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     

Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco

The plant hormone abscisic acid (ABA) participates in the control of several important physiological processes in plants such as stomata regulation, seed dormancy and stress tolerance. A new strategy was developed to study these phenomena by blocking abscisic acid with intracellularly expressed specific single-chain variable fragment (scFv) antibodies. Here evidence is presented that the expression of single-chain Fv antibodies against abscisic acid in the endoplasmic reticulum of transgenic tobacco cells leads to a wilty phenotype. Stomatal conductance is increased at high CO₂ concentrations dependent on the level of antibody expression in leaves. Symptoms of abscisic acid deficiency were generated in the transformants although they have even higher levels of abscisic acid than wild-type plants.     

Early stomatal closure in waterlogged pea plants is mediated by abscisic acid in the absence of foliar water deficits

Abstract Soil waterlogging decreased leaf conductance (interpreted as stomatal closure) of vegetative pea plants (Pisuin sativum L. cv. ‘Sprite’) approximately 24 h after the start of flooding, i.e. from the beginning of the second 16 h-long photo-period. Both adaxial and abaxial surfaces of leaves of various ages and the stipules were affected. Stomatal closure was sustained for at least 3 d with no decrease in foliar hydration measured as water content per unit area, leaf water potential or leaf water saturation deficit. Instead, leaves became increasingly hydrated in association with slower transpiration. These changes in the waterlogged plants over 3 d were accompanied by up to 10-fold increases in the concentration of endogenous abscisic acid (ABA). Waterlogging also increased foliar hydration and ABA concentrations in the dark. Leaves detached from non-waterlogged plants and maintained in vials of water for up to 3 d behaved in a similar way to leaves on flooded plants, i.e. stomata closed in the absence of a water deficit but in association with increased ABA content. Applying ABA through the transpiration stream to freshly detached leaflets partially closed stomata within 15 min. The extractable concentrations of ABA associated with this closure were similar to those found in flooded plants. When an ABA-deficient ‘wilty’ mutant of pea was waterlogged, the extent of stomatal closure was less pronounced than that in ordinary non-mutant plants, and the associated increase in foliar ABA was correspondingly smaller. Similarly, waterlogging closed stomata of tomato plants within 24 h, but no such closure was seen in ‘flacca’, a corresponding ABA-deficient mutant. The results provide an example of stomatal closure brought about by stress in the root environment in the absence of water deficiency. The correlative factor operating between the roots and shoots appeared to be an inhibition of ABA transport out of the shoots of flooded plants, causing the hormone to accumulate in the leaves.     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant: I. Abscisic acid content, light-dark changes in the stomatal conductance and genetic analysis

The present study was conducted to characterize geneticallyand physiologically a spontaneous mutation in sunflower whichconfers a wilty phenotype. The wilting condition of the mutantis due to abnormal stomatal behaviour. The mutant stomata resistclosure in darkness. This abnormality is associated with lowlevels of endogenous abscisic acid (ABA). By artificially elevatingthe ABA content of the mutant plants by spray treatments with10^– and 10^– M solutions it proved possible to effecta phenotypic reversion of the mutant. It has, therefore, beenproposed that the primary effect of this spontaneous mutationis to reduce the level of ABA. The genetic analysis has shownthat the willy phenotype is due to recessive nuclear mutationat a single locus. Key words: ABA, Helianthus annuus L, stomatal conductance, wilty mutant     

The effect of exogenous abscisic acid on stomatal development, stomatal mechanics, and leaf gas exchange in Tradescantia virginiana

Gas exchange parameters and stomatal physical properties were measured in Tradescantia virginiana plants grown under well-watered conditions and treated daily with either distilled water (control) or 3.0 mM abscisic acid (ABA). Photosynthetic capacity (CO(2) assimilation rate for any given leaf intercellular CO(2) concentration [c(i)]) and relative stomatal sensitivity to leaf-to-air vapor-pressure difference were unaffected by the ABA treatment. However, at an ambient CO(2) concentration (c(a)) of 350 micromol mol(-1), ABA-treated plants operated with significantly lower c(i). ABA-treated plants had significantly smaller stomata and higher stomatal density in their lower epidermis. Stomatal aperture versus guard cell pressure (P(g)) characteristics measured with a cell pressure probe showed that although the form of the relationship was similar in control and ABA-treated plants, stomata of ABA-treated plants exhibited more complete closure at P(g) = 0 MPa and less than half the aperture of stomata in control plants at any given P(g). Scaling from stomatal aperture versus P(g) to stomatal conductance versus P(g) showed that plants grown under ABA treatment would have had significantly lower maximum stomatal conductance and would have operated with lower stomatal conductance for any given guard cell turgor. This is consistent with the observation of lower c(i)/c(a) in ABA-treated plants with a c(a) of 350 micromol mol(-1). It is proposed that the ABA-induced changes in stomatal mechanics and stomatal conductance versus P(g) characteristics constitute an improvement in water-use efficiency that may be invoked under prolonged drought conditions.     

The role of abscisic acid in disturbed stomatal response characteristics of Tradescantia virginiana during growth at high relative air humidity

In this study, the role of abscisic acid (ABA) in altered stomatal responses of Tradescantia virginiana leaves grown at high relative air humidity (RH) was investigated. A lower ABA concentration was found in leaves grown at high RH compared with leaves grown at moderate RH. As a result of a daily application of 20 microM ABA to leaves for 3 weeks during growth at high RH, the stomata of ABA-treated leaves grown at high RH showed the same behaviour as did the stomata of leaves grown at moderate RH. For example, they closed rapidly when exposed to desiccation. Providing a high RH around a single leaf of a plant during growth at moderate RH changed the stomatal responses of this leaf. The stomata in this leaf grown at high RH did not close completely in response to desiccation in contrast to the stomata of the other leaves from the same plant. The ABA concentration on a fresh weight basis, though not on a dry weight basis, of this leaf was significantly lower than that of the others. Moreover, less closure of stomata was found in the older leaves of plants grown at high RH in response to desiccation compared with younger leaves. This was correlated with a lower ABA concentration in these leaves on a fresh weight basis, though not on a dry weight basis. Stomata of leaves grown at moderate RH closed in response to short-term application of ABA or sodium nitroprusside (SNP), while for leaves grown at high RH there was a clear difference in stomatal responses between the leaf margins and main-vein areas. The stomatal aperture in response to short-term application of ABA or SNP at the leaf margins of leaves grown at high RH remained significantly wider than in the main-vein areas. It was concluded that: (i) a long-term low ABA concentration in well-watered plants during growth at high RH could be a reason for less or no stomatal closure under conditions of drought stress; and (ii) the long-term ABA concentration on a fresh weight basis rather than on a dry weight basis is likely to be responsible for structural or physiological changes in stomata during leaf growth.     

Stomatal closure is induced rather by prevailing xylem abscisic acid than by accumulated amount of xylem-derived abscisic acid

We have studied the stomatal response in relation to the xylem-derived abscisic acid (ABA) accumulation in sunflower leaves. When ABA was introduced into detached leaves of the sunflower through xylem flux, stomatal conductance was regulated, water flux was changed as a result and at the same time the xylem-derived ABA was metabolised in the leaves. We computed the xylem-derived ABA accumulation in the leaves as a function of time by taking into account the variation of ABA flux into the leaves (the product of water flux and ABA concentration) and a continuing ABA metabolism. We found that ABA accumulation was rapid during an initial lag phase, much slowed down during the decreasing phase of stomatal conductance, but still substantial when stomatal conductance reached a new stable state. The results show a poor link between the kinetics of ABA-induced stomatal closure and the xylem-derived ABA accumulation. Xylem-derived ABA was metabolised rapidly in the leaves. Tetcyclacis, as an inhibitor, substantially inhibited this process. Two hours after ABA was fed into a leaf, about 70% of the fed ABA was metabolised, but when tetcyclacis was added into the feeding solution, less than 30% of ABA was metabolised, even after 24 h of incubation. The inhibition of ABA metabolism by tetcyclacis did not lead to more stomatal closure, which was still concentration-dependent. Since the accumulation of xylem-derived ABA was enhanced substantially by the presence of tetcyclacis, these results strongly indicate that stomata mainly respond to the prevailing ABA concentration in the xylem stream, rather than to the accumulated amount of xylem-derived ABA in the leaves.     

Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid

The tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) deficiency. The wild-type allele of notabilis, LeNCED1, encodes a putative 9-cis-epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline-inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10-fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric 'Super-Promoter'. Many of these plants were wilty, suggesting co-suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had significantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor norflurazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the first time that plant ABA content can be increased through manipulating NCED.     

Diurnal variations in abscisic acid content and stomatal response to applied abscisic acid in leaves of irrigated and non-irrigated Arbutus unedo plants under naturally fluctuating environmental conditions

Summary Endogenous abscisic acid content (ABA) of Arbutus unedo leaves growing under natural conditions in a macchia near Sobreda, Portugal, was very high (0.25 to 2.3 g g¹ fresh weight). Highest concentrations were found during the very early morning hours and at midday. During the late morning hours and in the late afternoon ABA concentrations decreased to between one-third and one-fourth of peak values. The samples for ABA content were obtained from both irrigated ( between-10 and-25 bar) and non-irrigated plants experiencing natural water stress during the dry season ( of-50 bar). During the course of the measurement day, stomatal conductance was relatively constant and conductance of watered plants was 50 to 100% greater than that of unwatered plants. No clear correlations between ABA content and stomatal conductance and/or xylem water potential were observed. Despite large differences in water potential and differences in degree of stomatal opening, absolute concentrations of ABA were not found to differ.Small quantities (8–14 pmoles cm² leaf area) of ABA were applied to leaves of irrigated and non-irrigated Arbutus unedo plants by injection into the petiole. These extremely small ABA doses resulted in transient reductions in stomatal conductance. The effectiveness with which injected ABA closed stomata was highest during the morning and decreased substantially at midday. Increased sensitivity to injected ABA may again occur in the late afternoon but recent measurements suggest that this may depend on long-term drought experience of the plants. The characteristics of the response to injected ABA were similar in irrigated and non-irrigated plants although irrigated plants responded in general more strongly.     

Stomatal behaviour and water movement through roots of wheat plants treated with abscisic acid

Abstract Experiments with isolated roots of wheat plants suggested that when water uptake rates are low, low concentrations of abscisic acid (ABA) may increase the flux of water into roots. This increase was recorded despite an ABA-stimulated reduction in the hydraulic conductance of the whole root system. Hydraulic conductances were measured under steady-state conditions. A system is described where the stomatal behaviour and water movement through roots of a single intact plant may be concurrently monitored. Experiments with intact plants confirmed that application of ABA could increase the rate of water movement into roots when uptake rates were low. No such increase was observed at high flux rates. Application of ABA to roots caused partial stomatal closure and caused conductance to oscillate around a reduced mean value. An ABA-stimulated increase in the turgor sensitivity of stomata is postulated and the significance of this effect is discussed.     

Stomatal response characteristics of Tradescantia virginiana grown at high relative air humidity

Plants produced at high relative air humidity (RH) show poor control of water loss after transferring to low RH, a phenomenon which is thought to be due to their stomatal behaviour. The stomatal anatomy and responses of moderate (55%) and high (90%) RH grown Tradescantia virginiana plants to treatments that normally induce stomatal closure, i.e. desiccation, abscisic acid (ABA) application and exposure to darkness were studied using attached or detached young, fully expanded leaves. Compared with plants grown at moderate RH the transpiration rate, stomatal conductance and aperture of high RH grown plants measured at the same condition (40% RH) were, respectively, 112, 139 and 132% in light and 141, 188 and 370% in darkness. Besides the differences in stomatal size (guard cell length was 56.7 and 73.3 µm for moderate and high RH grown plants, respectively), there was a clear difference in stomatal behaviour. The stomata responded to desiccation, ABA and darkness in both moderate and high RH grown plants, but the high variability of stomatal closure in high RH grown plants was striking. Some stomata developed at high RH closed in response to darkness or to a decrease in relative water content to the same extent as did stomata from moderate RH grown plants, whereas others closed only partly or did not close at all. Evidently, some as yet unidentified physiological or anatomical changes during development disrupt the normal functioning of some stomata in leaves grown at high RH. The failure of some stomata to close fully in response to ABA suggests that ABA deficiency was not responsible for the lack of stomatal closure in response to desiccation.     

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     

NFX1-LIKE2 (NFXL2) suppresses abscisic acid accumulation and stomatal closure in Arabidopsis thaliana

The NFX1-LIKE1 (NFXL1) and NFXL2 genes were identified as regulators of salt stress responses. The NFXL1 protein is a nuclear factor that positively affects adaptation to salt stress. The nfxl1-1 loss-of-function mutant displayed reduced survival rates under salt and high light stress. In contrast, the nfxl2-1 mutant, defective in the NFXL2 gene, and NFXL2-antisense plants exhibited enhanced survival under these conditions. We show here that the loss of NFXL2 function results in abscisic acid (ABA) overaccumulation, reduced stomatal conductance, and enhanced survival under drought stress. The nfxl2-1 mutant displayed reduced stomatal aperture under all conditions tested. Fusicoccin treatment, exposition to increasing light intensities, and supply of decreasing CO(2) concentrations demonstrated full opening capacity of nfxl2-1 stomata. Reduced stomatal opening presumably is a consequence of elevated ABA levels. Furthermore, seedling growth, root growth, and stomatal closure were hypersensitive to exogenous ABA. The enhanced ABA responses may contribute to the improved drought stress resistance of the mutant. Three NFXL2 splice variants were cloned and named NFXL2-78, NFXL2-97, and NFXL2-100 according to the molecular weight of the putative proteins. Translational fusions to the green fluorescent protein suggest nuclear localisation of the NFXL2 proteins. Stable expression of the NFXL2-78 splice variant in nfxl2-1 plants largely complemented the mutant phenotype. Our data show that NFXL2 controls ABA levels and suppresses ABA responses. NFXL2 may prevent unnecessary and costly stress adaptation under favourable conditions.     

The relationship between the methyl-erythritol phosphate pathway leading to emission of volatile isoprenoids and abscisic acid content in leaves

It was investigated whether the methyl-erythritol phosphate (MEP) pathway that generates volatile isoprenoids and carotenoids also produces foliar abscisic acid (ABA) and controls stomatal opening. When the MEP pathway was blocked by fosmidomycin and volatile isoprenoid emission was largely suppressed, leaf ABA content decreased to about 50% and leaf stomatal conductance increased significantly. No effect of fosmidomycin was seen in leaves with constitutively high rates of stomatal conductance and in plant species with low foliar ABA concentration. In all other cases, isoprene emission was directly associated with foliar ABA, but ABA reduction upon MEP pathway inhibition was also observed in plant species that do not emit isoprenoids. Stomatal closure causing a midday depression of photosynthesis was also associated with a concurrent increase of isoprene emission and ABA content. It is suggested that the MEP pathway generates a labile pool of ABA that responds rapidly to environmental changes. This pool also regulates stomatal conductance, possibly when coping with frequent changes of water availability. MEP pathway inhibition by leaf darkening, and its down-regulation by exposure to elevated CO2, was also associated with a reduction of foliar ABA content. However, stomatal conductance was reduced, indicating that stomatal aperture is not regulated by the MEP-dependent foliar ABA pool, under these specific cases.     

Diurnal change in the relationship between stomatal conductance and abscisic acid in the xylem sap of field-grown peach trees

To evaluate whether abscisic acid (ABA) in the xylem sap playsan important role in controlling stomatal aperture of field-grownPrunus persica trees under drought conditions, stomatal conductance(g) and xylem ABA concentrations were monitored both in irrigatedand non-irrigated trees, on two consecutive summer days (threetimes a day). Stomata1 conductance of non-irrigated trees hada morning maximum and declined afterwards. The changes in gduring the day, rather than resulting from variations in theconcentrations of ABA in the xylem sap or the delivery rateof this compound to the leaves, were associated with changesin the relationship between g and xylem ABA. The stomata ofwater-stressed trees opened during the first hours of the day,despite the occurrence of a high concentration of ABA in thexylem sap. However, stomatal responsiveness to ABA in the xylemwas enhanced throughout the day. As a result, a tight inverserelationship between g and the logarithm of xylem ABA concentrationwas found both at midday and in the afternoon. A similar relationshipbetween g and ABA was found when exogenous ABA was fed to leavesdetached from well-watered trees. These results indicate thatABA derived from the xylem may account for the differences ing observed between field-grown peach trees growing with differentsoil water availabilities. Several possible explanations forthe apparent low stomatal sensitivity to xylem ABA in the morning,are discussed, such as high leaf water potential, low temperatureand high cytokinin activity. Key words: Prunus persica L., stomata, xylem ABA, water deficits, root-to-shoot communication     

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     

Effects of patchy stomatal closure on gas exchange measurements following abscisic acid treatment

Gas exchange data and images of leaf fluorescence were collected concurrently as stomata responded to abscisic acid (ABA) application. When 10^?5kmolm^?3 ABA was applied to the transpiration stream in a short pulse, stomatal conductance (g_s), photosynthesis (A) and intercellular CO2 concentration (C_i) decreased rapidly after a short lag period and became approximately constant after 2h. There was an apparent reduction in the A versus c1 relationship as stomata closed, but the data returned to the A versus C1 curve while stomatal conductance was constant or slowly rising during the second hour after ABA treatment. Larger amounts of ABA administered during the pulse caused larger deviations from the A versus c1 relationship. When 10^?7kmolm^?3 ABA was applied continuously through the transpiration stream, gs, A and Cⁱ decreased, but there was no substantial deviation from the A versus c{ curve. Fluorescence images were patchy as stomata closed for all experiments, but became slowly more uniform during the time that gas exchange was returning to the A versus Cj curve. The distribution of con-ductance among patches was not bimodal, and larger devi-ations from the A versus ct curve had greater ranges of pixel values and more pixel values representing low values of Cj during stomatal closure than did experiments show-ing small or no deviation. Estimates of A and gs from fluo-rescence images compared favourably with measured val-ues in most cases, suggesting that the patchy distributions of fluorescence were caused by patchy distributions of stomatal conductance and that apparent reductions in the A versus ct relationship were the result of these patchy stomatai distributions and not direct effects of ABA on mesophyll functioning. The data show that stomatal patches can be temporary and that patchiness may not be reflected in gas exchange data if the range of stomatal con-ductances is not large. These observations may explain some of the discrepancies among previous studies concerning the effect of ABA on the A versus Cⁱ relationship.     

Species-dependent changes in stomatal sensitivity to abscisic acid mediated by external pH

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.     

Specificity and reversibility of the rapid stomatal response to abscisic acid

Summary Closure of stomata caused by low (10^-7M) concentrations of abscisic acid (ABA) is specific for cis-trans ABA, and is initiated within 5 minutes. Upon withdrawal of the hormone supply, reopening starts within 5 minutes. Gas analysis of leaves treated with ABA or DCMU allows one to distinguish effects on the stomatal apparatus from inhibition of photosynthesis and to conclude that ABA acts on stomata directly.