Stomatal lock-open, a consequence of epidermal cell death, follows transient suppression of stomatal opening in barley attacked by Blumeria graminis

Blumeria graminis f.sp. hordei (Bgh) attack disrupted stomatal behaviour, and hence leaf water conductance (g(l)), in barley genotypes Pallas and Ris?-S (susceptible), P01 (with Mla1 conditioning a hypersensitive response; HR), and P22 and Ris?-R (with mlo5 conditioning papilla-based penetration resistance). Inoculation caused some stomatal closure well before the fungus attempted infection. Coinciding with epidermal cell penetration, stomatal opening in light was also impeded, although stomata of susceptible and mlo5 lines remained largely able to close in darkness. Following infection, in susceptible lines stomata closed in darkness but opening in light was persistently impeded. In Ris?-R, stomata recovered nearly complete function by approximately 30 h after inoculation, i.e. after penetration resistance was accomplished. In P01, stomata became locked open and unable to close in darkness shortly after epidermal cells died due to HR. In the P22 background, mlo5 penetration resistance was often followed by consequential death of attacked cells, and here too stomata became locked open, but not until approximately 24 h after pathogen attack had ceased. The influence of epidermal cell death was localized, and only affected stomata within one or two cells distance. These stomata were unable to close not only in darkness but also after application of abscisic acid and in wilted leaves suffering drought. Thus, resistance to Bgh based on HR or associated with cell death may have previously unsuspected negative consequences for the physiological health of apparently 'disease-free' plants. The results are discussed in relation to the control of stomatal aperture in barley by epidermal cells.     

SNP markers linked to leaf rust and grain mold resistance in sorghum

Grain mold and rust are diseases that can significantly reduce sorghum grain yield. Breeding for resistance to these diseases is hindered by inefficient disease screening. A viable option to greatly improve breeding efficiency is to identify molecular markers or genes linked to the host resistance. In this study, we applied 14,739 single nucleotide polymorphism markers to the sorghum mini core of 242 accessions that had been evaluated for rust resistance in both greenhouse and field and for grain mold in the field for 2 years. Through association mapping we have identified two loci linked to grain mold resistance and five loci linked to rust resistance. Among the two loci linked to grain mold resistance, one contained a homolog of the maize nonhost resistance gene Rxo1. Two of rust-linked loci each contained the rust resistance gene homologous to the maize rust resistance gene Rp1-D which is the B locus (the A locus containing Pu was not linked in this study) and to the wheat rust resistance gene Lr1. The remaining loci contained genes important in other steps of the defense response, such as cyclophilins that mediate resistance response preceding hypersensitive response (HR) and Hin1 directly involved in producing HR. The results from this study will facilitate marker-assisted selection of host resistance to grain mold and rust in sorghum.     

Soybean adaptation to water stress at selected stages of growth

Soybean (Glycine max [L.] Merr. cv Braxton) plants were grown in sandy soil with only natural rainfall (N) or with supplemental irrigation (I). Water-stressed plants grew more extensive root systems, whereas irrigated plants developed larger shoots and smaller root systems. Maximum stomatal apertures were observed at the beginning of each photoperiod. Partial stomatal closure occurred each afternoon, but stomata of I plants remained open longer than those of N plants. Significant reductions in net carbon fixation rate generally accompanied decreases in stomatal aperture, which coincided with periods of high temperature, low relative humidity, maximum solar radiation, and water stress. Leaf water potential decreased from morning to afternoon, with a greater decrease observed for N plants. Midafternoon stomatal closure did not occur in N plants with very large root systems following a heavy rain which saturated the soil profile. With smaller root systems and greater evaporative demand from larger shoots, the I plants continued to show midafternoon stress following the heavy rain. The large root systems of the N plants absorbed sufficient water to meet shoot evaporative demand for several days following the rain. Root soil system resistance apparently contributed to the afternoon water stress in the I plants.     

Stomatal responses to changes in temperature at increasing water stress

Summary The response of stomata to a gradual increase in temperature at increasing plant water stress was studied in a hot desert habitat (Negev, Israel) in the field, but under controlled temperature and humidity conditions. Four native species (Zygophyllum dumosum, Artemisia herba-alba, Hammada scoparia, Reaumuria negevensis) and one cultivated plant (Prunus armeniaca) were used in these studies. The stomatal response to temperature was compared with the response in well-irrigated plants of the same species.At low water stress, the diffusion resistance for water vapour decreased in response to a gradual increase in temperature. Transpiration increased accordingly. This response was reversible. All species responded in the same way. The opening of stomata with increasing temperature was apparently independent of the stomatal response regulated by atmospheric humidity. At high plant water stress, the stomatal response was reversed, i.e., the stomata closed when temperature was gradually increased. This stomatal closure was also independent of the closure regulated by atmospheric humidity. The plant water potential at which the stomatal response to temperature was reversed, differed among the species investigated.     

Stomatal Diffusion Resistance of Snap Beans. II. Effect of Light

The effect of light on the stomatal resistance of abaxial and adaxial leaf surfaces of snap beans (Phaseolus vulgaris L.) was studied in the growth chamber and in the field. The adaxial stomata required more light to open than the abaxial stomata; the abaxial stomatal apertures were still about 50% open at 1% full sunlight and light-induced closure was never observed under daytime field conditions. A given value of abaxial stomatal resistance was obtained at a given illumination of the abaxial guard cells whether illumination was adaxial or abaxial.     

An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.     

Bacterial infection systemically suppresses stomatal density

Many plant pathogens gain entry to their host via stomata. On sensing attack, plants close these pores to restrict pathogen entry. Here, we show that plants exhibit a second longer term stomatal response to pathogens. Following infection, the subsequent development of leaves is altered via a systemic signal. This reduces the density of stomata formed, thus providing fewer entry points for pathogens on new leaves. Arabidopsis thaliana leaves produced after infection by a bacterial pathogen that infects through the stomata (Pseudomonas syringae) developed larger epidermal pavement cells and stomata and consequently had up to 20% reductions in stomatal density. The bacterial peptide flg22 or the phytohormone salicylic acid induced similar systemic reductions in stomatal density suggesting that they might mediate this effect. In addition, flagellin receptors, salicylic acid accumulation, and the lipid transfer protein AZI1 were all required for this developmental response. Furthermore, manipulation of stomatal density affected the level of bacterial colonization, and plants with reduced stomatal density showed slower disease progression. We propose that following infection, development of new leaves is altered by a signalling pathway with some commonalities to systemic acquired resistance. This acts to reduce the potential for future infection by providing fewer stomatal openings.     

Disease resistance to Pectobacterium carotovorum is negatively modulated by the Arabidopsis Lectin Receptor Kinase LecRK-V.5

Plant stomata function in disease resistance by restricting bacteria entry inside leaves. During plant-bacteria interactions, stomatal closure is initiated by the recognition of Microbe-Associated Molecular Patterns (MAMPs). Recently, we have shown that the Lectin Receptor Kinase V.5 (LecRK-V.5) negatively regulates bacterium- and MAMP-induced stomatal closure upstream of Reactive Oxygen Species (ROS) production mediated by abscisic acid signaling. Closed stomata in lecrk-V.5 mutants are correlated with constitutive high level of ROS in guard cells. Consequently, lecrk-V.5 mutants are more resistant to hemi-biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000). In this report, we further investigate the role of LecRK-V.5 in resistance against necrotrophic bacteria Pectobacterium carotovorum ssp. carotovorum (Pcc). Upon surface-inoculation lecrk-V.5 mutants exhibited enhanced resistance against Pcc whereas a wild-type level of resistance was observed using infiltration-inoculation, an inoculation method that bypasses the epidermal barrier. Enhanced resistance of dip-inoculated lecrk-V.5 mutants against necrotrophic bacteria, that induce different defense responses than hemi-biotrophic bacteria, further suggests a possible role for LecRK-V.5 in stomatal immunity.     

Calcium Effects on Stomatal Movement in Commelina communis L. : Use of EGTA to Modulate Stomatal Response to Light, KCl and CO(2)

Stomatal movements depend on both ion influx and efflux; attainment of steady state apertures reflects modulation of either or both processes. The role of Ca²⁺ in those two processes was investigated in isolated epidermal strips of Commelina communis, using the Ca²⁺ chelator EGTA to reduce apoplastic [Ca²⁺]. The results suggest that a certain concentration of Ca²⁺ is an absolute requirement for salt efflux and stomatal closure. EGTA (2 millimolar) increased KCl-dependent stomatal opening in darkness and completely inhibited the dark-induced closure of initially open stomata. Closure was inhibited even in a KCl-free medium. Thus, maintenance of stomata in the open state does not necessarily depend on continued K⁺ influx but on the inhibition of salt efflux. Opening in the dark was stimulated by IAA in a concentration-dependent manner, up to 15.4 micrometer without reaching saturation, while the response to EGTA leveled off at 9.2 micrometer. IAA did not inhibit stomatal closure to the extent it stimulated opening. The response to IAA is thus consistent with a primary stimulation of opening, while EGTA can be considered a specific inhibitor of stomatal closing since it inhibits closure to a much larger degree than it stimulates opening. CO₂ causes concentration-dependent reduction in the steady state stomatal aperture. EGTA completely reversed CO₂-induced closing of open stomata but only partially prevented the inhibition of opening.     

The Arabidopsis Lectin Receptor Kinase LecRK-V.5 Represses Stomatal Immunity Induced by Pseudomonas syringae pv. tomato DC3000

Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5) negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP) treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS) and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO₂ uptake and photosynthesis.     

Stomatal Response to Light of Solanum pennellii, Lycopersicon esculentum, and a Graft-induced Chimera

To learn how species differences in stomatal behavior are regulated, the response of epidermal and leaf diffusive resistance to light was investigated in Lycopersicon esculentum Mill., Solanum pennellii Corr., and a periclinal chimera having an S. pennellii epidermis and an L. esculentum mesophyll that was produced from a graft of the two species. S. pennellii has about 23% fewer stomata per square millimeter than does L. esculentum, and the two species have contrasting stomatal sensitivities to light. The abaxial stomata of L. esculentum open in dimmer light and to a greater extent than the adaxial stomata. The abaxial and adaxial stomata of S. pennellii respond similarly to light incident on the adaxial epidermis and are less open at all quantum flux densities than comparable stomata of L. esculentum. The patterns of response to light of the abaxial and adaxial stomata of the chimera were practically identical to those of L. esculentum, and quite unlike those of S. pennellii. Thus, the pattern of stomatal light response in the chimera was regulated by the L. esculentum mesophyll. The reduction in stomatal frequency of the chimera, which was regulated by the epidermis of S. pennellii, contributed to the 40% difference in leaf diffusive resistance between the plants in moderate light.     

Leaf Age as a Determinant in Stomatal Control of Water Loss from Cotton during Water Stress

The stomatal resistance of individual leaves of young cotton plants (Gossypium hirsutum L. var. Stoneville 213) was measured during a period of soil moisture stress under conditions of constant evaporative demand. When plants were subjected to increasing soil water stress, increases in stomatal resistance occurred first on the lower leaves and the stomata on the upper surfaces were the most sensitive to decreasing leaf-water potential. Stomatal closure proceeded from the oldest leaves to the youngest as the stress became more severe. This apparent effect of leaf age was not due to radiation differences during the stress period. Radiation adjustments on individual leaves during their development altered the stomatal closure potential for all leaves, but did not change the within-plant pattern. Our data indicate that no single value of leaf water potential will adequately represent a threshold for stomatal closure in cotton. Rather, the stomatal resistance of each leaf is uniquely related to its own water potential as modified by age and radiation regime during development. The effect of age on stress-induced stomatal closure was not associated with a loss of potassium from older leaves. Increases in both the free and bound forms of abscisic acid were observed in water-stressed plants, but the largest accumulations occurred in the youngest leaves. Thus, the pattern of abscisic acid accumulation in response to water stress did not parallel the pattern of stomatal closure induced by water stress.     

Stomatal morphology and physiology explain varied sensitivity to abscisic acid across vascular plant lineages

Abscisic acid (ABA) can induce rapid stomatal closure in seed plants, but the action of this hormone on the stomata of fern and lycophyte species remains equivocal. Here, ABA-induced stomatal closure, signaling components, guard cell K⁺ and Ca²⁺ fluxes, vacuolar and actin cytoskeleton dynamics, and the permeability coefficient of guard cell protoplasts (P_f) were analyzed in species spanning the diversity of vascular land plants including 11 seed plants, 6 ferns, and 1 lycophyte. We found that all 11 seed plants exhibited ABA-induced stomatal closure, but the fern and lycophyte species did not. ABA-induced hydrogen peroxide elevation was observed in all species, but the signaling pathway downstream of nitric oxide production, including ion channel activation, was only observed in seed plants. In the angiosperm faba bean (Vicia faba), ABA application caused large vacuolar compartments to disaggregate, actin filaments to disintegrate into short fragments and P_f to increase. None of these changes was observed in the guard cells of the fern Matteuccia struthiopteris and lycophyte Selaginella moellendorffii treated with ABA, but a hypertonic osmotic solution did induce stomatal closure in fern and the lycophyte. Our results suggest that there is a major difference in the regulation of stomata between the fern and lycophyte plants and the seed plants. Importantly, these findings have uncovered the physiological and biophysical mechanisms that may have been responsible for the evolution of a stomatal response to ABA in the earliest seed plants.

Physiological and biophysical evidence for insensitivity of stomata to abscisic acid in ferns and lycophytes supports stomatal responsiveness to abscisic acid evolved after the divergence of ferns.     

TaAbc1, a Member of Abc1-Like Family Involved in Hypersensitive Response against the Stripe Rust Fungal Pathogen in Wheat

To search for genes involved in wheat (Triticum aestivum L.) defense response to the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned a new wheat gene similar to the genes in the Abc1-like gene family. The new gene, designated as TaAbc1, encodes a 717-amino acid, 80.35 kD protein. The TaAbc1 protein contains two conserved domains shared by Abc1-like proteins, two trans-membrane domains at the C-terminal, and a 36-amino acid chloroplast targeting presequence at the N-terminal. Characterization of TaAbc1 expression revealed that gene expression was tissue-specific and could be up-regulated by biotic agents (e.g., stripe rust pathogen) and/or by an abiotic stress like wounding. High-fold induction was associated with the hypersensitive response (HR) triggered only by avirulent stripe rust pathotypes, suggesting that TaAbc1 is a rust-pathotype specific HR-mediator. Down-regulating TaAbc1 reduced HR but not the overall resistance level in Suwon11 to CYR23, suggesting TaAbc1 was involved in HR against stripe rust, but overall host resistance is not HR-dependent.     

The plant-specific actin binding protein SCAB1 stabilizes actin filaments and regulates stomatal movement in Arabidopsis

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H(2)O(2)-, and CaCl(2)-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.     

Phospholipase Dδ is involved in nitric oxide-induced stomatal closure

Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H₂O₂ and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H₂O₂ levels as the wild type in response to ABA. However, ABA- or H₂O₂-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H₂O₂ in ABA-induced stomatal closure.     

Water use strategy affects avoidance of ozone stress by stomatal closure in Mediterranean trees—A modelling analysis

Both ozone (O₃) and drought can limit carbon fixation by forest trees. To cope with drought stress, plants have isohydric or anisohydric water use strategies. Ozone enters plant tissues through stomata. Therefore, stomatal closure can be interpreted as avoidance to O₃ stress. Here, we applied an optimization model of stomata involving water, CO₂, and O₃ flux to test whether isohydric and anisohydric strategies may affect avoidance of O₃ stress by stomatal closure in four Mediterranean tree species during drought. The data suggest that stomatal closure represents a response to avoid damage to the photosynthetic mechanisms under elevated O₃ depending on plant water use strategy. Under high-O₃ and well-watered conditions, isohydric species limited O₃ fluxes by stomatal closure, whereas anisohydric species activated a tolerance response and did not actively close stomata. Under both O₃ and drought stress, however, anisohydric species enhanced the capacity of avoidance by closing stomata to cope with the severe oxidative stress. In the late growing season, regardless of the water use strategy, the efficiency of O₃ stress avoidance decreased with leaf ageing. As a result, carbon assimilation rate was decreased by O₃ while stomata did not close enough to limit transpirational water losses.     

Stomatal response of populus clones to light intensity and vapor pressure deficit

Responses of stomata of clones of Populus candicans Ait. × P. berolinensis Dipp. and Populus deltoides Bartr. × P. caudina (Ten.) Bugala to two levels of light intensity and vapor pressure deficit were studied in controlled environments. Significant stomatal responses to light and vapor pressure deficit were observed. Interactive effects of low light intensity and high vapor pressure deficit elicited greater stomatal closure than was obtained under low light or high vapor pressure deficit alone, indicating adaptation for increased water use efficiency under conditions unfavorable for photosynthesis relative to transpiration. Adaxial stomata of both clones were more sensitive than abaxial stomata to changing vapor pressure deficit and light intensity. Stomatal response to vapor pressure deficit appeared to be independent of bulk leaf water status. Stomata of P. candicans × P. berolinensis were more sensitive than stomata of P. deltoides × P. caudina to a change in vapor pressure deficit and less sensitive to a change in light intensity. The sensitivity of stomata of P. candicans × P. berolinensis to vapor pressure deficit may be related to drought resistance in its parentage (P. berolinensis).     

Ineffectiveness of Abscisic Acid in Stomatal Closure of Yellow Lupin, Lupinus luteus var. Weiko III

Stomata of yellow lupin leaves are remarkably insensitive toabscisic acid (ABA). Stomatal resistance was monitored usingboth a viscous now porometer and a diffusion porometer. Resultswere confirmed with scanning electron microscopy. When exogenousABA solutions were supplied via petioles, 10^–6 M solutionshad no effect on stomatal resistance. Upper (adaxial) stomatawere not affected by 10^–5 M ABA but lower stomata showed3-fold more resistance after 2 h. Stomata of both surfaces closedafter 30 min in 10^–4 M ABA. Isolated epidermal peels of lupin leaves were floated on ABAsolutions yet upper surface peels showed no stomatal closingin 10^–4 M ABA, while lower surface stomata closed to abarely significant extent. Stomata of intact leaves were not very sensitive to darkness,showing at most a doubling in resistance after 6 h darkness.Complete stomatal closure, however, was readily produced bywilting leaves. Hence, lupin stomata are physically capableof closing. Endogenous ABA levels of water-stressed leaves increased approximately10-fold, which corresponds to concentrations below 10 µMABA. It is concluded that ABA is unlikely to play a role incontrolling short-term stomatal response of lupins.     

Water Stress Reduces Ozone Injury via a Stomatal Mechanism

Various studies have shown that water-stressed plants are more tolerant of ozone exposures than are unstressed plants. Two probable explanations for this tolerance are (a) stomatal closure which reduces ozone uptake and (b) biochemical or anatomical changes within the leaves. Phaseolus vulgaris cv Pinto bean plants were established and transferred to membrane systems which controlled the osmotic potential around the roots at −35 or −80 kilopascals for 5 days prior to ozone treatment (0 or 1.0 microliters per liter for 2 hours). Both water-stressed and unstressed plants were sprayed with various concentrations of abscisic acid to close the stomata or with fusicoccin to induce stomata opening. The abaxial stomatal resistances of primary and trifoliate leaves were measured just prior to ozone exposure. Plant response to ozone was determined by stress ethylene production and chlorophyll loss. Both water stress and abscisic acid induced stomatal closure and reduced ozone injury. In water-stressed plants, fusicoccin induced stomatal opening and those plants were as sensitive to ozone as were the non-water-stressed plants. These data suggest that water stress protects plants from ozone injury mainly through its influence on stomatal aperture rather than through biochemical or anatomical changes.