent Pst strain, as well as in uninfected control leaves. A similar, strong induction of 3RA was observed in compatible but, again, not in incompatible interactions of Pst with its natural host, Lycopersicon esculentum. The strength of the effect and its confinement to compatible interactions suggests that it may be applicable as a diagnostic tool.Abbreviations Pst Pseudomonas syringae pathovar tomato - 3RA 3-O--d-ribofuranosyl adenosine     

Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves

The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9′-cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an ¹⁸O₂-containing atmosphere resulted in the synthesis of ABA with levels of ¹⁸O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA.     

ζ-Carotene<Emphasis Type="Italic"> cis</Emphasis> isomers as products and substrates in the plant poly-<Emphasis Type="Italic">cis</Emphasis> carotenoid biosynthetic pathway to lycopene

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and -carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9-tricis--carotene with 15,9-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by -carotene desaturase, the 15-cis double bond of 9,15,9-tricis--carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9-dicis--carotene is utilized by -carotene desaturase via 7,9,9-tricis-neurosporene to 7,9,7,9-tetracis-lycopene. Other -carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis--carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to -carotene. The 5-cis-lycopene and the 9-cis-and 13-cis--carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

Endogenous plant hormones of the broad bean,Vicia faba L. (-)-jasmonic acid,a plant growth inhibitor in pericarp

(-)-Jasmonic acid was identified as a plant growth inhibitor of the pericarp of Vicia faba by means of gas-liquid chromatography, high resolution mass spectrometry, ¹H-nuclear magnetic resonance (¹H-NMR), and ¹³C-NMR. Additionally, the pericarp contains very small amounts of abscisic acid (ABA) and 4-dihydrophaseic acid. The highest level of jasmonic acid was reached prior to full pericarp length. This amount (3 g g^-1 fresh weight) is similar to the maximal ABA content in the developing seed. Jasmonic acid is a plant growth inhibitor possessing a relative activity in the wheat seedling bioassay of 1–2.5%, compared to ABA. Contrary to ABA, jasmonic acid does not cause retardation of leaf emergence. The possible physiological role of jasmonic acid in the pericarp is discussed and compared with the assumed function of ABA in developing seeds.Abbreviations ABA abscisic acid - DPA 4-dihydrophaseic acid - DPAMeTMS methyl ester trimethylsilyl ether of DPA - EtOAc ethyl acetate - Et₂O ether - MS mass spectrometry - NMR nuclear magnetic resonance - GLC gas-liquid chromatography - TLC thin-layer chromatography - UV ultraviolet light     

On the use of Avena protoplasts to study chloroplast development

Different methods were tested to isolate protoplasts from etiolated, partially greened, and light-grown leaves of Avena sativa. Preparations with high yields and high photosynthetic capacities (time of illumination 4 h) were obtained when small transverse leaf segments were incubated for 2 h at 30°C in 2% cellulysin (Calbiochem), 0.6 M mannitol, and 0.5% bovine serum albumin (BSA) at pH 5.6, without shaking. As measured by light-dependent O₂ evolution or fixation of labeled bicarbonate, protoplasts exhibited rates of up to 124 mol per mg of chlorophyll per h at 20°C and saturating bicarbonate, which were nearly identical to those found with intact leaves. The assay conditions necessary for this activity were 0.6 M sorbitol, 50 mM N-2-hydroxy-ethylpiperazine-N-2-ethane sulfonic acid (pH 7.6), and 10 mM NaHCO₃. If plastids were isolated from these protoplasts, sorbitol was 0.45 M, including 10 mM ethylenediaminetetraacetate (EDTA). under these conditions, rates of photosynthesis were up to 125 (light-grown) and 71 (6 h illuminated) mol O₂ evolved or ¹⁴CO₂ fixed per mg of chlorophyll per h, compared to 3.5 mol·mg chl^-1·h^-1 obtained with mechanically isolated plastids. With this system, CO₂-dependent O₂ evolution was already detected after 3 h of illumination of etiolated tissue, but could only be observed at pH values between 7.6 and 8.6, in the presence of EDTA. At lower pH (7.3) or at pH 7.6 in the absence of EDTA, light-dependent O₂ evolution up to 24 h of greening was only measurable with 3-phosphoglycerate as the substrate. The possible effects of EDTA in this respect as well as the advantages of using protoplasts or plastids isolated from protoplasts for developmental studies are discussed.Abbreviations BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid - MES 2(N-morpholino) ethane sulphonic acid - PGA 3-phosphoglycerate     

The relationship of arginine groups to photosynthetic HCO 3 - uptake inUlva sp. mediated by a putative anion exchanger

The marine macroalgaUlva sp. can take up HCO ₃ ^- via a process which chemically resembles that of anion exchange in red blood cells (Drechsler et al. 1993, Planta191, 34–40). In this work we explore the possibility that high-pK amino-acid residues could be functionally involved in the binding/transport of HCO ₃ ^- . It was found that the specific arginyl-reacting agents phenylglyoxal and 2,3-butanedione inhibited photosynthesis ofUlva competitively with inorganic carbon at pH 8.2–8.4 (which is close to the pH of normal seawater), where HCO ₃ ^- was the predominant inorganic carbon form taken up. The inhibition by phenylglyoxal was irreversible at 32°C and high pH values, while that of butanedione became irreversible in the presence of borate. These interactions, as well as the protection of the irreversible phenylglyoxal-inhibition by inorganic carbon and by the membrane-impermeant agents 4,4-diisothiocyanostilbene 2,2-disulfonate and 4,4-dinitrostilbene-2,2-disulfonate indicate that arginine (and possibly also lysine) are involved in the HCO ₃ ^- uptake process, probably at the plasmalemma level. The photosynthetic affinity ofUlva to external inorganic carbon gradually decreased with increasing pH from 8.2 to 10.5, and this decrease parallels the decline in protonation of amino acids with a pK of around 10. Based on this information, as well as the inhibition studies, it is suggested that arginine and lysine residues are essential proteinaceous constituents involved in anionic inorganic carbon (HCO ₃ ^- and possibly also CO ₃ ^2- ) uptake into theUlva cells.Abbreviations AE1 anion exchanger 1 (of red blood cells) - BD 2,3-butanedione - CA carbonic anhydrase - CI inorganic carbon - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - DNDS 4,4-dinitrostilbene-2,2-disulfonate - PG phenylglyoxal This paper is in partial fulfillment of a Ph.D. study by R. Sharkia. Supported by the Israel Academy of Sciences, grant 441/93 (to S.B.), and by the Fund for Encouragement of Research, Histadrut, Israel (to R.S.).     

Gibberellic acid induces cytoplasmic acidification in maize coleoptiles

Indole-3-acetic acid (IAA), fusicoccin and weak acids all lower the cytoplasmic pH (pH_i) and induce elongation growth of maize (Zea mays L.) coleoptiles. Gibberellic acid (GA₃) also induces elongation growth and we have used confocal laser scanning microscopy to study the effects of GA₃ on pH_i employing the pH-indicator dyes, 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein and carboxy-semi-naphthorhodafluor-1. We confirm that GA₃ induces growth significantly in light-grown but only slightly or not at all in dark-grown coleoptiles. The growth induced by IAA treatment was similar in light- and dark-grown coleoptiles. The pH_i decreased by up to 0.6 units during the first 7 min of GA₃ or IAA treatment of both light- and dark-grown coleoptiles. Gibberellic acid inhibited IAA-induced growth of dark-grown coleoptiles. Hence, in dark-grown coleoptiles GA₃ may activate either directly or indirectly reactions that interfere with the signalling pathway leading to elongation growth. The possible role of pH_i in growth is discussed.Abbreviations ABA abscisic acid - AM acetoxymethyl ester - BCECF 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein - [Ca²⁺]_i cytoplasmic free calcium - GA_(n) gibberellin A_(n) - GA₃ gibberellic acid - IAA indole-3-acetic acid - PGR plant growth regulator - pH_i cytoplasmic pH - Pipes piperazine-N,N-bis[2-ethanesulfonic acid] - Snarf-1 carboxy-semi-naphthorhodafluor-1 We thank Dr R. King (CSIRO, Canberra) for providing the GA₁ and T. Phillips for processing the photographic material. H.R. Irving was supported by an Australian Research Council Research Fellowship and the work was supported by an Australian Research Council grant.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Correlation between loss of turgor and accumulation of abscisic acid in detached leaves

Mature leaves of Phaseolus vulgaris L. (red kidney bean), Xanthium strumarium L. (cocklebur), and Gossypium hirsutum L. (cotton) were used to study accumulation of abscisic acid (ABA) during water stress. The water status of individual, detached leaves was monitored while the leaves slowly wilted, and samples were cut from the leaves as they lost water. The leaf sections were incubated at their respecitive water contents to allow ABA to build up or not. At least 8 h were required for a new steady-state level of ABA to be established. The samples from any one leaf covered a range of known water potentials (), osmotic pressures (), and turgor pressures (p). The and p values were calculated from pressure-volume curves, using a pressure bomb to measure the water potentials. Decreasing water potential had little effect on ABA levels in leaves at high turgor. Sensitivity of the production of ABA to changes in progressively increased as turgor approached zero. At p=1 bar, ABA content averaged 4 times the level found in fully turgid samples. Below p=1 bar, ABA content increased sharply to as much as 40 times the level found in unstressed samples. ABA levels rose steeply at different water potentials for different leaves, according to the at which turgor became zero. These differences were caused by the different osmotic pressures of the leaves that were used; must cqual - for turgor to be zero. Leaves vary in , not only among species, but also between plants of one and the same species depending on the growing conditions. A difference of 6 bars (calculated at =0) was found between the osmotic pressures of leaves from two groups of G. hirsutum plants; one group had previously experienced periodic water stress, and the other group had never been stressed. When individual leaves were subsequently wilted, the leaves from stress-conditioned plants required a lower water potential in order to accumulate ABA than did leaves from previously unstressed plants. On the basis of these results we suggest that turgor is the critical parameter of plant water relations which controls ABA production in water-stressed leaves.Abbreviations ABA abscisic acid - me-ABA abscisic-acid methyl ester - leaf water potential - osmotic pressure - p volumeaveraged turgor - volumetric modulus of elasticity