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.     

Ethylene limits abscisic acid‐ or soil drying‐induced stomatal closure in aged wheat leaves

The mechanism of age‐induced decreased stomatal sensitivity to abscisic acid (ABA) and soil drying has been explored here. Older, fully expanded leaves partly lost their ability to close stomata in response to foliar ABA sprays, and soil drying which stimulated endogenous ABA production, while young fully expanded leaves closed their stomata more fully. However, ABA‐ or soil drying‐induced stomatal closure of older leaves was partly restored by pretreating plants with 1‐methylcyclopropene (1‐MCP), which can antagonize ethylene receptors, or by inoculating soil around the roots with the rhizobacterium Variovorax paradoxus 5C‐2, which contains 1‐aminocyclopropane‐1‐carboxylic acid (ACC)‐deaminase. ACC (the immediate biosynthetic precursor of ethylene) sprays revealed higher sensitivity of stomata to ethylene in older leaves than younger leaves, despite no differences in endogenous ACC concentrations or ethylene emission. Taken together, these results indicate that the relative insensitivity of stomatal closure to ABA and soil drying in older leaves is likely due to altered stomatal sensitivity to ethylene, rather than ethylene production. To our knowledge, this is the first study to mechanistically explain diminished stomatal responses to soil moisture deficit in older leaves, and the associated reduction in leaf water‐use efficiency.     

Involvement of root ABA and hydraulic conductivity in the control of water relations in wheat plants exposed to increased evaporative demand

We studied the possible involvement of ABA in the control of water relations under conditions of increased evaporative demand. Warming the air by 3°C increased stomatal conductance and raised transpiration rates of hydroponically grown Triticum durum plants while bringing about a temporary loss of relative water content (RWC) and immediate cessation of leaf extension. However, both RWC and extension growth recovered within 30 min although transpiration remained high. The restoration of leaf hydration and growth were enabled by increased root hydraulic conductivity after increasing the air temperature. The use of mercuric chloride (an inhibitor of water channels) to interfere with the rise on root hydraulic conductivity hindered the restoration of extension growth. Air warming increased ABA content in roots and decreased it in shoots. We propose this redistribution of ABA in favour of the roots which increased the root hydraulic conductivity sufficiently to permit rapid recovery of shoot hydration and leaf elongation rates without the involvement of stomatal closure. This proposal is based on known ability of ABA to increase hydraulic conductivity confirmed in these experiments by measuring the effect of exogenous ABA on osmotically driven flow of xylem sap from the roots. Accumulation of root ABA was mainly the outcome of increased export from the shoots. When phloem transport in air-warmed plants was inhibited by cooling the shoot base this prevented ABA enrichment of the roots and favoured an accumulation of ABA in the shoot. As a consequence, stomata closed.     

Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying

The hypothesis that ABA produced by roots in drying soil is responsible for stomatal closure was tested with grafted plants constructed from the ABA-deficient tomato mutants, sitiens and flacca and their near-isogenic wild-type parent. Three types of experiments were conducted. In the first type, reciprocal grafts were made between the wild type and sitiens or flacca. Stomatal conductance accorded with the genotype of the shoot, not the root. Stomates closed in all of the grafted plants in response to soil drying, regardless of the root genotype, i.e. regardless of the ability of the roots to produce ABA. In the second type of experiment, wild-type shoots were grafted onto a split-root system consisting of one wild-type root grafted to one mutant (flacca or sitiens) root. Water was withheld from one root system, while the other was watered well so that the shoots did not experience any decline in water potential or loss of turgor. Stomates closed to a similar extent when water was withheld from the mutant roots or the wild-type roots. In the third type of experiment, grafted plants with wild-type shoots and either wild-type or sitiens roots were established in pots that could be placed inside a pressure chamber, and the pressure increased as the soil dried so that the shoots remained fully turgid throughout. Stomates closed as the soil dried, regardless of whether the roots were wild type or sitiens. These experiments demonstrate that stomatal closure in response to soil drying can occur in the absence of leaf water deficit, and does not require ABA production by roots. A chemical signal from roots leading to a change in apoplastic ABA levels in leaves may be responsible for the stomatal closure.     

Can early wilting of old leaves account for much of the ABA accumulation in flooded pea plants?

When pea plants (Pisum sativum L.) were subjected to flooding,abscisic acid (ABA) content in shoots and roots increased upto 8-fold in the following days and stomatal conductance significantlydecreased. Although young leaves of flooded plants had a slightlyhigher water potential than those of the unflooded plants, oldleaves had lower water potential and lost turgor at the timewhen a substantial ABA increase was detected. In plants wherethe old leaves were clipped off, flooding did not cause anyABA increase during 7 d of the experimental period, except underconditions of higher transpiration demand, when the increasein ABA content was both delayed and small in scale (only I-fold).When intact plants were flooded and ABA was assayed separatelyin both old and young leaves, the ABA increase in old leavespreceded that in young leaves. Evidence here suggests that theflooding-induced ABA increase mainly results from the wiltingof old leaves. This suggests that young leaves may be protectedfrom wilting by ABA originating in old leaves under unfavourableenvironmental conditions. Key words: Waterlogging, soil flooding, ABA, leaf water relations, pea, Pisum sativum     

Stomatal responses to vapour pressure deficit are regulated by high speed gene expression in angiosperms

Plants dynamically regulate water use by the movement of stomata on the surface of leaves. Stomatal responses to changes in vapour pressure deficit (VPD) are the principal regulator of daytime transpiration and water use efficiency in land plants. In angiosperms, stomatal responses to VPD appear to be regulated by the phytohormone abscisic acid (ABA), yet the origin of this ABA is controversial. After a 20 min exposure of plants, from three diverse angiosperm species, to a doubling in VPD, stomata closed, foliar ABA levels increased and the expression of the gene encoding the key, rate‐limiting carotenoid cleavage enzyme (9‐cis‐epoxycarotenoid dioxygenase, NCED) in the ABA biosynthetic pathway was significantly up‐regulated. The NCED gene was the only gene in the ABA biosynthetic pathway to be up‐regulated over the short time scale corresponding to the response of stomata. The closure of stomata and rapid increase in foliar ABA levels could not be explained by the release of ABA from internal stores in the leaf or the hydrolysis of the conjugate ABA‐glucose ester. These results implicate an extremely rapid de novo biosynthesis of ABA, mediated by a single gene, as the means by which angiosperm stomata respond to natural changes in VPD.     

A hydraulic signal in root-to-shoot signalling of water shortage

Photosynthesis and biomass production of plants are controlled by the water status of the soil. Upon soil drying, plants can reduce water consumption by minimizing transpiration through stomata, the closable pores of the leaf. The phytohormone abscisic acid (ABA) mediates stomatal closure, and is the assigned signal for communicating water deficit from the root to the shoot. However, our study does not support ABA as the proposed long-distance signal. The shoot response to limited soil water supply is not affected by the capacity to generate ABA in the root; however, the response does require ABA biosynthesis and signalling in the shoot. Soil water stress elicits a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure. Attenuation of the hydraulic response in various plants prevented long-distance signalling of water stress, consistent with root-to-shoot communication by a hydraulic signal.     

Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling?

Exposing plants to low VPD reduces leaf capacity to maintain adequate water status thereafter. To find the impact of VPD on functioning of stomata, stomatal morphology and leaf anatomy, fava bean plants were grown at low (L, 0.23 kPa) or moderate (M, 1.17 kPa) VPDs and some plants that developed their leaves at moderate VPD were then transferred for 4 days to low VPD (M→L). Part of the M→L‐plants were sprayed with ABA (abscisic acid) during exposure to L. L‐plants showed bigger stomata, larger pore area, thinner leaves and less spongy cells compared with M‐plants. Stomatal morphology (except aperture) and leaf anatomy of the M→L‐plants were almost similar to the M‐plants, while their transpiration rate and stomatal conductance were identical to that of L‐plants. The stomatal response to ABA was lost in L‐plants, but also after 1‐day exposure of M‐plants to low VPD. The level of foliar ABA sharply decreased within 1‐day exposure to L, while the level of ABA‐GE (ABA‐glucose ester) was not affected. Spraying ABA during the exposure to L prevented loss of stomatal closing response thereafter. The effect of low VPD was largely depending on exposure time: the stomatal responsiveness to ABA was lost after 1‐day exposure to low VPD, while the responsiveness to desiccation was gradually lost during 4‐day exposure to low VPD. Leaf anatomical and stomatal morphological alterations due to low VPD were not the main cause of loss of stomatal closure response to closing stimuli.     

Fern and lycophyte guard cells do not respond to endogenous abscisic acid

Stomatal guard cells regulate plant photosynthesis and transpiration. Central to the control of seed plant stomatal movement is the phytohormone abscisic acid (ABA); however, differences in the sensitivity of guard cells to this ubiquitous chemical have been reported across land plant lineages. Using a phylogenetic approach to investigate guard cell control, we examined the diversity of stomatal responses to endogenous ABA and leaf water potential during water stress. We show that although all species respond similarly to leaf water deficit in terms of enhanced levels of ABA and closed stomata, the function of fern and lycophyte stomata diverged strongly from seed plant species upon rehydration. When instantaneously rehydrated from a water-stressed state, fern and lycophyte stomata rapidly reopened to predrought levels despite the high levels of endogenous ABA in the leaf. In seed plants under the same conditions, high levels of ABA in the leaf prevented rapid reopening of stomata. We conclude that endogenous ABA synthesized by ferns and lycophytes plays little role in the regulation of transpiration, with stomata passively responsive to leaf water potential. These results support a gradualistic model of stomatal control evolution, offering opportunities for molecular and guard cell biochemical studies to gain further insights into stomatal control.     

Stomatal behaviour and leaf water potential of chilled and water-stressed Solanum melongena, as influenced by growth history

Abstract Leaf diffusion resistance and leaf water potential of intact Solanum melongena plants were measured during a period of chilling at 6 °C. Two pretreatments, consisting of a period of water stress or a foliar spraying of abscisic acid (ABA), were imposed upon the plants prior to chilling. The control plants did not receive a pretreatment. In addition to intact plant studies, stomatal responses to water loss and exogenous abscisic acid were investigated using excised leaves, and the influence of the pretreatment observed. Chilled, control plants wilted slowly and maintained open stomata despite a decline in leaf water potential to –2.2 MPa after 2 d of chilling. In contrast plants that had been water stressed or had been sprayed with abscisic acid, prior to chilling, did not wilt and maintained a higher leaf water potential and a greater leaf diffusion resistance. In plants that had not received a pretreatment, abscisic acid caused stomatal closure at 35 °C, but at 6°C it did not influence stomatal aperture. The two pretreatments greatly increased stomatal sensitivity to both exogenous ABA and water stress, at both temperatures. Stomatal response to water loss from excised leaves was greatly reduced at 6°C. These results are discussed in relation to low temperature effects on stomata and the influence of preconditioning upon plant water relations.     

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.     

Stomatal responses to water stress and to abscisic Acid in phosphorus-deficient cotton plants

Cotton (Gossypium hirsutum L.) plants were grown in sand culture on nutrient solution containing adequate or growth-limiting levels of P. When water was withheld from the pots, stomata of the most recently expanded leaf closed at leaf water potentials of approximately −16 and −12 bars in the normal and P-deficient plants, respectively. Pressure-volume curves showed that the stomata of P-deficient plants closed when there was still significant turgor in the leaf mesophyll. Leaves of P-deficient plants accumulated more abscisic acid (ABA) in response to water stress, but the difference was evident only at low water potentials, after initiation of stomatal closure. In leaves excised from unstressed plants, P deficiency greatly increased stomatal response to ABA applied through the transpiration stream. Kinetin blocked most of this increase in apparent sensitivity to ABA. The effect of P nutrition on stomatal behavior may be related to alterations of the balance between ABA and cytokinins.     

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     

Dynamics of adaptation of stomatal behaviour to moderate or high relative air humidity in Tradescantia virginiana

Chlorophyll fluorescence imaging was used to measure stomatalclosure in response to desiccation of Tradescantia virginianaleaves grown under high (90%) and moderate (55%) relative humidities(RHs), or transferred between these humidities. Stomata in leavesgrown at high RH were less responsive to desiccation than thoseof leaves grown at moderate RH. Stomata of plants transferredfrom moderate RH conditions to high RH showed the same diminishedclosure in response to desiccation as did stomata that developedat high RH. This response was found both when the leaves werefully expanded and when still actively expanding during themoderate RH pre-treatment. Four days of exposure to high RHwas the minimal exposure time to induce the diminished closureresponse. When leaves were grown in high RH prior to a 10 dmoderate RH treatment, the reduced stomatal closure responseto desiccation was only reversed in leaves (regions) which wereactively expanding during moderate RH treatment. This indicatesthat with respect to stomatal responses to desiccation, highRH leaf regions have a limited capacity to adapt to moderateRH conditions. The decrease in responsiveness to desiccationof the stomata, induced by long-term exposure to high RH, wasnot due to osmotic adjustment in the leaves. Within 1 d aftertransferring moderate RH-grown plants to a high RH, the abscisicacid (ABA) concentration of their leaves decreased to the lowlevel of ABA found in high RH-grown leaves. The closure responsein leaves exposed to high RH for 5 d, however, could not befully restored by the application of ABA. Transferring plantsfrom high to moderate RH resulted in increased ABA levels within2 d without a recovery of the stomatal closing response. Itis discussed that the diminished stomatal closure in plantsexposed to high RH could be due to changes in the signallingpathway for ABA-related closure of stomata or to an increasedsequestration of ABA by mesophyll tissue or the symplast inthe epidermis, induced by a longer period (several days) ofa low ABA level. Key words: Abscisic acid, desiccation, PSII efficiency, relative water content, stomatal closure, vapour pressure deficit, water potential Received 8 October 2007; Revised 5 November 2007 Accepted 9 November 2007     

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.     

Effects of flooding and drought on stomatal activity, transpiration, photosynthesis, water potential and water channel activity in strawberry stolons and leaves

Transpiration, xylem water potential and water channel activity were studied in developing stolons and leaves of strawberry (Fragaria × ananassa Duch.) subjected to drought or flooding, together with morphological studies of their stomata and other surface structures. Stolons had 0.12 stomata mm^–2 and a transpiration rate of 0.6 mmol H₂O m^–2 s^–1, while the leaves had 300 stomata mm^–2 and a transpiration rate of 5.6 mmol H₂O m^–2 s^–1. Midday water potentials of stolons were always less negative than in leaves enabling nutrient ion and water transport via or to the strawberry stolons. Drought stress, but not flooding, decreased stolon and leaf water potential from –0.7 to –1 MPa and from –1 to –2 MPa, respectively, with a concomitant reduction in stomatal conductance from 75 to 30 mmol H₂O m^–2 s^–1. However, leaf water potentials remained unchanged after flooding. Similarly, membrane vesicles derived from stolons of flooded strawberry plants showed no change in water channel activity. In these stolons, turgor may be preserved by maintaining root pressure, an electrochemical and ion gradient and xylem differentiation, assuming water channels remain open. By contrast, water channel activity was reduced in stolons of drought stressed strawberry plants. In every case, the effect of flooding on water relations of strawberry stolons and leaves was less pronounced than that of drought which cannot be explained by increased ABA. Stomatal closure under drought could be attributed to increased delivery of ABA from roots to the leaves. However, stomata closed more rapidly in leaves of flooded strawberry despite ABA delivery from the roots in the xylem to the leaves being strongly depressed. This stomatal closure under flooding may be due to release of stress ethylene. In the relative absence of stomata from the stolons, cellular (apoplastic) water transport in strawberry stolons was primarily driven by water channel activity with a gradient from the tip of the stolon to the base, concomitant with xylem differentiation and decreased water transport potential from the stolon tip to its base. Reduced water potential in the stolons under drought are discussed with respect to reduced putative water channel activity.     

Stomatal Responses to Water Deficits and Abscisic Acid in Leaves of Sunflower Plants (Helianthus annuus L.) Grown under Different Conditions

The decrease in diffusive conductance of a leaf exposed to waterstress or to exogenous abscisic acid (ABA) was smaller in leavesof sunflower plants (Helianthus annuus L. cv. NK285) that hadbeen grown in a phytotron in humid air than in leaves of sunflowersgrown outdoors. Stomata of the phytotron-grown plants were slowerto close after detachment of a leaf than those of the outdoorplants. When stomata closed rapidly, as they did in detachedleaves and after treatment with ABA, the extent of closure wasvaried over the leaf's surface, in particular in the case ofphytotron-grown plants, and the extent of the heterogeneitywas greater in the phytotrongrown plants than in the outdoorplants. When stomata closed gradually, for example, under conditionsof limited moisture in the soil, closure occurred uniformlyover leaves of plants of both types. The smaller decrease indiffusive conductance of leaves from phytotron-grown plantsafter treatment with ABA resulted from the presence of patcheson the surface in which stomata remained open. The smaller decreaseof diffusive conductance in the phytotron-grown plants underconditions of limited moisture in the soil resulted from theuniformly lower responsiveness of stomata on a leaf to the decreasein water potential. When estimates are made of the intercellularconcentration of CO₂ (Ci) from gas-exchange measurements, heterogeneityin stomatal closure should be monitored when stomata close rapidly,in particular in plants grown in humid air, because heterogeneousstomatal closure can lead to overestimates of Ci. (Received April 18, 1994; Accepted May 25, 1995)     

Does transpiration control stomatal responses to water vapour pressure deficit?

Three types of observations were used to test the hypothesis that the response of stomatal conductance to a change in vapour pressure deficit is controlled by whole-leaf transpiration rate or by feedback from leaf water potential. Varying the leaf water potential of a measured leaf by controlling the transpiration rate of other leaves on the plant did not affect the response of stomatal conductance to vapour pressure deficit in Glycine max. In three species, stomatal sensitivity to vapour pressure deficit was eliminated when measurements were made at near-zero carbon dioxide concentrations, despite the much higher transpiration rates of leaves at low carbon dioxide. In Abutilon theophrasti, increasing vapour pressure deficit sometimes resulted in both decreased stomatal conductance and a lower transpiration rate even though the response of assimilation rate to the calculated substomatal carbon dioxide concentration indicated that there was no ‘patchy’ stomatal closure at high vapour pressure deficit in this case. These results are not consistent with stomatal closure at high vapour pressure deficit caused by increased whole-leaf transpiration rate or by lower leaf water potential. The lack of response of conductance to vapour pressure deficit in carbon dioxide-free air suggests that abscisic acid may mediate the response.     

Involvement of Abscisic Acid in Regulating Water Status in Phaseolus vulgaris L. during Chilling

During the first hours of chilling, bean (Phaseolus vulgaris L., cv Mondragone) seedlings suffer severe water stress and wilt without any significant increase in leaf abscisic acid (ABA) content (P. Vernieri, A. Pardossi, F. Tognoni [1991] Aust J Plant Physiol 18: 25-35). Plants regain turgor after 30 to 40 h. We hypothesized that inability to rapidly synthesize ABA at low temperatures contributes to chilling-induced water stress and that turgor recovery after 30 to 40 h is mediated by changes in endogenous ABA content. Entire bean seedlings were subjected to long-term (up to 6 d) chilling (3°C, 0.2-0.4 kPa vapor pressure deficit, 100 μmol·m⁻²·s⁻¹ photosynthetic photon flux density, continuous fluorescent light). During the first 24 h, stomata remained open, and plants rapidly wilted as leaf transpiration exceeded root water absorption. During this phase, ABA did not accumulate in leaves or in roots. After 24 h, ABA content increased in both tissues, leaf diffusion resistance increased, and plants rehydrated and regained turgor. No osmotic adjustment was associated with turgor recovery. Following turgor recovery, stomata remained closed, and ABA levels in both roots and leaves were elevated compared with controls. The application of ABA (0.1 mm) to the root system of the plants throughout exposure to 3°C prevented the chilling-induced water stress. Excised leaves fed 0.1 mm ABA via the transpiration stream had greater leaf diffusion resistance at 20 and 3°C compared with non-ABA fed controls, but the amount of ABA needed to elicit a given degree of stomatal closure was higher at 3°C compared with 20°C. These findings suggest that endogenous ABA may play a role in ameliorating plant water status during chilling.     

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.