Characterization of the ABA-deficient tomato mutant notabilis and its relationship with maize Vp14

The notabilis (not) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis--the oxidative cleavage of a 9-cis xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant, viviparous 14 (vp14) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt-related tomato cDNA library. A full-length cDNA clone was identified which is specific to the not gene locus. The ORFs of the tomato cDNA and maize Vp14 are very similar, apart from parts of their N-terminal sequences. The not mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that not is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by not mutant homozygotes.     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Control of abscisic acid synthesis

The abscisic acid (ABA) biosynthetic pathway involves the formation of a 9-cis-epoxycarotenoid precursor. Oxidative cleavage then results in the formation of xanthoxin, which is subsequently converted to ABA. A number of steps in the pathway may control ABA synthesis, but particular attention has been given to the enzyme involved in the oxidative cleavage reaction, i.e. 9-cis-epoxycarotenoid dioxygenase (NCED). Cloning of a gene encoding this enzyme in maize was first reported in 1997. Mapping and DNA sequencing studies indicated that a wilty tomato mutant was due to a deletion in the gene encoding an enzyme with a very similar amino acid sequence to this maize NCED. The potential use of this gene in altering ABA content will be discussed together with other genes encoding ABA biosynthetic enzymes.     

Structural insights into maize viviparous14, a key enzyme in the biosynthesis of the phytohormone abscisic acid

The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-Å resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.     

The rea (red embryonic axis) phenotype describes a new mutation affecting the response of maize embryos to abscisic acid and osmotic stress

During a screen for mutants with defective germination, a newphenotype was observed consisting of red pigmentation of theembryonic axis in the dormant seed. Segregation ratios, as determinedin F₂ and back-crossed progeny, indicate that the phenotypeis due to a recessive single gene mutation that has been symbolizedrea to denote red embryonic axis. A closer inspection of therea phenotype revealed that the mutant is occasionally viviparous,indicating a defect in abscisic acid (ABA) metabolism. The mutationprobably affects ABA sensitivity since no difference in ABAcontent was detected in mutant versus normal tissues. Moreover,when immature mutant and wild-type embryos were incubated onmedia containing 10 M ABA, only the mutants germinated. ABA-regulatedgene expression in rea embryos differed from that of embryosof the viviparous mutant vp1 which does not respond to the inhibitoryaction of ABA at the level of immature embryo germination. Theseresults, therefore, indicate that the two genes exert a differentrole in the control of embryogenesis. Key words: Zea mays L, embryo dormancy, ABA     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Growth and Graviresponsiveness of Primary Roots of Zea mays Seedlings Deficient in Abscisic Acid and Gibberellic Acid

Moore, R. and Dickey, K. 1985. Growth and graviresponsivenessof primary roots of Zea mays seedlings deficient in abscisicacid and gibberellic acid.—J. exp. Bot. 36: 1793–1798. The objective of this research was to determine if gibberellicacid (GA) and/or abscisic acid (ABA) are necessary for graviresponsivenessby primary roots of Zea mays. To accomplish this objective wemeasured the growth and graviresponsiveness of primary rootsof seedlings in which the synthesis of ABA and GA was inhibitedcollectively and individually by genetic and chemical means.Roots of seedlings treated with Fluridone (an inhibitor of ABAbiosynthesis) and Ancymidol (an inhibitor of GA biosynthesis)were characterized by slower growth rates but not significantlydifferent gravicurvatures as compared to untreated controls.Gravicurvatures of primary roots of d-5 mutants (having undetectablelevels of GA) and vp-9 mutants (having undetectable levels ofABA) were not significantly different from those of wild-typeseedlings. Roots of seedlings in which the biosynthesis of ABAand GA was collectively inhibited were characterized by gravicurvaturesnot significantly different from those of controls. These results(1) indicate that drastic reductions in the amount of ABA andGA in Z. mays seedlings do not significantly alter root graviresponsiveness,(2) suggest that neither ABA nor GA is necessary for root gravicurvature,and (3) indicate that root gravicurvature is not necessarilyproportional to root elongation. Key words: Abscisic acid, Ancymidol, Fluridone, gibberellic acid, root gravitropism, Zea mays     

Abscisic acid regulates TSRF1-mediated resistance to Ralstonia solanacearum by modifying the expression of GCC box-containing genes in tobacco

Although recent studies have established a significant regulatoryrole for abscisic acid (ABA) and ethylene response factor (ERF)proteins in plant pathogen resistance, it is not clear whetherand how ABA performs this role. Previously, it was reportedthat an ERF protein, TSRF1, activates the expression of GCCbox-containing genes and significantly enhances the resistanceto Ralstonia solanacearum in both tobacco and tomato plants.Here, it is reported that TSRF1-regulated pathogen resistanceis modified by ABA application. TSRF1 activates the expressionof ABA biosynthesis-related genes, resulting in the increaseof ABA biosynthesis, which further stimulates ethylene production.More interestingly, ABA application decreases, while the inhibitorof ABA biosynthesis fluridone increases, the TSRF1-enhancedresistance to R. solanacearum. This observation is further supportedby the finding that ABA and fluridone reversibly modify theability of TSRF1 to bind the ethylene-responsive GCC box, consequentlyaltering the expression of element-controlled genes. These resultstherefore establish that TSRF1-regulated resistance to R. solanacearumcan be modified in tobacco by ABA. Key words: Abscisic acid, ERF protein TSRF1, GCC box-containing genes, Ralstonia solanacearum, tobacco     

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. ¹⁸O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.     

Graviresponsiveness and abscisic-acid content of roots of carotenoid-deficient mutants of Zea mays L.

The abscisic-acid (ABA) content of roots of the carotenoid-deficient w-3, vp-5, and vp-7 mutants of Z. mays was analyzed using gas chromatography-mass spectrometry with an analysis sensitivity of 6 ng ABA g^–1 fresh weight (FW). Roots of normal seedlings of the same lines were characterized by the following amounts of ABA (as ng ABA g^–1 FW,±standard deviation): w-3, 279±43; vp-5, 237±26; vp-7, 338±61. We did not detect any ABA in roots of any of the mutants. Thus, the lack of carotenoids in these mutants correlated positively with the apparent absence of ABA. Primary roots of normal and mutant seedlings were positively gravitropic, with no significant differences in the curvatures of roots of normal as compared with mutant seedlings. These results indicate that ABA 1) is synthesized in maize roots via the carotenoid pathway, and 2) is not necesary for positive gravitropism by primary roots of Z. mays.Abbreviation ABA abscisic acid     

Abscisic Acid and C10 Dicarboxylic Acids in Wilty Tomato Mutants

Linforth, R. S. T., Taylor, I. B. and Hedden, P. 1987. Abscisicacid and C10 dicarboxylic acids in wilty tomato mutants.—J.exp. Bot. 38: 1734–1740. The concentration of C₁₀ dicarboxylic acids in wilty tomatomutants was investigated. Three of the genotypes studied (flacca,sitiens and the double mutant homozygote flacca/sitiens) werefound to have higher concentrations of 2,7-dimethyl-2,4-octadienedioicacid (ODA) than the isogenic normal form. In contrast, the othergenotypes (notabilisand the double mutant homozygotes notabilis/flaccaand notabilis/sitiens) were found to have lower concentrationsof ODA than the isogenic normal form. The concentration of ODAin flacca plants was increased by water stress and reduced byexogenously applied abscisic acid (ABA). A second structurallyrelated compound, 2,7-dimethyl-4-octenedioic acid (OEA) wasalso quantified, but it showed no clear genotype-related pattern. The concentration of ABA in the wilty tomato mutants was alsoinvestigated. As expected in the light of previously publishedresults, it was reduced in the single mutants relative to theisogenic control plants. In the double mutant flacca/sitiensABA levels were similar to those of the single mutant sitiens.However, in the two double mutants notabilis/flacca and notabilis/sitiensABA was substantially lower than those in any other genotypeinvestigated. Key words: Abscisic acid, 2,7-dimethyl-2,4-octadienedioic acid, 2,7-dimethyl-4-octenedioc acid, tomato, wilty mutants     

Root Growth, Secondary Root Formation and Root Gravitropism in Carotenoid-deficient Seedlings of Zea mays L.

The effect of ABA on root growth, secondary-root formation androot gravitropism in seedlings of Zea mays was investigatedby using Fluridone-treated seedlings and a viviparous mutant,both of which lack carotenoids and ABA. Primary roots of seedlingsgrown in the presence of Fluridone grew significantly slowerthan those of control (i.e. untreated) roots. Elongation ofFluridone-treated roots was inhibited significantly by the exogenousapplication of 1 mM ABA. Exogenous application of 1 µMand 1 nM ABA had either no effect or only a slight stimulatoryeffect on root elongation, depending on the method of application.The absence of ABA in Fluridone-treated plants was not an importantfactor in secondary-root formation in seedlings less than 9–10d old. However, ABA may suppress secondary-root formation inolder seedlings, since 11-d-old control seedlings had significantlyfewer secondary roots than Fluridone-treated seedlings. Rootsof Fluridone-treated and control seedlings were graviresponsive.Similar data were obtained for vp-9 mutants of Z. mays, whichare phenotypically identical to Fluridone-treated seedlings.These results indicate that ABA is necessary for neither secondary-rootformation nor for positive gravitropism by primary roots. Zea mays, gravitropism, carotenoid-deficient, Fluridone, root growth, vp-9 mutant     

The Metabolism of cis ABA-aldehyde by the Wilty Mutants of Potato, Pea and Arabidopsis thaliana

RS-[²H₁] cis ABA-aldehyde was fed to ABA-deficient mutants ofpotato (droopy), pea (wilty) and Arabidopsis thaliana (aba¹)along with appropriate non-mutant controls. Both the wilty andaba¹ mutants readily oxidized the monodeuterated ABA-aldehydeto ABA. The incorporation of label into ABA by these two mutantswas indistinguishable from that detected in the non-mutant controls.In contrast, the droopy mutants poorly incorporated the labelledprecursor into ABA. Instead they reduced and isomerized RS-[²H₁] cis ABA-aldehyde to a mixture of 2, cis and 2, trans ABA-alcohols.Thus the droopy mutant affects the last step in ABA biosynthesis,a position it shares with the tomato mutants, flacca and sitiens.Genetic evidence suggesting that droopy and sitiens may be correspondinggene loci is discussed. Key words: ABA metabolism, wilty mutants, pea, potato, Arabidopsis     

Metabolism of xylem-delivered ABA in relation to ABA flux and concentration in leaves of maize and Commelina communis

When detached maize leaves were fed with an ABA solution viathe xylem, the relationship between the relative stomatal inhibitionand ABA concentrations was similar under different humidityconditions, but the relationship between such inhibition andABA flux was different according to changes of humidity. Tounderstand whether such stomatal behaviour was related to theway through which xylem-delivered ABA was metabolized, detachedleaves of maize and Commelina were fed with tritium-labelled(³H)-ABA at concentrations similar to that found in xylem ofdroughted plants and it was found that xylem-delivered ABA wasmetabolized rapidly in both species. The half-life of ABA metabolism,calculated from the time-related ABA disappearance curve, was42 and 64 min for maize and Commelina, respectively. The veryshort half-life suggests that there is a large capacity in leavesto metabolize xylem-delivered ABA and that metabolism is a majorfactor in the control of ABA accumulation in leaves. When ABAwas fed at different fluxes, either through changing the feedingconcentrations or through manipulating the rates of leaf transpiration(i.e. the volume flux), ABA was metabolized at rates that wereproportional to the amount that was delivered. The absoluterate of ABA metabolism was, therefore, linearly related to theamount of ABA that had arrived. It was found that xylem-deliveredABA reached the epidermis of Commelina, and was metabolizedat the same pattern as that in mesophyll tissues, i.e. at asimilar half-life and at rates constantly related to the amountthat was delivered. The role of the rapid ABA metabolism wasdiscussed in the context of stomatal control by either concentrationor flux of xylem-carried ABA. Key words: Abscisic acid, ABA metabolism, xylem-delivered ABA, maize, Commelina     

C-ABI3, the Carrot Homologue of the Arabidopsis ABI3, is Expressed during Both Zygotic and Somatic Embryogenesis and Functions in the Regulation of Embryo-Specific ABA-Inducible Genes

A carrot gene homologous to the ABI3 gene of Arabidopsis wasisolated from a carrot somatic embryo cDNA library and designatedC-ABI3. The sequence of C-ABI3 was very similar to those ofABI3 of Arabidopsis and VP1 of maize in certain conserved regions.The expression of C-ABI3 was detected specifically in embryogeniccells, somatic embryos and developing seeds. Thus, expressionof C-ABI3 was limited to tissues that acquired desiccation tolerancein response to endogenous or exogenous abscisic acid (ABA).Endogenous levels of ABA in seeds increased transiently andthen desiccation of seeds started. The expression of C-ABI3in developing seeds was observed prior to the increase in levelsof endogenous ABA that was followed by desiccation of seeds.In transgenic mature leaves in which C-ABI3 was ectopicallyexpressed, expression of ECP31, ECP63 and ECP40 was inducedby treatment with ABA, which indicates that the expression ofECP genes was controlled by the pathway(s) that involved C-ABI3and ABA. This suggests that C-ABI3 has the same function asVP1/ABI3 factor in carrot somatic embryos. (Received March 4, 1998; Accepted September 4, 1998)     

Regulation of IBA synthetase from maize (Zea mays L.) by drought stress and ABA

The rate of indole-3-butyric acid (IBA) synthesis in maize seedlingsis dependent on the culture conditions of the plants. When theseedlings were grown on filter paper soaked with different amountsof water, the activity of IBA synthetase differed strongly.High amounts of water (150 and 200 ml per bowl) inhibited IBAsynthesis completely in vitro, whereas 30 and 50 ml water perbowl increased the activity dramatically. Under conditions whereIBA synthetase was inhibited (150 ml H₂O), an increase of enzymeactivity was observed when abscisic acid (ABA) was exogenouslyadded in concentrations between 510^–4 to 510^–7M. Under ‘drought’ conditions (50 ml H₂O per bowl)the same ABA concentrations were inhibitory. Jasmonic acid andsalicylic acid also enhanced IBA synthetase activity to someextent, whereas indole-3-acetic acid (IAA) and kinetin had noeffect. Activity could also be enhanced by osmotic stress (NaCIand sorbitol), but not under temperature stress. In accompanyinginvestigations the endogenous contents of IAA, IBA, and ABAunder the different culture conditions have been determinedas well as the energy charge of the seedlings. Similar observationshave been made with Amaranthus, wheat and pea seedlings Key words: Abscisic acid, Amaranthus paniculatus, drought stress, inole-3-butyric acid biosynthesis, Pisum sativum, Triticum aestivum, Zea mays     

Inactivation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase,which catalyses the final step in the biosynthesis of ethylene,showed a non-linear time-course in vitro and activity decayedwith a half-life of around 14 min. This loss of activity wasstudied using tomato ACC oxidase purified from Escherichia coiltransformed with the cDNA clone pTOM13. Inactivation was notdue to end-product inhibition by dehydroascorbic acid or cyanide.Preincubatlon of enzyme in the combined presence of Fe²⁺ ascorbateand ACC, which together allowed catalytic turnover, resultedin almost total loss of ACC oxidase activity. Enzyme Inactivatedby catalysis could not be reactivated by passage through SephadexG-25 or by treating with combina tions of DTT and CO₂ A non-lineartime-course and inactivation in the presence of all substratesand cofactors was also shown for the enzyme assayed in vivowith melon fruit discs. Using the purified tomato enzyme a distinctascorbate-dependent inactivation was also observed, which occurredIn the absence of catalysis and was prevented, although notreversed, by catalase. This ascorbate-dependent inactivationmay thus be due to H₂O₂ attack on ACC oxidase. Key words: 1-aminocyclopropane-1-carboxylate (ACC) oxidase, catalase, catalytic inactivation, ethylene