Bulk Leaf Abscisic Acid Research Articles

ABA, Hydraulics, and Gas Exchange of Split-rooted Apple Trees

Approach-grafted 1-year-old `Gala'/M7 apple trees were grown with both tops for the remainder of the 2003 season in a greenhouse. Trees were supplied with >100% (control, PRD100) or 50% (PRD50, DI50) of daily ETc either applied to one root compartment only (PRD100, PRD50) or divided evenly across both root compartments (control and DI50). ETc was estimated from gravimetric measurements, and irrigation was switched between wet and dry root compartments several times throughout the experiment. Soil moisture was measured both gravimetrically (tripod) and volumetrically (time-domain reflectometry). Predawn leaf water potential (υpd) and single leaf gas exchange (photosynthesis, stomatal conductance, and transpiration) were recorded daily, and sap flow in stems and roots was monitored continuously using the heat-pulse technique. Leaves were collected for abscisic acid (ABA) determination following gas exchange measurements. Regardless of irrigation placement (i.e., PRD or DI), both 50% ETc treatments experienced similar declines in υpd and single leaf gas exchange relative to control levels. In addition, ABA concentrations were similar for PRD50 and DI50, and were significantly higher than the control and PRD100 treatments. PRD100 trees had similar υpd as control trees; however, gas exchange was reduced >25% compared to the control. Bulk leaf ABA concentration did not differ significantly from control levels and does not by itself explain the down regulation of stomata with PRD100.

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.

QTL analysis of drought-related traits and grain yield in relation to genetic variation for leaf abscisic acid concentration in field-grown maize

deficit (Zeevaart and Creelman, 1988; Bray, 1997). ABA modulates the expression of a large number of genes Abscisic acid (ABA) concentration is a quantitatively whose products may protect the cell from the harmful inherited trait which plays a pivotal role in the eVects of an excessive water loss (Bray, 1993; Close, 1996, response of plants to drought stress. A recent study 1997; Ingram and Bartels, 1996). It has been hypothesized identified 17 quantitative trait loci (QTLs) controlling that ABA may be involved in controlling leaf expansion bulk-leaf ABA concentration (L-ABA) in a maize (Zea during water deficit via modulation of cell wall-associated mays L.) population of 80 F 4 random families tested peroxidase activity (Bacon et al., 1997). Under conditions for two years under droughted field conditions. Sixteen of water deficit, an increased ABA concentration has also of the QTL regions influencing L-ABA also harboured been shown to increase the root to shoot ratio (Sharp, QTLs for one or more of the following traits: stomatal 1996), another important trait influencing drought tolerconductance, a drought sensitivity index, leaf temper- ance. The adaptive role of an increased ABA concentraature, leaf relative water content, anthesis-silking tion is well represented by the consequent reduction in interval, and grain yield. The analysis of the effects of stomatal conductance and in water lost by transpiration each QTL region on the investigated traits indicated ( Trejo et al., 1995). Even though it has been shown that that L-ABA mainly represented an indicator of the level ABA in the xylem sap more tightly controls stomatal of drought stress experienced by the plant at the time conductance than ABA in the bulk-leaf tissue (Davies of sampling because an increase in L-ABA was most and Zhang, 1991; Tardieu et al., 1992, 1993; Jackson, commonly associated with a decrease in both stomatal 1993), the diYculty in collecting a large number of conductance and grain yield as well as an increase in samples of xylem sap has led many authors to focus on leaf temperature. Opposite results were observed at bulk-leaf ABA concentration (L-ABA). one QTL region on chromosome 7 near the RFLP locus Although the above-mentioned findings clearly indicate asg8. A model is presented to interpret these con- a positive role of ABA in increasing plant survival under trasting results in terms of pleiotropic effects. drought, it remains diYcult to predict the global eVect which genetic variation in L-ABA may have on crop

Root signalling and osmotic adjustment during intermittent soil drying sustain grain yield of field grown wheat

A field study was conducted to investigate the effect of intermittent soil drying on resulting non-hydraulic and hydraulic root signals, leaf gas exchange, leaf growth, day of heading, leaf osmotic adjustment and yield of wheat grown in sand and loam soils in lysimeters. A 40-day-drought treatment was imposed when the flag leaf started to emerge and was terminated close to maturity. Soil water content and soil water potential of various soil layers were measured using the neutron moderation method and tensiometers, respectively. Soil drying in the top soil layers induced increase in both xylem and bulk-leaf abscisic acid (ABA) content and reduced the stomatal conductance and leaf growth even before a measurable change in leaf water potential could be detected in droughted plants when compared with fully watered plants. Further, heading and flowering occurred 4 days earlier in the droughted than in the well-watered plants before any loss in leaf water potential had occurred as compared with the fully watered plants. When more severe drought reduced the leaf water status, further accumulation of leaf ABA occurred and transpiration decreased in addition to gradual osmotic adjustment and senescence of older leaves. The osmotic adjustment sustained leaf turgor pressure during soil drying. At severe drought, the osmotic adjustment at full turgor in the flag leaves was 0.85 MPa. In sand, the kernel dry weight increased and as a result similar grain yield was obtained in both the treatments. In loam which had more water available than sand, no significant reduction in the final yield was induced by the drought. It is concluded that (1) non-hydraulic root signals caused early drought adaptation at mild water stress by reducing leaf growth and stomatal conductance and hastening of heading and flowering; (2) osmotic adjustment sustained turgor maintenance and hence the yield-forming processes during moderate and severe water stress.

Abscisic acid immunolocalization and ultrastructural changes in water‐stressed lavender (Lavandula stoechas L.) plants

The relationship between the bulk abscisic acid (ABA) content, ABA compartmental redistribution, and chloroplast ultrastructural changes was studied in leaves of lavender (Lavandula stoechas L.) plants subjected to water stress. ABA was uniformly distributed in the cytosol, nucleus, chloroplasts, and cell walls of mesophyll cells in well‐watered plants. In plants subjected to water stress (−2.6 MPa water potential) the bulk leaf ABA increased from 900 to 3 600 pmol g−1 fresh weight. At the ultrastructural level, the first indication of this rise in ABA was a 4‐fold increase in ABA immunolabeling in the cell wall in which the highest labeling values were recorded. This increase in apoplastic ABA in lavender was not attributable to ABA release from the chloroplast, because a simultaneous increase in ABA labeling was observed in both the chloroplast and nucleus (2‐ and 3‐fold, respectively). Water stress induced a progressive increase in bulk leaf ABA concentration to 13 600 pmol g−1 fresh weight coincident, with the highest immunolabeling of ABA in the nucleus and chloroplast. Under severe water stress, the chloroplast membrane broke down, resulting in leakage of ABA from the chloroplast. The stress‐induced increase of ABA in chloroplasts and nuclei may serve a function other than affecting stomatal movement.

Xylem Sap pH Increase: A Drought Signal Received at the Apoplastic Face of the Guard Cell That Involves the Suppression of Saturable Abscisic Acid Uptake by the Epidermal Symplast.

Drought increased the pH of Commelina communis xylem sap from 6.1 to 6.7. Conductances of transpiring leaves were 50% lower in pH 7.0 than in pH 6.0 buffers, but bulk leaf abscisic acid (ABA) concentration and shoot water status were unaffected by pH. Stomatal apertures of isolated abaxial epidermis incubated on simple buffers increased with external pH, so in vivo this must be overridden by alternative pH effects. Reductions in leaf transpiration rate at pH 7.0 were dependent on the presence of 10-8 mol dm-3 ABA in the xylem stream. We inferred that at pH 7.0 leaf apoplastic ABA concentrations increased: pH did not affect distributions of ABA among leaf tissues, but isolated epidermis and mesophyll tissue took up more 3H-ABA from pH 6.0 than from pH 7.0 buffers. The apoplastic ABA increase at pH 7.0 may result from reduced symplastic sequestration. A portion of 3H-ABA uptake by the epidermis was saturable at pH 6.0 but not at pH 7.0. An ABA uptake carrier may contribute to ABA sequestration by the leaf symplast of well-watered plants, and its inactivity at pH 7.0 may favor apoplastic ABA accumulation in draughted plants. Effects of external pH on stomatal apertures in the isolated epidermis indicate that published data supporting a role for internal guard cell ABA receptors should be reassessed.

Stomatal Closure in Flooded Tomato Plants Involves Abscisic Acid and a Chemically Unidentified Anti-Transpirant in Xylem Sap.

We address the question of how soil flooding closes stomata of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants within a few hours in the absence of leaf water deficits. Three hypotheses to explain this were tested, namely that (a) flooding increases abscisic acid (ABA) export in xylem sap from roots, (b) flooding increases ABA synthesis and export from older to younger leaves, and (c) flooding promotes accumulation of ABA within foliage because of reduced export. Hypothesis a was rejected because delivery of ABA from flooded roots in xylem sap decreased. Hypothesis b was rejected because older leaves neither supplied younger leaves with ABA nor influenced their stomata. Limited support was obtained for hypothesis c. Heat girdling of petioles inhibited phloem export and mimicked flooding by decreasing export of [14C]sucrose, increasing bulk ABA, and closing stomata without leaf water deficits. However, in flooded plants bulk leaf ABA did not increase until after stomata began to close. Later, ABA declined, even though stomata remained closed. Commelina communis L. epidermal strip bioassays showed that xylem sap from roots of flooded tomato plants contained an unknown factor that promoted stomatal closure, but it was not ABA. This may be a root-sourced positive message that closes stomata in flooded tomato plants.

Carbon Isotope Discrimination, Gas Exchange, and Yield of Spring Wheat Selected for Abscisic Acid Content

Because of its role in controlling stomatal closure, abscisic acid (ABA) may be a useful selection criterion for improving drought resistance of wheat (Triticum aestivum L.). Spring wheat lines previously selected for low and high dehydration-induced ABA accumulation in laboratory tests were studied in the field to determine if ABA accumulation potential is related to leaf water-use efficiency (WUE), carbon isotope discrimination (Δ), and grain yield. Plants were grown in irrigated or drought-stressed regimes under a rain shelter in 1987 and 1988. In the field, there was a trend for class differences in bulk-leaf ABA content to be the reverse of those obtained in the laboratory drought test. The class originally disignated low-ABA (had in general a slightly higher ABA content in the field) and tended to have lower stomatal conductance (gs), higher WUE, and a much greater biomass than the original high-ABA class. The original labels were retained, for consistency in reporting. The ABA classes differed significantly in yield components and harvest index. In one experiment with only two low- and two high-ABA selections, grain yields did not differ overall, but in another experiment with 20 low- and 20 high-ABA selections, grain yield of the low-ABA class was significantly higher. Biomass decreased more under drought in the low-ABA class than the high. Although drought resulted in lower ∆, indicating higher WUE, the average difference in ∆ between ABA classes was not significant in either the irrigated or drough-stressed regimes. However, slopes of regressions of grain yield on ∆, which were positive, differed significantly (nearly two-fold) between high- and low-ABA classes, with low-ABA selections having a steeper regression slope under drought conditions. The possible role of ABA in determining the results in discussed.

Leaf Gas Exchange and Water Relations of Lupins and Wheat. III. Abscisic Acid and Drought-Induced Stomatal Closure

Changes in the content of endogenous abscisic acid (ABA) were followed in glasshouse experiments during stomatal closure induced by drought in leaves of lupin (Lupinus cosentinii Guss. cv. Eregulla) and wheat (Triticum aestivum L. cvv. Gamenya and Warigal), species which differ in stomatal sensitivity to changes in leaf water potential. Increases in bulk leaf ABA concentration were closely correlated with decreases in leaf conductance in both species. In lupin, substantial increases in ABA and decreases in conductance occurred over a very narrow range of leaf water potential. ABA concentrations in wheat leaves were highly negatively correlated with bulk leaf turgor, but there was no significant relationship between ABA and turgor in lupin. However, ABA accumulated progressively in the leaves of both species as soil water content decreased. Stomatal closure in lupin could be induced by supplying exogenous ABA to detached leaves via the transpiration stream at concentrations of 10-4 to 10-2 mol m-3 of (+)-ABA. Abaxial stomata closed more readily than those on the adaxial surface in response to both drought and applied ABA. Stomatal response to ABA was not affected by the presence of the cytokinin zeatin, and zeatin by itself had no effect on conductance. When treatments designed to reduce endogenous cytokinin concentrations were imposed (prolonged leaf detachment or prior drought), stomatal response to low concentrations of ABA was enhanced. However, such treatments did not significantly change the stomatal response to high ABA concentrations, nor affect the stomatal conductance of leaves supplied with water alone. It is concluded that drought-induced stomatal closure could be mediated by ABA in both wheat and lupin, despite the initially small change in leaf water status in the latter species.

Water-stress-induced changes in the abscisic acid content of guard cells and other cells of Vicia faba L. leaves as determined by enzyme-amplified immunoassay

A highly sensitive, solid-phase, enzyme-amplified immunoassay for the plant growth regulator (+)-abscisic acid (ABA) was developed. The assay sensitivity (0.2-10 fmol) was sufficient for analyzing free ABA in homogeneous tissue samples dissected from Vicia faba L. leaves. Eight hours after detached leaves had been desiccated to 10% loss of fresh weight, the bulk leaf ABA content increased from </=0.2 to 6.2 ng.(mg dry weight)(-1). Epidermal tissue, spongy parenchyma cells, and palisade parenchyma cells from this water-stressed leaf had the following ABA contents, respectively: 4.8, 9.4, and 9.0 ng.(mg dry weight)(-1). Guard cells, which respond to exogenous ABA by losing solutes and volume, were also assayed. When they were dissected from control (fully hydrated) leaves, their ABA content was approximately 0.7 fg.(cell pair)(-1) [[unk]0.2 ng.(mg dry weight)(-1)]. In contrast, the ABA content of guard cells of water-stressed leaves was approximately 17.7 fg.(cell pair)(-1). These results indicate that ABA accumulation in a highly stressed V. faba leaflet is generalized; guard cells contain only 0.15% of bulk leaf ABA. The time course for loss of ABA from guard cells of a floating epidermal peel was studied. There was little loss within 30 min, but after 4 hr, the ABA content was only 17% of the original value. These results indicate that the bulk of guard cell ABA is not readily diffusible (i.e., probably not apoplastic). The results also indicate that common laboratory procedures results in lowered guard cell ABA content.

Control of Stomatal Behaviour by Abscisic Acid which Apparently Originates in the Roots

Zhang, J., Schurr, U. and Davies, W. J. 1987. Control of stomatal behaviour by abscisic acid which apparently originates in the roots.—J. exp. Bot. 38: 1174-1181. Two experiments indicate that abscisic acid (ABA) may influence stomatal behaviour of Commelina communis L. Stomatal conductance could not be correlated with bulk leaf ABA content but when the abaxial epidermis was assayed for ABA, small increases in ABA content correlated well with limitations of leaf conductance. Restricted conductance of the abaxial surface of leaves was associated with an increase of approximately 40 amole ABA per stomatal complex. This agrees with previously published figures. When roots of individual plants were split between two containers, drying the soil around one part of the root system restricted leaf conductance, even though leaf water relations were not affected. Increased ABA content of the epidermis coincided with increased ABA content of the roots in drying soil. Other roots of the same plant in moist soil did not show increased ABA content. These results suggest that in drying soil, ABA can move from the roots to the epidermis and restrict stomatal aperture even when leaf water potentials and turgors remain constant. The importance of this mechanism in providing a sensitive foliar response to decreasing soil moisture is discussed. Key words—Soil drying, ABA, roots, stomata, water relations. Correspondence to: Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LAI 4YQ, U.K.

Abscisic Acid and Water Relations of Rice (Oryza sativa L.): Sequential Responses to Water Stress in the Leaf

Water stress was imposed by withholding water at an early vegetative stage from plants of two rice cultivars (IR20 and 63–83) grown in pots. As stress intensified the following sequence of responses of the leaves was observed: (i) rise in abscisic acid (ABA) content, (ii) closure of stomata, (iii) initiation of leaf rolling. In both cultivars, turgor (ψp) declined linearly with total water potential (ψ) of the leaf. Bulk leaf ABA content increased linearly as ψp declined, and attained twice the control (unstressed) level following a reduction in ψp of about 0.12 MPa. Stomatal conductance exhibited a sigmoidal relationship to ψp, declining abruptly when a particular ‘critical’ ψp was reached (threshold response). The critical potentials varied considerably between experiments, but were closely correlated with control potentials and with the potentials at which ABA concentration doubled relative to controls. Leaf rolling was initiated at ψs near to zero ψp. Increases in the ratio of adaxial to abaxial conductance were associated with rolling. Variations in the above responses could be accounted for by variations in the rate of stress development, which in terms of ψ reduction ranged from 0.38 to 0.86 MPa day−1. Fast drying rates resulted in: (a) reduced osmotic adjustment, (b) increased amounts of ABA in the leaf at a given level of ψ or ψp, (c) an increase in the ABA concentration present at 50 per cent stomatal closure, and (d) initiation of leaf rolling at a higher ψ.

Changes in abscisic acid content during stomatal closure in pearl millet ( Pennisetum americanum (L.) Leeke)

The hypothesis that abscisic acid (ABA) mediates stomatal response to water stress was examined in pearl millet ( Pennisetum americanum (L.) Leeke) under conditions favouring rapid water loss. Following severing of the shoot from the root system and its exposure in situ in light at low atmospheric humidity, there was a rapid reduction in leaf water potential and, following an initial transient, ‘hydropassive’ opening, a rapid reduction in stomatal aperture. While closure of stomates was virtually complete within 20 min from shoot excision, an increase in bulk leaf ABA content was not detected until 25–30 min. Thereafter, ABA content increased linearly with time at a mean rate of 3.5 ng g −1 initial fresh wt./min. The patterns, timing and extent, both of stomatal closure and ABA increase, were identical for two genotypes, BJ 104 and Serere 39. Although such results cast doubt on the involvement of ABA in the rapid response of stomata to water stress it is concluded that an internal redistribution of the hormone could easily initiate closure.

Water Relations in Apple Seedlings: Changes in Water Potential Components, Abscisic Acid Levels and Stomatal Conductances under Irrigated and Non-irrigated Conditions1

Abstract Stomatal conductance, abscisic acid levels, and water potential components (water, osmotic, and rurgor potentials) were measured in irrigated and non-irrigated open pollinated seedlings of ‘Northern Spy’ apples (Malus domestica Borkh.). Although non-irrigated seedlings typically displayed water potentials of 0.2 to 0.6 MPa lower than those of irrigated seedlings, turgor potentials remained comparable in both groups, Because diurnal osmotic adjustment was also greater for non-irrigated seedlings. Abscisic acid (ABA) levels in the leaf increased linearly in response to changes in leaf turgor, rather than water potential. Stomatal conductance was independent of bulk leaf ABA levels and was poorly correlated with leaf turgor potential above a critical value of 0.7 MPa.