Epidermal Strips Research Articles

Putative involvement of cytosolic Ca 2+ and GTP-binding proteins in cyclic-GMP-mediated induction of stomatal opening by auxin in Commelina communis L.

To investigate whether cyclic GMP (cGMP) would mediate, in an intracellular Ca2+ -dependent manner, coupling of auxin to stomatal opening, the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) were compared in epidermal strips of Commelina communis. In this comparison were studied possible effects of intracellular Ca2+ modulators, GTP-binding protein (G-protein) modulators and selective inhibitors of enzymatic reactions which use or generate cGMP. The stomatal response to IBA was almost similarly reversed by the Ca2+ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), the intracellular Ca2+-release inhibitors ruthenium red and procaine, the inactive cGMP analog Rp-8-bromoguanosine 3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPS), the inhibitor of cGMP-producing guanylyl cyclase LY 83583, the G-protein inhibitor mas17 and the G-protein antagonist pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2. Comparison with stomatal opening in response to 8-Br-cGMP, which was almost completely suppressed by either BAPTA, ruthenium red, procaine or Rp-8-Br-cGMPS, strongly suggests that cGMP acts downstream of G-protein activation as a second messenger for IBA signal transduction and that the cGMP pathway likely depends on cytosolic Ca2+signaling.

Blue light‐induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases

The phytohormone abscisic acid (ABA) inhibits blue light‐induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA‐insensitive (abi) mutants of Arabidopsis thaliana. Apoplastic acidification was monitored with a flat tipped pH‐electrode placed on epidermal strips, in which only guard cells were viable. Blue light‐induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane‐bound H+‐ATPases. In wild type epidermal strips, ABA reduced blue light‐induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca2+ concentration in guard cells, since factors that increase the Ca2+ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2. The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K‐252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2. The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.

Phototropism of Conidial Germ Tubes of Botrytis cinerea and Its Implication in Plant Infection Processes.

The germ tubes of Botrytis cinerea showed negative phototropism to near ultraviolet (NUV) and blue (300 to 520 nm) light followed by far-red (700 to 810 nm), whereas red light (600 to 700 nm) induced positive phototropism significantly. Minimum germ tube growth occurred during exposure to negative phototropism-inducing wavelengths, whereas it was maximum under positive phototropism-inducing wavelengths. NUV radiation and blue light that induced negative phototropism of B. cinerea promoted infection-hypha formation on both onion scale and broad bean (Vicia faba) leaf epidermal strips, whereas positive phototropism-inducing red light suppressed it, resulting in a high proportion of germ tubes without infection hyphae. In broad bean leaf infection, the number of infection points and area of necrosis per drop of conidial suspension were higher under NUV radiation and blue light than that of a dark control or leaflets pretreated with NUV radiation and blue light. In contrast, lower numbers of infection points and very small necrotic lesions developed under red light. In the case of red-light-pretreated leaflets, the number of infection points developed were higher, but areas of necrosis did not increase significantly compared with leaflets kept under red light without pretreatment. These results show the importance of phototropism of conidium germ tubes in plant infection.

CO 2 sensing in stomata of abi1-1 and abi2-1 mutants of Arabidopsis thaliana

Stomatal responses to CO 2 were investigated in Arabidopsis thaliana wild-type and ABA insensitive mutants ( abi1-1 and abi2-1). Although stomata from abi1 and abi2 mutant plants failed to respond to ABA and were originally wide open, they still remained sensitive to illumination and atmospheric CO 2-suppression, showing at least 50 %. of wild-type response. Similar results were obtained studying either stomatal movements in epidermal strip bioassays or changes in leaf conductance in whole plant experiments. The previously reported CO 2 insensitivity of stomata from abi1 and abi2 mutant plants probably has to be ascribed to the experimental conditions used by the authors. Thus, in abi1 and abi2 mutant plants, total impairment in the response to ABA is accompanied by a limited inhibition in stomatal response to CO 2 and light. These results do not support a total convergence of ABA and CO 2 signalling pathways in guard cells.

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH.

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pHi) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pHi, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152-163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1.

Disturbances in stomatal behaviour caused by air pollutants

Many atmospheric pollutants, even when present at relatively low concentrations, may interfere with the control of stomatal aperture, and they thus have the potential to upset the water balance of the leaf or the whole plant. Although at high concentrations pollutants such as SO2 and O3 usually cause stomatal closure, at low concentrations stomatal conductance is often increased. As well as creating a risk of loss of control of water relations, this is likely to increase the dose of the pollutant entering the mesophyll. It is, however, difficult to generalize about the nature of the physiological disturbances caused by pollutants because of variation in the responses between plants. In some cases the effects may be peculiar to one, or just a few, species. Two mechanisms underlying the interference with stomatal control have recently been identified, one involving O3 and the other CO2. In Aster tripolium (sea aster) stomata in detached epidermal strips close as the external Na+ concentration is increased, and it has been proposed that this phenomenon is involved in the regulation of salt loading of shoot tissues. Ozone has been shown to have the capacity to interfere with Na+-induced stomatal closure, and the possibility that it therefore disrupts an aspect of salinity tolerance in this species is worthy of further research. Elevated CO2, on the other hand, has been found to interfere with the control of water relations of beech (Fagus sylvatica): for a given degree of drought, stomatal conductance and rates of soil water depletion were significantly higher in elevated CO2 than in ambient air. It is normally assumed that atmospheric CO2-enrichment will lead to increased plant productivity and improved water economy, while also providing some protection against other atmospheric pollutants through partial stomatal closure. However, the response of beech indicates that in some species there may also be detrimental effects of CO2 enrichment on plant-water relations.

Enlargement urethroplasty using retrospongious epidermal flap

Free or pedunculated skin flaps may be used to repair stenoses of the anterior urethra. The authors prefer using free preputial flaps in reconstructing the bulbar urethra which, having a spongy body, guarantees an adequate vascular and mechanical support for the transplanted flaps. The bulbar reconstruction technique uses an epidermal epithelial strip along the dorsal face of the urethra. The dorsal approach to the urethral channel prevents mechanical yielding of the transplanted area and consequent formation of symptomatic urethrocele. Dorsal enlargement urethroplasty has the advantage that invasion of both the area of skin removal and the urethral channel is reduced to a minimum.

Determination of Epidermal Transpiration in Four Cultivars of Nicotiana Tabacum L. Using Epidermal Strips in a Quasi-Steady State System

A quasi-steady state method is presented for quantifying epidermal transpiration of epidermal strips where simple relations between transmembrane fluxes and parameters of diffusibility of penetrating compounds hold. Contrary to most permeability studies, we did not use astomatous, enzymatically isolated, or dried cuticular membranes, because these procedures are largely responsible for the problems cited in the literature. Instead, we used freshly harvested stomatous epidermal strips, thus avoiding the sorption of lipids by the cuticular membranes during enzymatic isolation. Our approach allowed estimation of amounts and composition of intracuticular soluble lipids. Diffusion coefficients (D-values) were calculated with smaller associated standard deviations and an order of magnitude lower than previously reported; the fresh material sorption of the diffusing compound by the membrane and hydration of the cuticular pores was greatly reduced. In the present study the hold-up time (te) ranged from 66.2 ± 0.3 to 110.3 ± 0.9sec. Furthermore, 0.1 μm thick membranes were used, contrary to previous studies of water permeability that used cuticles more than 2 μm thick. Because a small but constant flow of penetrant could be detected during the first half of the steady flow to te, small holes probably did not influence the reported permeability. Permeability coefficients (Pd) in the order of 0.65 × 10−9 ms−1 were calculated. Pd values in the order of 5.68 × 10−3 ms−1 were calculated when incomplete stomatal closure occurred, while when areas of mass flow were detected, Pd values in the order of 1.26 × 10−2 ms−1 were calculated. The degree of contamination of the epidermal strips by cellular debris was quantified and expressed as the total chlorphyll content per exposed surface area of the epidermal strip, and an average of 8.7% contamination was observed compared to the total leaf chlorophyll content. Leakage from the system was calculated to be approximately 0.18 × 10−10 ms−1, which represents an average 2.7% experimental variability. These results are discussed in terms of the limitations associated with using composite membranes that are stomatous and have trichomes, for possible application in drought tolerance selection.

Evidence for the existence of a sulfonylurea-receptor-like protein in plants: modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium channel openers.

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.

Blue light‐promoted stomatal opening in abaxial epidermis of Commelina benghalensis is maximal at low calcium

The requirement for calcium in blue light‐promoted stomatal opening, in comparison with that in red light, was studied in epidermal strips of Commelina benghalensis L. Blue light promoted stomatal opening in the presence of a low level of calcium, whereas in red light opening was relatively tolerant to calcium. Stomatal opening under blue light was restricted by external calcium (above 5 μM) or abscisic acid. When present in the incubation medium, EGTA increased the extent of stomatal opening under blue light. Verapamil (a calcium‐channel blocker) and trifluoperazine (TFP, a calmodulin antagonist) reduced the stimulation of stomatal opening by blue light. Lanthanum, an external calcium‐channel antagonist, had no significant effect on stomatal opening under either blue or red light. These observations indicate that blue light‐promoted stomatal opening preferentially occurs at low levels of calcium, and modulation by calmodulin is strongly suggested. We conclude that a fine‐tuning of the calcium level within guard cells is essential during the transduction of the blue light signal.

The effect of elevated CO 2 concentrations on K + and anion channels of Vicia faba L. guard cells

The effects of elevated CO2 concentrations on stomatal movement, anion- and K+-channel activities were examined in guard cells from epidermal strips of Vicia faba. Membrane voltage was measured using intracellular, double-barrelled microelectrodes and ion-channel currents were recorded under voltage clamp during exposure to media equilibrated with ambient (350 μl · l−1), 1000 μl · l−1 and 10 000 μl · l−1 CO2 in 20% O2 and 80% N2. The addition of 1000 μl · l−1 CO2 to the bathing solution caused stomata to close with a halftime of approx. 40 min, and with 10 000 μl · l−1 CO2 closure occurred with a similar time course. Under voltage clamp, exposure to 1000 μl · l−1 and 10 000 μl · l−1 CO2 resulted in a rapid increase (mean, 1.5 ± 0.2-fold, n = 8; range 1.3- to 2.5-fold) in the magnitude of current carried by outward-rectifying K+ channels (IK,out). The effect of CO2 on IK,out was essentially complete within 30 s and was independent of clamp voltage, but was associated with 25–40% (mean, 30 ± 4%) decrease in the halftime for current activation. Exposure to CO2 also resulted in a four-fold increase in background current near the free-running membrane voltage, recorded as the instantaneous current at the start of depolarising and hyperpolarising voltage steps, and a decrease in the magnitude of current carried by inward-rectifying K+ channels (IK,in). The effect of CO2 on IK,in was generally slower than on IK,out; it was allied with a transient acceleration of its activation kinetics during the first 60–120 s of treatment; and it was associated with a negative shift in the voltage-sensitivity of gating over a period of 3–5 min. Measurements carried out to isolate the background currents attributable to anion channels (ICl), using tetraethylammonium chloride and CsCl, showed that CO2 also stimulated ICl and dramatically altered its relaxation kinetics. Within the timeframe of CO2 action at the membrane, no significant effect was observed on cytosolic pH, measured using the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyflourescein (BCECF) and ratio fluorescence microphotometry. These results are broadly consistent with the pattern of guard-cell response to abscisic acid, and indicate that guard cells control both anion and K+ channels to achieve net solute loss in CO2. By contrast with the effects of abscisic acid, however, the data indicate that CO2 action is not mediated through changes in cytosolic pH and thereby implicate new and, as yet, unidentified pathway(s) for channel regulation in the guard cells.

Differential gene transcript accumulation in barley leaf epidermis and mesophyll in response to attack byBlumeria graminisf.sp.hordei(syn.Erysiphe graminisf.sp.hordei)

A number of barley cDNAs isolated by differential screening represent gene transcripts which accumulate during fungal pathogen attack. The encoded proteins comprise species commonly encountered in plant–pathogen interactions, e.g. PR proteins, but also protein species not previously reported from plant-pathogen interactions: a novel flavonoidO-methyltransferase, a 14-3-3 protein, an endoplasmatic chaperone (GRP94), an oxalate oxidase, an oxalate oxidase-like protein, and some species with no known homologues. One of these latter sequences BH72-Q3, encoding a small proline-rich protein, is presented here. Transcripts of 15 gene families accumulated in the leaves following inoculation withBlumeria graminisf.sp.hordei(syn.Erysiphe graminisf.sp.hordei) in both compatible and incompatible interactions. During the first 24 h after inoculation no clear differences were observed among different interaction types. At later stages transcript accumulation reflected the different infection types, with strong late accumulation in compatible interactions, a somewhat earlier accumulation in an incompatible multi-cell HR interaction, and only weak accumulation in an incompatible single cell HR interaction. Experiments using isolated epidermal strips and the residual mesophyll material showed that the oxalate oxidase-like gene transcript accumulated exclusively in the epidermis, whereas the 14-3-3, GRP94, and BH6-12 transcripts accumulated in both epidermis and mesophyll. All other gene transcripts appeared to accumulate preferentially in the mesophyll cells, however, with increased epidermal accumulation late after inoculation. The exception is the oxalate oxidase transcript, which accumulated exclusively in the mesophyll. The response of the barley plant to attack by the mildew fungus thus appears to consist of a complex of interconnected reactions in the epidermis and the underlying mesophyll cells.

Both rubisco and phosphoenolpyruvate carboxylase are beneficial for stomatal function in epidermal strips of Commelina benghalensis

The relative importance of the Calvin cycle and β-carboxylation, through phosphoenolpyruvate carboxylase (PEPC), pathways was assessed in relation to stomatal opening in epidermal strips of Commelina benghalensis, using d,l-glyceraldehyde (an inhibitor of the Calvin cycle) and 3,3-dichloro-2-dihydroxyphosphinoyl-methyl-2-propenoate (DCDP, an inhibitor of PEPC) or metabolites related to the Calvin cycle or PEPC. Stomatal opening was markedly inhibited by both DCDP and d,l-glyceraldehyde. In the absence of these inhibitors, exogenous metabolites such as PEP or ribose-5-P (R-5-P) had little or no effect on stomatal opening. On the other hand, in the presence of DCDP or d,l-glyceraldehyde, metabolites related to not only PEPC (PEP or malate) but also the Calvin cycle (R-5-P or PGA) relieved the inhibition of stomatal opening. We suggest that both Calvin cycle and β-carboxylation activity are beneficial for stomatal opening, particularly when either of these pathways is restricted.

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh..

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K+ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (Em) was not sensitive to the external K+ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K(+)-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235-246). The activation and inactivation times and the activation potential, all depended on the reversal potential (Erev) of the s-ORC conductance. The s-ORC was blocked by Ba2+ (K1/2 = 0.3-1.3 mM) and verapamil (K1/2 = 15-20 microM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K+ concentration. The slow activation of the IRC (t1/2 approximately 0.5 s) and its negative activation potential (Vthreshold = -155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701-2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes.

Calcium-calmodulin signalling is involved in light-induced acidification by epidermal leaf cells of pea,Pisum sativumL.

Pathways of signal transduction of red and blue lightdependent acidification by leaf epidermal cells were studied using epidermal strips of the Argenteum mutant of Pisum sativum. In these preparations the contribution of guard cells to the acidification is minimal. The hydroxypyridine nifedipine, a Ca2 +-channel blocker, partly inhibited the response to both blue and red light, while the phenylalkylamine, verapamil, a Ca2 +-channel blocker that has been shown in plant cells also to block K+-channels, caused nearly complete inhibition. The Ca2 + -channel activator S(-)Bay K 8644 induced acidification when added in the dark and diminished the light-induced lowering of the extracellular pH. The Ca2 + -ionophores, ionomycin and A23187, also reduced the light response. Furthermore, the light-induced acidification was inhibited by the calmodulin antagonists W-7 and trifluoperazine, but not by W-5. These calmodulin inhibitors completely inhibited the red light-induced acidification, but inhibited the response to blue light by only 60-70%. In general, inhibition by compounds affecting Ca-calmodulin signalling was always stronger on the red light response than that on the blue light response (with the exception of verapamil that blocked both the red and blue light responses equally well). This differential effect on red and blue light-induced responses indicates a role for Ca2 + CaM signalling in both the red and blue light responses, while a second process, independent of Ca2+ is activated by blue light.

Stereoselectivity in the biosynthetic conversion of xanthoxin into abscisic acid

All stereoisomers of xanthoxin (XAN) and abscisic aldehyde (ABA-aldehyde) were prepared from (R) and (S)-4-hydroxy-β-cyclogeraniol via asymmetric epoxidation. Their stomatal closure activities were measured on epidermal strips of Commelina communis L. Natural (S)-ABA-aldehyde showed strong activity comparable to that of (S)-abscisic acid (ABA). Natural (1′S, 2′R, 4′S)XAN and (1′S, 2′R, 4′R)-epi-XAN also induced stomatal closure at high concentrations. On the other hand, unnatural (1′R)-enantiomers of XAN, epi-XAN, and ABA-aldehyde were not effective. To further examine the Stereoselectivity on the biosynthetic pathway to ABA, deuterium-labeled substrates were prepared and fed to Lycopersicon esculentum Mill, under non-stressed or water-stressed conditions. Substantial incorporations into ABA were observed in the cases of natural (1′S, 2′R, 4′S)-XAN, (1′S, 2′R, 4′R)-epi-XAN and both enantiomers of ABA-aldehyde, leading to the following conclusions. The negligible effect of unnatural (1′R)-enantiomers of XAN, epi-XAN and ABA-aldehyde can be explained by their own biological inactivity and/or their conversion to inactive (R)-ABA. Even in the isolated epidermal strips, putative aldehyde oxidase activity is apparently sufficient to convert ABA-aldehyde to ABA while the activity of XAN dehydrogenase seems very weak. The stereochemistry of the 1′, 2′-epoxide is very important for the XAN-dehydrogenase while this enzyme is less selective regarding the 4′-hydrdxyl group of XAN and converts both (1′S, 2′R, 4′S)-XAN and (1′S, 2′R, 4′R)-epi-XAN to (S)-ABA-aldehyde. Abscisic aldehyde oxidase can nonstereoselectively convert both (S) and (R)-ABA-aldehyde to biologically active (S) and inactive (R)-ABA, respectively.

A reassessment of the intervention of calmodulin in the regulation of stomatal movement

Commelina cammunis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by 45Ca2+‐overlays. CaM was found to be 3‐ to 7‐fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca2+‐dependent manner were detected by gold‐labelled CaM overlays. Using bioassays with epidermal strips, different CaM‐antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ≅ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ≅ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca2+‐CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca2+‐ or a Ca2+‐CaM‐dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.

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.

Syringopeptins, Pseudomonas syringae pv. syringae phytotoxins, resemble syringomycin in closing stomata

The recent finding that syringomycin (SR) and syringotoxin (ST)‐producing isolates of Pseudomonas syringae pv. syringae also synthesize syringopeptins (SPs), another class of phytotoxic lipodepsipeptides, prompted studies of the biological properties and comparisons of the activities of the two groups of metabolites. The present paper reports the effects of two forms of SP on stomatal movement in detached leaves of Xanthium strumarium and in epidermal strips of Vicia faba and shows that these phytotoxins, as does the previously investigated SR, promote stomatal closure. SPs are at least 10‐fold more efficient than SR in both tissues. In epidermal strips, the toxin‐induced closure was not reversed by fusicoccin, a fungal metabolite that reversed the closing effects of abscisic acid. As reported in previous papers, these amphiphilic toxins affect several functions of biological membranes. A marked decrease in the rate of photosynthesis recorded in X. strumarium leaves treated with SPs is consistent with effects on biological membranes.

How Do Stomata Read Abscisic Acid Signals?

When abscisic acid (ABA) was fed to isolated epidermis of Commelina communis L., stomata showed marked sensitivity to concentrations of ABA lower than those commonly found in the xylem sap of well-watered plants. Stomata were also sensitive to the flux of hormone molecules across the epidermal strip. Stomata in intact leaves of Phaseolus acutifolius were much less sensitive to ABA delivered through the petiole than were stomata in isolated epidermis, suggesting that mesophyll tissue and/or xylem must substantially reduce the dose or activity of ABA received by guard cells. Delivery of the hormone to the leaf was varied by changing transpiration flux and/or concentration. Varying delivery by up to 7-fold by changing transpiration rate had little effect on conductance. At a given delivery rate, variation in concentration by 1 order of magnitude significantly affected conductance at all but the highest concentration fed. The results are discussed in terms of the control of stomatal behavior in the field, where the delivery of ABA to the leaf will vary greatly as a function of both the concentration of hormone in the xylem and the transpiration rate of the plant.