Wilty Mutant Research Articles

The ecological implications of interplant drought cuing

Abstract Plants can perceive, integrate and respond to multiple signals and cues informative of imminent threats and opportunities. Here, we tested the hypothesis that unstressed plants are able to perceive and adaptively respond to stress cues emitted from their drought‐stressed neighbours, and to induce adaptive responses in additional unstressed plants. Triplets of split‐root Stenotaphrum secundatum plants were grown in rows. One root of the first plant was subjected to drought while its other root shared its pot with one of the roots of an unstressed target neighbour, which in turn shared its other pot with an additional unstressed target neighbour. Cuing from drought‐stressed plants increased survival in both proximate directly cued and in more distant relayed‐cued target plants under drought. Drought cuing lowered plant performance under benign conditions. Experiments with a Wilty mutant and fluridone treatments showed that drought cuing was greatly reduced in ABA‐deficient Pisum Sativum plants. Synthesis. Our findings demonstrate for the first time the possible adaptive implications of both direct and relayed stress cuing among neighbouring plants, the possible cost of plant responsiveness to drought cues under benign conditions and the involvement of ABA in interplant drought cuing. The results suggest that interplant root communication of drought cues could have novel implications for plant interactions, survival and performance under both natural and agricultural settings.

Molecular characterization of a mutation affecting abscisic acid biosynthesis and consequently stomatal responses to humidity in an agriculturally important species.

Mutants deficient in the phytohormone abscisic acid (ABA) have been instrumental in determining not only the biosynthetic pathway for this hormone, but also its physiological role in land plants. The wilty mutant of Pisum sativum is one of the classical, well-studied ABA-deficient mutants; however, this mutant remains uncharacterized at a molecular level. Using a candidate gene approach, we show that the wilty mutation affects the xanthoxin dehydrogenase step in ABA biosynthesis. To date, this step has only been represented by mutants in the ABA2 gene of Arabidopsis thaliana. Functional ABA biosynthesis appears to be critical for normal stomatal responses to changes in humidity in angiosperms, with wilty mutant plants having no increase in foliar ABA levels in response to a doubling in vapour pressure deficit, and no closure of stomata. Phylogenetic analysis of the ABA2 gene family from diverse land plants indicates that an ABA-biosynthesis-specific short-chain dehydrogenase (ABA2) evolved in the earliest angiosperms. The relatively recent origin of specificity in this step has important implications for both the evolution of ABA biosynthesis and action in land plants.

Identification of the tomato ABA-deficient mutant sitiens as a member of the ABA-aldehyde oxidase gene family using genetic and genomic analysis

The sitiens (sit) wilty mutant of tomato (Solanum lycopersicum L.) is deficient in functional enzyme activity at the final step in abscisic acid (ABA) biosynthesis. The biochemical lesion is believed to be an impaired aldehyde oxidase (AO). Molecular mapping using various interspecies crosses has previously shown sit to co-map with a cluster of unresolved RFLP markers on the short arm of chromosome 1. Here, the utilisation of bridging lines to produce interspecies mapping populations involving a self-compatible S. peruvianum accession (LA2157) allowed the fine mapping of sit within this cluster. Identification of a novel AO gene, within the region now known to contain the sit locus, was confirmed by analysis of the tomato whole genome shotgun sequence assembly. This novel AO protein shares 76-78% identity at the amino acid level with the previously characterised tomato AO proteins. The DNA sequence of this putative sit gene was characterised in wild type and in two allelic sit mutants (sit and sitw): changes in DNA sequence were identified in these mutant alleles that cause a truncation of exon 2 and the deletion of exon 7, respectively. These results establish the identity of the tomato sit gene and are consistent with its proposed function of encoding the ABA aldehyde oxidase apoenzyme.

A Wilty Mutant of Rice has Impaired Hydraulic Conductance

The rice CM2088 mutant is the wilty phenotype and wilts markedly under well-watered sunny conditions. The leaf water potential and epidermal (mainly stomatal) conductance of CM2088 plants decreased significantly under conditions that induced intense transpiration, as compared with those of wild-type plants, revealing that the wilty phenotype was not the result of abnormal stomatal behavior but was due to an increase in resistance to water transport. The resistance to water transport was dramatically elevated in the node and the sheath and blade of a leaf of the mutant, but not in the root or stem. The diameter of xylem vessels in the large vascular bundles of the leaf sheath and the internode tended to be small, and the numbers of vessel elements with narrowed or scalariform perforation plates in the leaf blade and sheath were greater in the mutant than in the wild type. Most xylem vessels were occluded, with air bubbles in the leaf sheath of the mutant during the midday hours under intense transpiration conditions, while no bubbles were observed in plants that were barely transpiring, revealing that the significant increase in resistance to water transport was a result of the cavitation. The additive effects of cavitation in xylem vessels and the decreased diameter and deformed plates of vessel elements might be responsible for the wilty phenotype of CM2088.

Cation currents in protoplasts from the roots of a Na+ hyperaccumulating mutant of Capsicum annuum

A wilty mutant (scabrous diminutive, sd) of Capsicum annuum L. hyperaccumulates Na+ in all tissues and has a lower K+ content in the roots. This has been shown to be due to a greater efflux of (86)Rb+ (K+) and influx of (22)Na+ in the mutant. In this study, the transporters responsible for these fluxes were investigated by applying patch clamp techniques to protoplasts derived from root cortical cells. Inwardly rectifying K+ currents were comparable in the two genotypes, but a characteristically bigger outward K+ current was observed in protoplasts from mutant roots, correlating with a bigger efflux of (86)Rb+ from mutant plants. Whole-cell currents due to the movement of Na+ have also been studied in both genotypes. The magnitude of the time-independent inward currents that conduct Na+ at hyperpolarizing voltages were comparable in both genotypes. However, microelectrode measurements of membrane potentials in cortical cells of roots in high Na+ conditions revealed that the membrane potentials of the root cells in the mutants were approximately 60 mV more negative than in wild-type root cells. Quantitatively, this hyperpolarization is calculated to be sufficient to account for the increased Na+ influx in the mutants.

Wild barley eibi1 mutation identifies a gene essential for leaf water conservation.

Drought is a major abiotic stress that limits plant growth and crop productivity. A spontaneous wilty mutant (eibi1) hypersensitive to drought was identified from wild barley (Hordeum spontaneum Koch). eibi1 showed the highest relative water loss rate among the known wilty mutants, which indicates that eibi1 is one of the most drought-sensitive mutants. eibi1 had the same abscisic acid (ABA) level, the same ability to accumulate stress-induced ABA, and the same stomatal movement in response to light, dark, drought, and exogenous ABA as the wild type, revealing that eibi1 was neither an ABA-deficient nor an ABA-insensitive mutant. The eibi1 leaves had a larger chlorophyll efflux rate in 80% ethanol than the wild-type leaves; and the transpiration rate of eibi1 was more closely related to chlorophyll efflux rate than to stomatal density, demonstrating that the cuticle of eibi1 was defective. eibi1 will be a promising candidate to study the actual barrier layer in the cuticle that limits water loss of the plant. Exogenous ABA reduced leaf length growth in eibi1 more than in the wild type, implying an interaction on plant growth of ABA signal transduction and the eibi1 product. One may infer that the eibi1 product may reverse the growth inhibition induced by ABA.

The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis

The pathway of biosynthesis of abscisic acid (ABA) can be considered to comprise three stages: (i) early reactions in which small phosphorylated intermediates are assembled as precursors of (ii) intermediate reactions which begin with the formation of the uncyclized C40 carotenoid phytoene and end with the cleavage of 9'-cis-neoxanthin (iii) to form xanthoxal, the C15 skeleton of ABA. The final phase comprising C15 intermediates is not yet completely defined, but the evidence suggests that xanthoxal is first oxidized to xanthoxic acid by a molybdenum-containing aldehyde oxidase and this is defective in the aba3 mutant of Arabidopsis and present in a 1-fold acetone precipitate of bean leaf proteins. This oxidation precludes the involvement of AB-aldehyde as an intermediate. The oxidation of the 4'-hydroxyl group to the ketone and the isomerization of the 1',2'-epoxy group to the 1'-hydroxy-2'-ene may be brought about by one enzyme which is defective in the aba2 mutant and is present in the 3-fold acetone fraction of bean leaves. Isopentenyl diphosphate (IPP) is now known to be derived by the pyruvate-triose (Methyl Erythritol Phosphate, MEP) pathway in chloroplasts. (14C)IPP is incorporated into ABA by washed, intact chloroplasts of spinach leaves, but (14C)mevalonate is not, consequently, all three phases of biosynthesis of ABA occur within chloroplasts. The incorporation of labelled mevalonate into ABA by avocado fruit and orange peel is interpreted as uptake of IPP made in the cytoplasm, where it is the normal precursor of sterols, and incorporated into carotenoids after uptake by a carrier in the chloroplast envelope. An alternative bypass pathway becomes more important in aldehyde oxidase mutants, which may explain why so many wilty mutants have been found with this defect. The C-1 alcohol group is oxidized, possibly by a mono-oxygenase, to give the C-1 carboxyl of ABA. The 2-cis double bond of ABA is essential for its biological activity but it is not known how the relevant trans bond in neoxanthin is isomerized.

Expression of a dehydrin gene during embryo development and drought stress in ABA-deficient mutants of sunflower (Helianthus annuus L.).

The synthesis of a particular class of proteins, the dehydrins, is a common response to drought in plants. Dehydrins are known to be synthesized by the cell in response to abscisic acid, which represents a link between environment and nuclear activity, though dehydrin genes may be expressed even constitutively. We have investigated the relationship between abscisic acid (ABA) and accumulation of a dehydrin mRNA in sunflower, in which a dehydrin cDNA (HaDhnla) was isolated. In particular, we studied changes in the steady-state level of dehydrin transcripts in two mutants for ABA synthesis and accumulation: nd-1 (an albino, non-dormant and lethal mutant with a very low ABA content and no ABA accumulation in response to stress) and w-1 (a wilty mutant, with reduced ABA accumulation) during embryo and plantlet development and drought stress. Differences between genotypes were observed through embryogenesis: w-1 shows a lower content of dehydrin transcripts in the early stages compared to control plants, indicating that ABA affects dehydrin mRNA accumulation; however, dehydrin transcripts level appears independent of ABA content in late embryogenesis. Also during drought stress in w-1 adult leaves, ABA is not quantitatively related to the steady-state level of the HaDhn1a transcripts. Finally, data on nd-1 mutant show a high level of dehydrin transcripts after drought stress in plantlet cotyledons and leaflets. These results indicate the existence of two regulation pathways of HaDhn1a transcripts accumulation, an ABA-dependent and an ABA-independent one, which may have cumulative effects.

Seed germination and seedling growth in a wilty mutant of sunflower ( Helianthus annuus L.): Effect of abscisic acid and osmotic potential

The effect of abscisic acid (ABA) on seed germination and seedling growth of w−1, a wilty, ABA-deficient sunflower ( Helianthus annuus L.) mutant, is described in comparison with the control line (W-1). Exogenous ABA inhibited seed germination in both genotypes; however, for some ABA concentrations, whole achenes of w-1 mutant were slightly more inhibited than the wild type; naked seeds of w-1 were significantly less inhibited than the wild type. To investigate the sensitivity to osmotic stress in relation to ABA levels, naked seeds were subjected to different PEG solutions. Mutant seeds showed a higher osmotic resistance, germinating at more negative osmotic potentials; moreover, at low osmotic potentials, germination of w-1 seeds occurred much earlier than control seeds. Growth inhibition experiments indicated that shoot and root growth of w-1 seedlings is less sensitive to ABA with respect to control line. These differences may be related to the ABA deficiency of the w-1 genotype, but we cannot exclude the possibility that the wilty mutant may have a complex alteration of either ABA synthesis and the reception and/or transduction of the hormonal signal.

Cell neutrality in sodium chloride‐treated leaves and calli of the wilty mutantlycopersicon esculentumflacca and the wild specieslycopersicon peruvianum

Abstract Some parameters involved in cell neutrality (inorganic solute, cation‐anion balance, proline and organic acid contents linked to oxidative respiration, and PEPCase activity) were determined in salinized plant leaves and leaf calli from two tomato species, the wilty mutant Lycopersicon esculentum cv flacca, and the wild relative L. peruvianum. Callus sensitivity was associated to membrane integrity and cell neutrality; while whole plant sensitivity was linked to nutritional unbalance and energy supply, both factors affecting the maintenance of the cell neutrality by means of ion translocation/compartimentalization.

Abscisic‐acid‐deficient mutants at the aba gene locus of Arabidopsis thaliana are impaired in the epoxidation of zeaxanthin

Abstract. The xanthophyll content of wild type and abscisic acid (ABA) ‐ deficient mutants of pea and Arabidopsis thaliana was determined. The wilty mutant of pea was indistinguishable from the non‐mutant control. In contrast, plants homozygous for mutant alleles at the aba locus of Arabidopsis were very different from wild type. In these mutants, zeaxanthin accumulated to abnormally high levels. The major carotenoids, violaxanthin and 9′‐cis‐neoxanthin were virually absent from the mutant chromatograms. It was concluded that the aba genetic lesion impairs the epoxidation of zeaxanthin to violaxanthin and that this results in an inability to accumulate ABA. This provides clear evidence that zeaxanthin is a precursor of ABA.

An homogeneous radioimmunoassay for abscisic acid using a scintillation proximity assay technique

AbstractAn homogeneous radioimmunoassay (RIA) for the plant growth regulator abscisic acid (ABA) has been developed by modifying an established heterogeneous assay. The introduction of a scintillation proximity reagent has enabled the production of a one‐step RIA in which radioactivity associated with antibody‐bound ABA can be counted in the presence of the unbound radiolabelled ABA and without the addition of a liquid scintillation cocktail. The assay has been validated by comparing the results with those obtained by combined gas chromatography/mass spectrometry and the precision of each method determined. The homogeneous RIA has a working range of 0.165–13.2 ng ABA and has been used to determine ABA concentration in extracts of leaves of tomato, Lycopersicon esculentum Mill. cv. Ailsa Craig (wild type) and three wilty mutants, notabilis, flacca and sitiens.

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.

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.

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.

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

Using (13)C-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 [(13)C]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 [(2)H]ABA-t-diol to ABA. Incubation of stressed leaves in the presence of (18)O2 provided evidence consistent with Xan and ABA originating via oxidative cleavage of a xanthophyll such as violaxanthin.

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.

Abscisic acid relations and the response of Populus trichocarpa stomata to leaf water potential.

The role of abscisic acid (ABA) in the mediation of stomatal responses to low leaf water potential was examined with intact plants and epidermal strips of Populus trichocarpa Torr. & A. Gray. Clones of this species grown under well-watered conditions maintain a high leaf conductance when the foliage wilts. However, foliar ABA concentration in P. trichocarpa increased manyfold in response to water stress as it did also in P. deltoides Bartr. ex Marsh. and P. trichocarpa x deltoides hybrids. Application of ABA to epidermal strips appeared to cause solute leakage, however stomata of P. trichocarpa remained partially open even when the guard cells were plasmolyzed. Foliar application of ABA induced closure of stomata in young expanding leaves, but not in fully expanded foliage. Ten days after ABA application, stomata on young leaves were open at high water potential but closed at low water potential. These characteristics are discussed with respect to wilty mutants of tomato and potato, which also have stomata unresponsive to leaf wilting.

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.

Abscisic Acid and C10Dicarboxylic Acids in Wilty Tomato Mutants

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