Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.     

The Physiology of a Wilty Pea: Abscisic Acid Production under Water Stress

‘Wilty’ (JI 1069), a mutant of Pisum sativum, hasbeen examined for its ability to produce abscisic acid (ABA)under water stress. ABA was measured using combined gas chromatography-massspectrometry and multiple-ion-monitoring employing a deuteratedinternal standard. In intact droughted plants, ‘Wilty’produced less ABA than a non-wilty line (JI 1194) and maximumproduction was delayed. Detached leaves of the wilty mutantlost significantly more water than control leaves but did notshow an increase in ABA content. Non-stressed mutant materialfrom both intact plants and isolated leaves contained less ABAthan control tissue. Key words: Pea, Wilty mutant, Abscisic acid     

Wild barley<Emphasis Type="Italic"> eibi1</Emphasis> 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.Abbreviation ABA Abscisic acid     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant: III. Phenotypic interaction in reciprocal grafts from wilty mutant and wild-type plants

The present study was conducted to evaluate phenotypic interactionin reciprocal grafts between wilty (w-1) sunflower mutant andnormal (W-1) plants. The w-1 genotype is a ‘leaky’ABA-deficient mutant, characterized by high stomatal conductance,in both light and dark conditions, and high transpiration rate. In well-watered conditions, mutant scions grafted on to normalrootstock (w-1/W-1) showed higher leaf relative water content,leaf water potential and ABA levels than those of control grafts(w-1/w-1). In addition, detached leaves of w-1/W-1 exhibitedlower water loss than w-1/w-1 grafts, while mutant rootstockdid not affect the transpiration rate of detached W-1 leaves.When drought stress was imposed to potted plants by withholdingwater, the mutant scions grafted on to normal roots showed apartial phenotypic reversion. A rapid stomatal closure and arise in ABA levels in response to a small decrease in leaf waterpotential was observed. By contrast, in w-1/w-1 grafts significantreductions in stomatal conductance and ABA accumulation weredetected only in conjunction with a severe water deficit. W-1scions on mutant stocks (W-1/w-1) maintained the normal phenotypeof control wild-type grafts (W1/W-1). Key words: ABA, grafting, Helianthus annuus, stomatal conductance, water relations, wilty mutant     

Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis

To examine the cross talk between the abscisic acid (ABA) and ethylene signal transduction pathways, signaling events during ABA-induced stomatal closure were examined in Arabidopsis (Arabidopsis thaliana) wild-type plants, in an ethylene-overproducing mutant (eto1-1), and in two ethylene-insensitive mutants (etr1-1 and ein3-1). Using isolated epidermal peels, stomata of wild-type plants were found to close within a few minutes in response to ABA, whereas stomata of the eto1-1 mutant showed a similar but less sensitive ABA response. In addition, ABA-induced stomatal closure could be inhibited by application of ethylene or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In contrast, stomata of the etr1-1 and ein3-1 mutants were able to close in response to concomitant ABA and ACC application, although to a lesser extent than in wild-type plants. Moreover, expression of the ABA-induced gene RAB18 was reduced following ACC application. These results indicate that ethylene delays stomatal closure by inhibiting the ABA signaling pathway. The same inhibitive effects of ethylene on stomatal closure were observed in ABA-irrigated plants and the plants in drought condition. Furthermore, upon drought stress, the rate of transpiration was greater in eto1-1 and wild-type plants exposed to ethylene than in untreated wild-type control plants, indicating that the inhibitive effects of ethylene on ABA-induced stomatal closure were also observed in planta.     

An Investigation into the Stomatal Behaviour of a Wilty Mutant of Pisum sativum

The water relations and stomatal behaviour of a wilty line ofpea (JI 1069) were investigated and compared with those of severalnon-wilty lines (JI 1180, JI 1194, and JI 74). The leaves ofthe wilty line were found to have a lower percent water content,water potential and diffusive resistance and the dimensionsof the stomatal cells were larger than those of the non-wiltytypes. The aperture of stomata on epidermal samples taken from plantsin the light or dark period of a diurnal rhythm was consistentlylarger for the wilty pea than for the non-wilty lines, however,their stomatal responses on detached epidermis to light, CO₂and KC1 concentration were similar. There was no differencein response to ABA of stomata on detached epidermis of wiltyor non-wilty types of pea. Key words: Pisum sativum, Wilty mutant, Water relations, Stomatal behaviour     

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     

The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis

The movement of guard cells in stomatal complexes controls water loss and CO(2) uptake in plants. Examination of the dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) revealed that it is expressed and functions in Arabidopsis guard cells. CHL1 promoter-beta-glucuronidase and CHL1 promoter-green fluorescent protein constructs showed strong expression in guard cells, and immunolocalization experiments with anti-CHL1 antibody confirmed these results. To assess CHL1 function, chl1 mutant plants grown in the presence of nitrate were examined. Compared with wild-type plants, chl1 mutants had reduced stomatal opening and reduced transpiration rates in the light or when deprived of CO(2) in the dark. These effects result in enhanced drought tolerance in chl1 mutants. At the cellular level, chl1 mutants showed reduced nitrate accumulation in guard cells during stomatal opening and failed to show nitrate-induced depolarization of guard cells. In wild-type guard cells, nitrate induced depolarization, and nitrate concentrations increased threefold during stomatal opening. These results identify an anion transporter that functions in stomatal opening and demonstrate that CHL1 supports stomatal function in the presence of nitrate.     

Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation

In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2-1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1-1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.     

Tissue-specific localization of an abscisic acid biosynthetic enzyme, AAO3, in Arabidopsis

Arabidopsis aldehyde oxidase 3 (AAO3) is an enzyme involved in abscisic acid (ABA) biosynthesis in response to drought stress. Since the enzyme catalyzes the last step of the pathway, ABA production sites may be determined by the presence of AAO3. Here, AAO3 localization was investigated using AAO3 promoter:AAO3-GFP transgenic plants and by an immunohistochemical technique. AAO3-GFP protein exhibited an activity to produce ABA from abscisic aldehyde, and the transgene restored the wilty phenotype of the aao3 mutant. GFP-fluorescence was detected in the root tips, vascular bundles of roots, hypocotyls and inflorescence stems, and along the leaf veins. Intense immunofluorescence signals were localized in phloem companion cells and xylem parenchyma cells. Faint but significant GFP- and immuno-fluorescence signals were observed in the leaf guard cells. In situ hybridization with antisense AAO3 mRNA showed AAO3 mRNA expression in the guard cells of dehydrated leaves. These results indicate that the ABA synthesized in vascular systems is transported to various target tissues and cells, and also that the guard cells themselves are able to synthesize ABA.     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant II. Water relations, stomatal conductance, abscisic acid content in leaves and xylem sap of plants subjected to water deficiency

The response of w-1, a wilty sunflower (Helianthus annuus L.)mutant, to water stress is described in comparison with thecontrol line (W-1). Detached leaves of w-1 strongly dehydratedduring the first 30 min without significant changes in leafconductance, whereas W-1 responded rapidly to water loss byreducing stomatal aperture. After 2 h stress ABA increased slightlyin w-1, while W-1 leaves showed a 20-fold increase. When waterstress was imposed to potted plants by water withholding, w-1quickly dehydrated, and lost turgor, while W-1 maintained positiveturgor values for a longer period. Wild-type plants respondedto small changes in leaf water potential by accumulating ABAand by closing stomata, whereas in the mutant significant changesin ABA content and in stomatal conductance were found only atvery low water potentials. In another experiment in which waterwas withheld under high relative humidity, when soil water contentstarted to decrease W-1 rapidly closed stomata in the absenceof any change in leaf water status and the reduction in conductancewas paralleled by a rise in xylem sap ABA concentration. Bycontrast the mutant started to accumulate ABA in the xylem sapand to close stomata when soil water content and leaf waterpotential were dramatically reduced. The low endogenous ABAlevels and the inability to synthesize the hormone rapidly eitherin the leaves or in the roots seem to be responsible for thehigh sensitivity of w-1 to water stress. Key words: ABA, Helianthus annuus L, water relations, stomatal conductance, drought, wilty mutant     

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 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 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.     

Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H(2)O(2) production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H(2)O(2) production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H(2)O(2) production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.     

Phospholipase D δ knock-out mutants are tolerant to severe drought stress

Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance.     

Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection

Oxalic acid is a virulence factor of several phytopathogenic fungi, including Sclerotinia sclerotiorum (Lib.) de Bary, but the detailed mechanisms by which oxalic acid affects host cells and tissues are not understood. We tested the hypothesis that oxalate induces foliar wilting during fungal infection by manipulating guard cells. Unlike uninfected leaves, stomatal pores of Vicia faba leaves infected with S. sclerotiorum are open at night. This cellular response appears to be dependent on oxalic acid because stomatal pores are partially closed when leaves are infected with an oxalate-deficient mutant of S. sclerotiorum. In contrast to oxalate-deficient S. sclerotiorum, wild-type fungus causes an increase in stomatal conductance and transpiration as well as a decrease in plant biomass. Green fluorescent protein-tagged S. sclerotiorum emerges through open stomata from the uninfected abaxial leaf surface for secondary colonization. Exogenous application of oxalic acid to the detached abaxial epidermis of V. faba leaves induces stomatal opening. Guard cells treated with oxalic acid accumulate potassium and break down starch, both of which are known to contribute to stomatal opening. Oxalate interferes with abscisic acid (ABA)-induced stomatal closure. The Arabidopsis (Arabidopsis thaliana) L. Heynh. mutants abi1, abi3, abi4, and aba2 are more susceptible to oxalate-deficient S. sclerotiorum than wild-type plants, suggesting that Sclerotinia resistance is dependent on ABA. We conclude that oxalate acts via (1) accumulation of osmotically active molecules to induce stomatal opening and (2) inhibition of ABA-induced stomatal closure.