Accumulation In Guard Cells Research Articles

The apparent feed‐forward response to vapour pressure deficit of stomata in droughted, field‐grown Eucalyptus globulus Labill

ABSTRACTThis study tested a multiplicative model of stomatal response to environment for drought‐affected trees of Eucalyptus globulus Labill. growing in southern Australia. The model incorporates a feed‐forward response to vapour pressure deficit of ambient air (δea) and performed well if evaluated using reduced major axis regression and log‐transformed data. There was strong evidence from gas‐exchange data, leaf water potentials and sapflow measurements of the feed‐forward response by stomata to leaf‐to‐air vapour pressure deficit (δel). The response of stomata to δel was irreversible. Stomatal conductance and the rate of net photosynthesis were highly correlated and declined, together with the rate of transpiration, throughout the afternoon as δea increased despite increasing leaf water potentials. The concentration of CO2 inside leaves (ci) increased as stomatal conductance declined indicating increasing non‐stomatal limitations to photosynthesis. The stomatal response to δel of E. globulus in the field is best described as an ‘apparent feed‐forward response’ that probably results from both slowly reversible depression of net photosynthesis and abscisic acid accumulation in guard cells. We suggest that the stomatal response to ci may strengthen the link between photosynthetic capacity and stomatal conductance during leaf drying as a result of either drought or large δ el.

Hydrogen peroxide is a common signal for darkness- and ABA-induced stomatal closure in Pisum sativum.

The requirement for hydrogen peroxide (H2O2) generation and action during stomatal closure induced by darkness and abscisic acid (ABA) was investigated in pea (Pisum sativum L.). Stomatal closure induced by darkness or ABA was inhibited by the H2O2-scavenging enzyme catalase or the antioxidant N-acetyl cysteine (NAC), or by diphenylene iodonium (DPI), an inhibitor of the H2O2-generating enzyme NADPH oxidase. Exogenous H2O2 induced stomatal closure in a dose- and time-dependent manner, and H2O2 was also required for ABA-inhibition of stomatal opening in the light. H2O2 accumulation in guard cells was increased by darkness or ABA, as assessed with the fluorescent dye dichlorodihydrofluorescein diacetate (H2-DCFDA) and confocal microscopy. Such increases were inhibited by catalase, NAC or DPI, consistent with the effects of these compounds on stomatal apertures. Employing polymerase chain reaction (PCR) with degenerate oligonucleotide primers, several NADPH oxidase homologues were identified from pea genomic DNA that had substantial identity to the Arabidopsis thaliana (L.) Heynh. rboh (respiratory burst oxidase homologue) genes. Furthermore, an antibody raised against the tomato rboh identified immunoreactive proteins in epidermal, mesophyll and guard cells.

MAP kinase specifically mediates the ABA-induced H2O2 generation in guard cells ofVicia faba L.

Mitogen-activated protein (MAP) kinase is involved in ABA-or H2O2-signaling, and H2O2 acts as intermediate in the downstream of ABA signal transduction pathway, which has recently emerged as a secondary messenger of ABA signaling. Using an epidermal strip bioassay and laser scanning confocal microscope, we provided the first evidence that MAP kinase plays an important role in H2O2 signal initial, amplification and specific targeting in response to stimuli in guard cells. ABA-or H2O2-inducedVicia faba stomatal closure was inhibited or reversed by the specific inhibitor PD98059 of MEK1/2; the guard cells were pre-incubated or -microinjected by 10 μmol · L−1 PD98059, ABA could not enhance the fluorescence intensity of H2O2 probe dichlorofluorescein (DCF). Meanwhile, after ABA induced the H2O2 accumulation in guard cells, the exogenous or intracellular PD98059 could reduce the DCF fluorescence intensity. Most interestingly, on the contrary to ABA, the DCF fluorescence intensity of guard cells treated by 100 μmol · L−1 salicylic acid (SA) was not down-regulated by PD98059, yet PD98059 did not regulate the stomatal movement being induced by light, dark or salicylic acid. These results suggest that MEK1/2 could mediate stomatal closure by abolishing the ABA-induced H2O2 generation/accumulation in the specific manner.

The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis.

The movement of guard cells in stomatal complexes controls water loss and CO(2) uptake in plants. Examination of the dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) revealed that it is expressed and functions in Arabidopsis guard cells. CHL1 promoter-beta-glucuronidase and CHL1 promoter-green fluorescent protein constructs showed strong expression in guard cells, and immunolocalization experiments with anti-CHL1 antibody confirmed these results. To assess CHL1 function, chl1 mutant plants grown in the presence of nitrate were examined. Compared with wild-type plants, chl1 mutants had reduced stomatal opening and reduced transpiration rates in the light or when deprived of CO(2) in the dark. These effects result in enhanced drought tolerance in chl1 mutants. At the cellular level, chl1 mutants showed reduced nitrate accumulation in guard cells during stomatal opening and failed to show nitrate-induced depolarization of guard cells. In wild-type guard cells, nitrate induced depolarization, and nitrate concentrations increased threefold during stomatal opening. These results identify an anion transporter that functions in stomatal opening and demonstrate that CHL1 supports stomatal function in the presence of nitrate.

Phot1 and phot2 mediate blue light regulation of stomatal opening.

The stomatal pores of higher plants allow for gaseous exchange into and out of leaves. Situated in the epidermis, they are surrounded by a pair of guard cells which control their opening in response to many environmental stimuli, including blue light. Opening of the pores is mediated by K(+) accumulation in guard cells through a K(+) channel and driven by an inside-negative electrical potential. Blue light causes phosphorylation and activation of the plasma membrane H(+)-ATPase that creates this potential. Thus far, no blue light receptor mediating stomatal opening has been identified, although the carotenoid, zeaxanthin, has been proposed. Arabidopsis mutants deficient in specific blue-light-mediated responses have identified four blue light receptors, cryptochrome 1 (cry1), cryptochrome 2 (cry2), phot1 and phot2. Here we show that in a double mutant of phot1 and phot2 stomata do not respond to blue light although single mutants are phenotypically normal. These results demonstrate that phot1 and phot2 act redundantly as blue light receptors mediating stomatal opening.

Pronounced differences between the native K+ channels and KAT1 and KST1 alpha-subunit homomers of guard cells.

Stomatal opening is the result of K(+)-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K(+)-selective ion channels. Here we compare the invitro properties of two guard-cell K(+)-channel alpha-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K(+)-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K(+)-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K+ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs+, 50% of the guard-cell K(+)-uptake channels (GCKClin) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to -90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as -155 mV were required. In contrast, compared to the K+ channels in vivo the functional alpha-subunit homomers almost lacked a voltage-dependent block by extracellular Ca2+. Similar to the block by Cs+ and Ca2+, the acid activation of the alpha-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCLin in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K(+)-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K(+)-uptake channels result either from the heterometric assembly of different alpha-subunits or evolve from cell-type specific posttranslational modification.

Sugar and Organic Acid Accumulation in Guard Cells of Vicia faba in Response to Red and Blue Light.

Changes in neutral sugar and organic acid content of guard cells were quantitated by high-performance liquid chromatography during stomatal opening in different light qualities. Sonicated Vicia faba epidermal peels were irradiated with 10 [mu]mol m-2 s-1 of blue light, a fluence rate insufficient for the activation of guard cell photosynthesis, or 125 [mu]mol m-2 s-1 of red light, in the presence of 1 mM KCl, 0.1 mM CaCl2. The low-fluence-rate blue light stimulated an average net stomatal opening of 4.7 [mu]m in 2 h, whereas the saturating fluence rate of red light stimulated an average net opening of 3.8 [mu]m in 2 h. Under blue light, the malate content of guard cells increased to 173% of the initial level during the first 30 min of opening and declined as opening continued. Sucrose levels continuously rose throughout the blue light-stimulated opening, reaching 215% of the initial level after 2 h. The starch hydrolysis products maltose and maltotriose remained elevated at all times. Under red light, guard cells showed very little increase in organic acid or maltose levels, whereas sucrose levels increased to 208% of the initial level after 2 h. Total measured organic metabolite concentrations were correlated with stomatal apertures in all cases except where substantial malate increases occurred. These results support the hypothesis that light quality modulates alternative mechanisms of osmotic accumulation in guard cells, including potassium uptake, photosynthetic sugar production, and starch breakdown.

Calcium and ABA-induced stomatal closure

Calcium and ABA-induced stomatal closure

Abaxial and Adaxial Stomatal Responses to Light of Different Wavelengths and to Phenylacetic Acid on Isolated Epidermes ofCommelina communisL.

Abaxial and adaxial stomatal responses to light of different wavelengths and to phenylacetic acid (PAA), a molecule known to form complexes with irradiated flavins, were examined on isolated epidermes of Commelina communis L. Blue light was superior to red and green in promoting opening. Potassium accumulation and malate production were common to both abaxial and adaxial stomatal cells, but the photosensitivity was markedly higher in the former than in the latter. PAA suppressed opening and potassium accumulation in guard cells, but hardly affected the level of epidermal malate; C02-free air failed to reverse these suppressions. The PAA-effect was more substantial in blue light than in red, green or darkness; thus, a flavin photoreceptor is indicated. Because of the overall effect of PAA under all conditions it is suggested that, in addition to its interaction with blue light reception, PAA also has a more general effect on guard cells.

Relationship of Temperature to Stomatal Aperture and Potassium Accumulation in Guard Cells of Vicia faba

Epidermal strips of Vicia faba were floated on 10 millimolar KCl at various temperatures and for several time periods. The diameter of the stomatal aperture was determined microscopically and K(+) content was estimated and expressed as the per cent of the guard cell stained. Stomatal opening was associated with increased K(+) in guard cells, but the quantitative association was modified both by time and temperature. At low temperatures (0-20 C) there was a prolonged Spannungsphase while at higher temperatures (30-45 C) motorphase was exhibited. During the motorphase there was a rapid opening of the stomates which was highly correlated with K(+) influx. At treatment periods of 360 minutes and temperatures higher than 25 C there appeared to be a maintenance phase during which K(+) concentration of the guard cells decreased without an equivalent decrease in aperture.

Organic acid and potassium accumulation in guard cells during stomatal opening

Leaflets of Vicia faba L. with either open or closed stomata were quick-frozen and freeze-dried. Individual guard cell pairs and pure samples of palisade parenchyma, spongy parenchyma, and epidermis lacking guard cells were dissected from the leaflets, weighed, and assayed for organic acids or K(+).K(+) was measured by a new enzymatic method. In guard cells of open stomata, as compared to closed stomata, K(+) was 2- to 4-fold higher, malic acid 6-fold higher, and citric acid 3-fold higher. Both aspartic and glutamic acids were also higher, but the amounts present were low compared to malic and citric acids. Isocitric acid was significantly higher in one experiment, but not in another. Glyceric acid was not increased. Succinic acid was too low to detect by the method used; but in guard cells of open stomata the concentration must have been less than 2% of that of malic acid. Malic acid was higher in the palisade parenchyma from the leaflet with open stomata. The ion balance shows that malic and citric acids provide much of the counter ion for the K(+) taken up during stomatal opening.

Stomatal movement and potassium transport in epidermal strips of Zea mays: The effect of CO2

The correlation between stomatal action and potassium movement in the epidermis of Zea mays was examined in isolated epidermal strips floated on distilled water. Stomatal opening in the isolated epidermis is reversible in response to alternate periods of light or darkness, and is always correlated with a shift in the potassium content of the guard cells. K accumulates in guard cells during stomatal opening, and moves from the guard cells into the subsidiary cells during rapid stomatal closure. When epidermal strips are illuminated in normal air, as against CO2-free air, the stomata do not open and there is a virtually complete depletion of K from the stomatal apparatus. In darkness CO2-containing air inhibits stomatal opening and K accumulation in guard cells, but does not lead to a depletion of K from the stomata as observed in the light.

Direct Determination of Potassium Ion Accumulation in Guard Cells in Relation to Stomatal Opening in Light

Understanding the mechanism of stomatal opening in leaves is important because stomata are the avenues for CO, and H.,O diffusion. Stomata in most species open in the light when the guard cells that encompass the pores take up water and increase in turgor relative to adjacent epidermal cells (1). Thus stomatal opening is likely an osmotic phenomenon that depends upon the accumulation of solute in the guard cells, and during opening tllis accumulation has been reported to range from 0.: M to 0.5 M in various experiments (2). For over 100 years botanists have believed that the primlary solute accumulating in the light was soluble carbohydrate produced during photosynthesis, because guard cells possess chloroplasts and other epidermal cells do not. However careful quantitative work [reviewed in (3)] failed to provide strong support for this view.