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     

Phenotypic interactions between abscisic acid deficient tomato mutants

Summary A series of double mutant homozygotes have been produced from three wilty tomato mutants; flacca, sitiens and notabilis. The phenotypic interaction between the mutant genes has been studied. The severity of phenotype in the double mutants does not correspond to that predicted from the single mutant homozygotes. The results are discussed in relation to the probable involvement of the mutants in abscisic acid metabolism.     

ABA-deficiency results in reduced plant and fruit size in tomato

Abscisic acid (ABA) deficient mutants, such as notabilis and flacca, have helped elucidating the role of ABA during plant development and stress responses in tomato (Solanum lycopersicum L.). However, these mutants have only moderately decreased ABA levels. Here we report on plant and fruit development in the more strongly ABA-deficient notabilis/flacca (not/flc) double mutant. We observed that plant growth, leaf-surface area, drought-induced wilting and ABA-related gene expression in the different genotypes were strongly correlated with the ABA levels and thus most strongly affected in the not/flc double mutants. These mutants also had reduced fruit size that was caused by an overall smaller cell size. Lower ABA levels in fruits did not correlate with changes in auxin levels, but were accompanied by higher ethylene evolution rates. This suggests that in a wild-type background ABA stimulates cell enlargement during tomato fruit growth via a negative effect on ethylene synthesis.     

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.     

2-trans-ABA alcohol accumulation in the wilty tomato mutants flacca and sitiens

Abstract Two wilty tomato mutants, flacca and sitiens, fail to increase their endogenous ABA concentration in response to water stress. Instead, a compound accumulates which has been identified as 2-trans-ABA alcohol. Levels of this compound have been estimated for three wilty mutants and the control; both before and after water stress. When the compound was biosynthesized in the presence of ¹⁸O₂, one atom appeared to be incorporated into the primary alcohol group. The possible implications of this for the ABA biosynthetic pathway are discussed.     

Xanthoxin Metabolism in Cell-free Preparations from Wild Type and Wilty Mutants of Tomato

Extracts prepared from the turgid and water-stressed leaves of wild-type tomato (Lycopersicon esculentum Mill cv Ailsa Craig) and the wilty mutants sitiens, notabilis, and flacca were tested for their ability to metabolize xanthoxin to ABA. Extracts from wild type and notabilis converted xanthoxin at similar rates, while extracts from sitiens and flacca showed little or no activity. We also observed no activity when extracts of sitiens and flacca were mixed. Similar results were obtained when ABA aldehyde was used as a substrate, in that extracts from wild type and notabilis were equally active, but extracts from flacca and sitiens showed little activity. None of the tomato extracts showed significant activity with xanthoxin acid, xanthoxin alcohol, or ABA-1′,4-′Trans-diol as substrates. Extracts from bean leaves (Phaseolus vulgaris L. cv Blue Lake) were similar to the wild-type tomato extracts in their ability to convert the various substrates to ABA, although excised bean leaves did convert ABA-1′,4′-trans-diol and xanthoxin alcohol to ABA when these substances were taken up through the petiole. These results are consistent with a role for xanthoxin as a normal intermediate on the ABA biosynthetic pathway, and they suggest that ABA aldehyde is the final ABA precursor.     

Dynamic Properties of Endogenous Phytochrome A in Arabidopsis Seedlings

The flacca tomato (Lycopersicon esculentum) mutant displays a wilty phenotype as a result of abscisic acid (ABA) deficiency. The Mo cofactor (MoCo)-containing aldehyde oxidases (AO; EC 1.2.3.1) are thought to play a role in the final oxidation step required for ABA biosynthesis. AO and related MoCo-containing enzymes xanthine dehydrogenase (XDH; EC 1.2.1.37) and nitrate reductase (EC 1.6.6.1) were examined in extracts of the flacca tomato genotype and of wild-type (WT) roots and shoots. The levels of MoCo were found to be similar in both genotypes. No significant XDH or AO (MoCo-containing hydroxylases) activities were detected in flacca leaves; however, the mutant exhibited considerable MoCo-containing hydroxylase activity in the roots, which contained notable amounts of ABA. Native western blots probed with an antibody to MoCo-containing hydroxylases revealed substantial, albeit reduced, levels of cross-reactive protein in the flacca mutant shoots and roots. The ABA xylem-loading rate was significantly lower than that in the WT, indicating that the flacca is also defective in ABA transport to the shoot. Significantly, in vitro sulfurylation with Na₂S reactivated preexisting XDH and AO proteins in extracts from flacca, particularly from the shoots, and superinduced the basal-level activity in the WT extracts. The results indicate that in flacca, MoCo-sulfurylase activity is impaired in a tissue-dependent manner.     

2,7-Dimethylocta-2,4-dienedioic acid is not a by-product of abscisic acid biosynthesis

The cause of the permanent wilted state of some mutant tomato (Lycopersicon esculentum Mill.) strains has been shown to be an insufficiency of abscisic acid (ABA). The mutants contain larger than normal amounts of a C₁₀ dienoic dicarboxylic acid (2,7-dimethylocta-2,4-dienoic acid (ODA) whose structure suggests that it is the central residue of a carotenoid from which two potential carbon skeletons of ABA have been removed. When tomato plants were supplied with ²H₂O for 6 days the ABA was found to be labelled with up to 11 deuterium atoms while the ODA was unlabelled. ODA, therefore, cannot be a by-product of the biosynthesis of ABA.     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Abscisic Acid Production and Water Relations in Wilty Tomato Mutants Subjected to Water Deficiency

Neill, S. J. and Horgan, R. 1985. Abscisic acid production andwater relations in wilty tomato mutants subjected to water deficiency.—J.exp. BoL 36: 1222-1231. Abscisic acid (ABA) concentrations were determined in shootsof Lycopersicon esculentum Mill. cv. Ailsa Craig wild type andthe three wilty mutants notabilis (not), flacca (flc) and sitiens(sit). ABA content of unstressed wild type leaves was 1.5 nmolg^–1 fr. wt.; concentrations in not, flc and sit were 49,26 and 15% of this respectively. Gradual water stress was imposed on potted plants and a morerapid stress imposed on detached leaves. Leaves of the wildtype and not responded to both stresses by increasing theirABA content but leaves of flc and sit did not produce any moreABA under stress. Transpiration rates of flc plants were three times greater thanthose of the wild type and stomatal resistances correspondinglylower. Stomata of both flc and the wild type responded to darknessand externally supplied ABA by closing. However, only wild typestomata responded to water stress by dosing; those of flc leavesremained open until the leaves were severely desiccated. Thus,there was some relationship between the lack of stomatal responseto water stress and the failure to synthesize ABA. Key words: ABA, biosynthesis, stomata, water shortage, wilty mutants     

Abscisic-acid metabolism in a wilty mutant of Nicotiana plumbaginifolia

A mutant of Nicotiana plumbaginifolia, CKR1, isolated on the basis of its enhanced resistance to cytokinins was found to have a greater tendency to wilt than the wild type (Blonstein et al., 1991, Planta 183, 244–250). Further characterisation has shown that the wiltiness in the mutant is not caused by an insensitivity to abscisic acid (ABA) because the external application of ABA leads to stomatal closure and phenotypic reversion. The basal ABA level in the mutant is < 20% of that in the wild type. Following stress, the ABA level in wild-type leaves increases by approx 9-to 10-fold while the mutant shows only a slight increase. This deficiency in ABA is unlikely to be the consequence of accelerated catabolism as the levels of two major metabolites of ABA, phaseic and dihydrophaseic acid, are also much reduced in the mutant. The qualitative and quantitative distributions of carotenoids, the presumed presursors of ABA, are the same for the leaves of both wild type and mutant. Biosynthesis of ABA at the C15 level was investigated by feeding xanthoxin (Xan) to detached leaves. Wild-type leaves convert between 9–19% of applied Xan to ABA while the mutant converts less than 1%. The basal level of trans-ABA-alcohol (t-ABA-alc) is 3-to 10-fold greater in the mutant and increases by a further 2.5-to 6.0-fold after stress. This indicates that the lesion in the wilty mutant of N. plumbaginifolia affects the conversion of ABA-aldehyde to ABA, as in the flacca and sitiens mutants of tomato and the droopy mutant of potato (Taylor et al., 1988, Plant Cell Environ. 11, 739–745; Duckham et al., 1989, J. Exp. Bot. 217, 901–905). Wild-type tomato and N. plumbaginifolia leaves can convert trans-Xan into t-ABA-alc, and Xan into ABA, while those of flacca and the wilty N. plumbaginifolia mutant convert both Xan and t-Xan to t-ABA-alc.     

The wilty tomato mutants flacca and sitiens are impaired in the oxidation of ABA-aldehyde to ABA

Abstract. Deuterium-labelled ABA-aldehyde was fed to various tomato genotypes. Normal and notabilis mutant plants incorporated substantial amounts of the label into ABA. In contrast, two ABA-deficient mutants, flacca and sitiens , reduced ABA-aldehyde to a mixture of cis- and trans -ABA alcohol rather than oxidizing it to ABA. It was concluded that ABA-aldehyde is the immediate precursor of ABA in higher plants. It appears that the flacca and sitiens lesions both act to block the last step of the ABA biosynthetic pathway. The mutant gene loci are likely to be involved in coding for different sub-units of the same dehydrogenase enzyme.     

Phenotypic expression of wild-type tomato and three wilty mutants in relation to abscisic Acid accumulation in roots and leaflets of reciprocal grafts

Lycopersicon esculentum Mill. cv Rheinlands Ruhm (RR) and cv Moneymaker and the three wilty mutants flacca (flc), sitiens (sit), and sitiens^w (sit^w), together with most reciprocal grafts, were grown in pots and in solution culture. Detached leaflets, and control and steam-girdled intact plants, were left turgid or were wilted in air. Detached leaflets and the leaflets and roots of the intact plants were analyzed for their abscisic acid (ABA) content. Turgid RR leaflets contained about 2.9 ng ABA per milligram dry weight. On average, the flc and sit leaflets contained 33 and 11% of this amount, respectively. The lack of ABA approximately correlated with the severity of the mutant phenotype. Mutant roots also contained less ABA than wild-type roots. Wild-type scions on mutant stocks (wild type/mutant) maintained the normal phenotype of ungrafted plants. Mutant scions grafted onto wild-type stocks reverted to a near wild-type phenotype. After the wild-type leaves were excised from solution culture-grown mutant/wild-type plants, the revertive morphology of the mutant scions was maintained, although endogenous ABA levels in the leaflets fell to typical mutant levels and the leaflets became wilty again. When stressed in air, both leaflets and roots of RR plants produced stress-induced ABA, but the mutant leaflets and roots did not. The roots and leaflets of the grafted plants behaved according to their own genotype, with the notable exception of mutant roots grown with wild-type scions. Roots of flc and sit^w recovered the ability to accumulate stress-induced ABA when grafted with RR scions before the stress was imposed.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

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     

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.     

Abnormal Stomatal Behaviour and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: EFFECT OF ABSCISIC ACID AND AUXIN ON STOMATAL BEHAVIOUR AND PEROXIDASE ACTIVITY

The wilty tomato mutant flacca and the normal variety RheinlandsRuhm were compared in terms of: (1) potassium transport intoand out of the guard cells, (2) cell wall properties which includeprotein, hydroxyproline and peroxidase activity, and (3) activityof indol-3yl-acetic acid oxidase. Also studied were the effectsof auxin on stomatal behaviour and peroxidase activity whenapplied to normal plants during development, and the short-termeffect of abscisic acid on the resistance of flacca stomatato closure under plasmolysis. Potassium transport, wall protein and hydroxyproline all seemedto be equal in mutant and normal plants. Peroxidase activitywas higher in the soluble and wall fractions of the mutant,and decreased toward normal in the mutant treated with abscisicacid. More stomata were open and peroxidase activity was higherin normal plants treated with auxin during development. Thepercentage of open stomata under plasmolysis was lower and theiraperture size was smaller in the epidermal strips taken fromabscisic-acid-treated mutant plants than from control mutantplants.     

The genetic relationship between the tomato mutants,flacca and lateral suppressor,with reference to abscisic acid accumulation

Two tomato mutants, Lycopersicon esculentum flacca and lateral suppressor, are assigned to map position 59 of chromosome 7. The tight linkage between these two gene loci was detected as a result of attempts to establish whether they would exhibit phenotypic interaction. The possibility that both mutants result in abnormalities of abscisic acid (ABA) accumulation is considered. ABA analysis supports the suggestion that plants homozygous for flacca have a substantially lower concentration but indicates that lateral suppressor homozygotes do not differ from normal in ABA content. An attempt is made to reconcile the results with those of Tucker (1976, New. Phytol. 77, 561–568) by suggesting that lateral suppressor plants may accumulate high levels of an ABA metabolite which is indistinguishable from ABA using the Commelina epidermal strip bioassay.Abbreviations ABA abscisic acid - flc flacca - ls lateral suppressor - La Lanceolate     

Abnormal stomatal behavior and root resistance,and hormonal imbalance in three wilty mutants of tomato

Three wilty mutants of tomato, flacca, sitiens, and notabilis, were compared with two control normal cultivars, Rheinlands Ruhm and Lukullus, for concentration of abscisic acid and root resistance to water flow. In addition, the three mutants treated with abscisic acid during development were compared with control mutant plants for stomatal opening and root resistance. The hormonal concentration was estimated by coleoptile assay and gas chromatography. Stomatal opening was estimated by measuring rate of transpiration and by examining leaf imprints. Root resistance was estimated by measuring the amount of exudate from roots of decapitated plants and the difference between the osmotic pressure of the exudate and the root medium. A lower level of abscisic acid was found in all three mutants as compared with the control normal plants. In addition, root resistance to water flow was higher in the three mutants than in the control normal types. All three mutants completely reverted to normal phenotypically, including stomatal and root resistances, when treated with abscisic acid. It has been suggested that the first hormonal change in the mutants is that of abscisic acid, and from it proceed the other changes.This work was supported in part by a research grant from the Ford Foundation (Ford-6, A-VII).     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: IV. Effect of Abscisic Acid and Water Content on RNase Activity and RNA

Plants of the wilty tomato (Lycopersicum esculentum) mutant, flacca, and of the normal cultivar Rheinlands Ruhm growing under either “normal” or high humidity were used in this research. Under normal humidity, RNase activity was much higher in mutant plants in which abscisic acid (ABA) and water content were lower than in the normal plant. The mutant also contained less RNA and protein per cell and less soluble RNA relative to ribosomal RNA as compared with the normal genotype. In ABA-treated mutant plants, RNase activity decreased while RNA, protein, the ratio of soluble to ribosomal RNA and water content increased.