Reversal of blue light-stimulated stomatal opening by green light.

Blue light-stimulated stomatal opening in detached epidermis of Vicia faba is reversed by green light. A 30 s green light pulse eliminated the transient opening stimulated by an immediately preceding blue light pulse. Opening was restored by a subsequent blue light pulse. An initial green light pulse did not alter the response to a subsequent blue light pulse. Reversal also occurred under continuous illumination, with or without a saturating red light background. The magnitude of the green light reversal depended on fluence rate, with full reversal observed at a green light fluence rate twice that of the blue light. Continuous green light given alone stimulated a slight stomatal opening, and had no effect on red light-stimulated opening. An action spectrum for the green light effect showed a maximum at 540 nm and minor peaks at 490 and 580 nm. This spectrum is similar to the action spectrum for blue light-stimulated stomatal opening, red-shifted by about 90 nm. The carotenoid zeaxanthin has been implicated as a photoreceptor for the stomatal blue light response. Blue/green reversibility might be explained by a pair of interconvertible zeaxanthin isomers, one absorbing in the blue and the other in the green, with the green absorbing form being the physiologically active one.

Stomata from npq1, a zeaxanthin-less Arabidopsis mutant, lack a specific response to blue light.

The Arabidopsis mutant npq1, which cannot accumulate zeaxanthin because of a defective violaxanthin deepoxidase, was used to investigate the role of zeaxanthin in the stomatal response to blue light. Neither dark-adapted nor light-treated guard cells or mesophyll cells of the npq1 mutant contained detectable zeaxanthin. In contrast, wild-type guard cells had a significant zeaxanthin content in the dark and accumulated large amounts of zeaxanthin when illuminated. The well-documented red light enhancement of blue light-stimulated stomatal opening, in which increasing fluence rates of background red light result in increased response to blue light, was used to probe the specific blue light response of Arabidopsis stomata. Stomata from the npq1 mutant did not have a specific blue light response under all fluence rates of background red light tested. On the other hand, stomata from leaves of hy4 (cry 1), an Arabidopsis mutant lacking blue light-dependent inhibition of hypocotyl elongation, had a typical enhancement of the blue light response by background red light. The lack of a specific blue light response in the zeaxanthinless npq1 mutant provides genetic evidence for the role of zeaxanthin as a blue light photoreceptor in guard cells.

Stomata from growth-chamber-grown Vicia faba have an enhanced sensitivity to CO2.

Abaxial stomata from Vicia faba leaves grown in a growth chamber under constant light, temperature and humidity showed an elaborate pattern of aperture changes over the course of a light cycle. These aperture changes were tightly correlated with changes in chamber CO2 concentration (r2=0.83). Changes in chamber [CO2] resulted, in turn, from substantial daily fluctuations in ambient [CO2], typical of the Los Angeles environment, with a constant offset caused by photosynthesis and respiration of the plants within the chamber. The dominant role of the stomatal response to CO2 in the control of aperture was confirmed by manipulation of chamber [CO2]. Fast (15 min) increases and decreases in [CO2] caused rapid decreases and increases in aperture, while constant [CO2] resulted in constant aperture. In contrast, aperture changes in comparable plants grown under greenhouse conditions were tightly correlated with changes in incident solar radiation (r2=0.80), and poorly correlated with changes in [CO2] (r2=0.09). Greenhouse-grown plants transferred to growth chamber conditions showed no apparent response to CO2. These data indicate that growth-chamber-grown V. faba leaves provide an experimental system optimally suited for the study of the stomatal response to CO2, and suggest that acclimation to environmental conditions alters the sensitivity of stomata to CO2.

Central Roles for Potassium and Sucrose in Guard-Cell Osmoregulation.

Osmoregulation in guard cells of intact, attached Vicia faba leaves grown under growth chamber and greenhouse conditions was studied over a daily light cycle of stomatal movements. Under both growth conditions guard cells had two distinct osmoregulatory phases. In the first (morning) phase, opening was correlated with K+ uptake and, to a lesser extent, sucrose accumulation. In the second (afternoon) phase, in which apertures were maximal, K+ content declined and sucrose became the dominant osmoticum. Reopening of the stomata after a CO2-induced closure was accompanied by accumulation of either K+ or sucrose, depending on the time of day, indicating that a single environmental signal may use multiple osmoregulatory pathways. Malate accumulation, correlated with K+ uptake, was detected under growth chamber but not greenhouse conditions, whereas Cl- was the main K+ counterion in the greenhouse. These results indicate that guard-cell osmoregulation in the intact leaf depends on at least two different osmoregulatory pathways, K+ transport and sucrose metabolism. Furthermore, the relative importance of the K+ counterions malate and Cl- appears to be environment-dependent.

Differential changes in size distribution of xyloglucan in the cell walls of gravitropically responding Pisum sativum epicotyls.

Growth-related change in the size distribution of hemicellulosic wall polymers during the gravitropic curvature response of intact pea (Pisum sativum L. cv Alaska) epicotyls was examined by gel-filtration chromatography. The gravitropic response was characterized by the appearance of curvature 20 to 30 min after horizontal placement, with 35 degrees of curvature attained by 80 min. Correlated with the onset of curvature, on the upper side of the epicotyl, there was a conspicuous transient increase in the abundance of relatively large hemicellulosic xyloglucan polymers, similar to increases previously found under conditions where diminished wall extensibility was expected. On the lower side there was a moderate, slower, and longer-term increase in abundance of small xyloglucan, similar to changes previously found in connection with auxin-stimulated growth responses. Both shifts occurred primarily in the epidermis. They appear to represent two coordinated physiological mechanisms contributing to differential growth.

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.

Molecular size and separability features of pea cell wall polysaccharides : implications for models of primary wall structure.

Relative molecular size distributions of pectic and hemicellulosic polysaccharides of pea (Pisum sativum cv Alaska) third internode primary walls were determined by gel filtration chromatography. Pectic polyuronides have a peak molecular mass of about 1100 kilodaltons, relative to dextran standards. This peak may be partly an aggregate of smaller molecular units, because demonstrable aggregation occurred when samples were concentrated by evaporation. About 86% of the neutral sugars (mostly arabinose and galactose) in the pectin cofractionate with polyuronide in gel filtration chromatography and diethylaminoethyl-cellulose chromatography and appear to be attached covalently to polyuronide chains, probably as constituents of rhamnogalacturonans. However, at least 60% of the wall's arabinan/galactan is not linked covalently to the bulk of its rhamnogalacturonan, either glycosidically or by ester links, but occurs in the hemicellulose fraction, accompanied by negligible uronic acid, and has a peak molecular mass of about 1000 kilodaltons. Xyloglucan, the other principal hemicellulosic polymer, has a peak molecular mass of about 30 kilodaltons (with a secondary, usually minor, peak of approximately 300 kilodaltons) and is mostly not linked glycosidically either to pectic polyuronides or to arabinogalactan. The relatively narrow molecular mass distributions of these polymers suggest mechanisms of co- or postsynthetic control of hemicellulose chain length by the cell. Although the macromolecular features of the mentioned polymers individually agree generally with those shown in the widely disseminated sycamore cell primary wall model, the matrix polymers seem to be associated mostly noncovalently rather than in the covalently interlinked meshwork postulated by that model. Xyloglucan and arabinan/galactan may form tightly and more loosely bound layers, respectively, around the cellulose microfibrils, the outer layer interacting with pectic rhamnogalacturonans that occupy interstices between the hemicellulose-coated microfibrils.

Changes in molecular size of previously deposited and newly synthesized pea cell wall matrix polysaccharides : effects of auxin and turgor.

Effects of indoleacetic acid (IAA) and of turgor changes on the apparent molecular mass (M(r)) distributions of cell wall matrix polysaccharides from etiolated pea (Pisum sativum L.) epicotyl segments were determined by gel filtration chromatography. IAA causes a two- to threefold decline in the peak M(r) of xyloglucan, relative to minus-auxin controls, to occur within 0.5 hour. IAA causes an even larger decrease in the peak M(r) concurrently biosynthesized xyloglucan, as determined by [(3)H]fucose labeling, but this effect begins only after 1 hour. In contrast, IAA does not appreciably affect the M(r) distributions of pectic polyuronides or hemicellulosic arabinose/galactose polysaccharides within 1.5 hours. However, after epicotyl segments are cut, their peak polyuronide M(r) increases and later decreases, possibly as part of a wound response. Xyloglucan also undergoes IAA-independent changes in its M(r) distribution after cutting segments. In addition, the peak M(r) of newly deposited xyloglucan increases from about 9 kilodaltons shortly after deposition to about 30 kilodaltons within 0.5 hour. This may represent a process of integration into the cell wall. A step increase in turgor causes the peak M(r) of previously deposited xyloglucan (but not of the other major polymers) to increase about 10-fold within 0.5 hour, returning to its initial value by 1.5 hours. This upshift may comprise a feedback mechanism that decreases wall extensibility when the rate of wall extension suddenly increases. IAA-induced reduction of xyloglucan M(r) might cause wall loosening that leads to cell enlargement, as has been suggested previously, but the lack of a simple relation between xyloglucan M(r) and elongation rate indicates that loosening must also involve other wall factors, one of which might be the deposition of new xyloglucan of much smaller size. Although the M(r) shifts in polyuronides may represent changes in noncovalent association, and for xyloglucan this cannot be completely excluded, xyloglucan seems to participate in a dynamic process that can both decrease and increase its chain length, possible mechanisms for which are suggested.

Effect of Indoleacetic Acid- and Fusicoccin-Stimulated Proton Extrusion on Internal pH of Pea Internode Cells.

Using (31)P nuclear magnetic resonance spectroscopy, we followed cytoplasmic and vacuolar pH in pea (Pisum sativum cv Alaska) internode segments during treatment with indoleacetic acid (IAA) or fusicoccin (FC) in continuously perfused, oxygenated buffer. Although IAA and FC induced normal H(+) extrusion, elongation, and glucan synthase activity responses during the measurements, neither the cytoplasmic nor the vacuolar pH showed significant change at any time between 5 minutes and 1 to 3 hours of treatment. Changes in cytoplasmic pH as small as about 0.04 pH unit were detected after treatment with 1-naphthyl acetate. Therefore, cytoplasmic pH changes do not appear to mediate IAA or FC stimulation of H(+) extrusion or other metabolic responses to these effectors.