Starch In Guard Cells Research Articles

Starch metabolism in guard cells: At the intersection of environmental stimuli and stomatal movement.

Starch metabolism in guard cells plays a central role in regulating stomatal movement in response to light, elevated ambient CO2 and potentially other abiotic and biotic factors. Here, we discuss how various guard cell signal transduction pathways converge to promote rearrangements in guard cell starch metabolism for efficient stomatal responses, an essential physiological process that sustains plant productivity and stress tolerance. We suggest manipulation of guard cell starch dynamics as a previously overlooked strategy to improve stomatal behaviour under changing environmental conditions.

Hydrogen peroxide is required for light-induced stomatal opening across different plant species

Stomatal movement is vital for plants to exchange gases and adaption to terrestrial habitats, which is regulated by environmental and phytohormonal signals. Here, we demonstrate that hydrogen peroxide (H2O2) is required for light-induced stomatal opening. H2O2 accumulates specifically in guard cells even when plants are under unstressed conditions. Reducing H2O2 content through chemical treatments or genetic manipulations results in impaired stomatal opening in response to light. This phenomenon is observed across different plant species, including lycopodium, fern, and monocotyledonous wheat. Additionally, we show that H2O2 induces the nuclear localization of KIN10 protein, the catalytic subunit of plant energy sensor SnRK1. The nuclear-localized KIN10 interacts with and phosphorylates the bZIP transcription factor bZIP30, leading to the formation of a heterodimer between bZIP30 and BRASSINAZOLE-RESISTANT1 (BZR1), the master regulator of brassinosteroid signaling. This heterodimer complex activates the expression of amylase, which enables guard cell starch degradation and promotes stomatal opening. Overall, these findings suggest that H2O2 plays a critical role in light-induced stomatal opening across different plant species.

Arabidopsis Sucrose Synthase 3 (SUS3) regulates starch accumulation in guard cells at the end of day

ABSTRACT Starch in the stomatal guard cells is largely synthesized using carbon precursors originating from sugars imported from the leaf mesophyll. Such heterotrophic nature of guard cell starch synthesis prompted us to investigate the role of cytosolic sucrose synthases (SUS) in this pathway. Out of the six members of the Arabidopsis SUS gene family, SUS3 was the most highly expressed isoform in guard cells. The Arabidopsis sus3 mutant displayed changes in guard cell starch contents comparable to the Wild Type (WT) up until 6 h into the day. After this time point, sus3 guard cells surprisingly started to accumulate starch at very high rates, reaching the end of the day with significantly more starch than WT. Based on the phenotype of the sus3 mutant, we suggest that in guard cells, SUS3 is involved in the regulation of carbon fluxes towards starch synthesis during the second half of the day. SUS3 may be part of a previously predicted guard cell futile cycle of metabolic reactions, in which sucrose is re-synthesized from UDP-glucose to avoid excessive starch synthesis toward the end of the day. This is in contrast to typical storage organs, in which cytosolic SUS is required to produce ADP-glucose for starch synthesis.

Guarding the gates: TOR mediates guard cell starch degradation to control stomatal opening.

Guarding the gates: TOR mediates guard cell starch degradation to control stomatal opening.

TOR promotes guard cell starch degradation by regulating the activity of β-AMYLASE1 in Arabidopsis.

Starch is the main energy storage carbohydrate in plants and serves as an essential carbon storage molecule for plant metabolism and growth under changing environmental conditions. The TARGET of RAPAMYCIN (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrient, hormone, and stress signaling to regulate growth in all eukaryotes. Here, we demonstrate that TOR promotes guard cell starch degradation and induces stomatal opening in Arabidopsis thaliana. Starvation caused by plants growing under short photoperiod or low light photon irradiance, as well as inactivation of TOR, impaired guard cell starch degradation and stomatal opening. Sugar and TOR induce the accumulation of β-AMYLASE1 (BAM1), which is responsible for starch degradation in guard cells. The plant steroid hormone brassinosteroid and transcription factor BRASSINAZOLE-RESISTANT1 play crucial roles in sugar-promoted expression of BAM1. Furthermore, sugar supply induced BAM1 accumulation, but TOR inactivation led to BAM1 degradation, and the effects of TOR inactivation on BAM1 degradation were abolished by the inhibition of autophagy and proteasome pathways or by phospho-mimicking mutation of BAM1 at serine-31. Such regulation of BAM1 activity by sugar-TOR signaling allows carbon availability to regulate guard cell starch metabolism and stomatal movement, ensuring optimal photosynthesis efficiency of plants.

Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and β-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.

Peeling back the layers of crassulacean acid metabolism: functional differentiation between Kalanchoë fedtschenkoi epidermis and mesophyll proteomes.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that offers the potential to engineer improved water-use efficiency (WUE) and drought resilience in C3 plants while sustaining productivity in the hotter and drier climates that are predicted for much of the world. CAM species show an inverted pattern of stomatal opening and closing across the diel cycle, which conserves water and provides a means of maintaining growth in hot, water-limited environments. Recent genome sequencing of the constitutive model CAM species Kalanchoë fedtschenkoi provides a platform for elucidating the ensemble of proteins that link photosynthetic metabolism with stomatal movement, and that protect CAM plants from harsh environmental conditions. We describe a large-scale proteomics analysis to characterize and compare proteins, as well as diel changes in their abundance in guard cell-enriched epidermis and mesophyll cells from leaves of K.fedtschenkoi. Proteins implicated in processes that encompass respiration, the transport of water and CO2 , stomatal regulation, and CAM biochemistry are highlighted and discussed. Diel rescheduling of guard cell starch turnover in K.fedtschenkoi compared with that observed in Arabidopsis is reported and tissue-specific localization in the epidermis and mesophyll of isozymes implicated in starch and malate turnover are discussed in line with the contrasting roles for these metabolites within the CAM mesophyll and stomatal complex. These data reveal the proteins and the biological processes enriched in each layer and provide key information for studies aiming to adapt plants to hot and dry environments by modifying leaf physiology for improved plant sustainability.

Brassinosteroid and Hydrogen Peroxide Interdependently Induce Stomatal Opening by Promoting Guard Cell Starch Degradation.

Starch is the major storage carbohydrate in plants and functions in buffering carbon and energy availability for plant fitness with challenging environmental conditions. The timing and extent of starch degradation appear to be determined by diverse hormonal and environmental signals; however, our understanding of the regulation of starch metabolism is fragmentary. Here, we demonstrate that the phytohormone brassinosteroid (BR) and redox signal hydrogen peroxide (H2O2) induce the breakdown of starch in guard cells, which promotes stomatal opening. The BR-insensitive mutant bri1-116 accumulated high levels of starch in guard cells, impairing stomatal opening in response to light. The gain-of-function mutant bzr1-1D suppressed the starch excess phenotype of bri1-116, thereby promoting stomatal opening. BRASSINAZOLE-RESISTANT1 (BZR1) interacts with the basic leucine zipper transcription factor G-BOX BINDING FACTOR2 (GBF2) to promote the expression of β-AMYLASE1 (BAM1), which is responsible for starch degradation in guard cells. H2O2 induces BZR1 oxidation, enhancing the interaction between BZR1 and GBF2 to increase BAM1 transcription. Mutations in BAM1 lead to starch accumulation and reduce the effects of BR and H2O2 on stomatal opening. Overall, this study uncovers the critical roles of BR and H2O2 in regulating guard cell starch metabolism and stomatal opening.

Starch biosynthesis by AGPase, but not starch degradation by BAM1/3 and SEX1, is rate-limiting for CO2 -regulated stomatal movements under short-day conditions.

Starch in guard cells functions in osmoregulation during stomatal movements. Starch metabolism is controlled by the circadian clock. We investigated the role of starch metabolism in stomatal responses to CO2 under different photoperiodic conditions. Guard cell starch levels correlate with low/high [CO2 ] exposure. Starch biosynthesis-deficient AGPase (ADG1) mutants but, unexpectedly, not the starch degradation-deficient BAM1, BAM3, and SEX1 mutants alone, are rate-limiting for stomatal conductance responses to [CO2 ]-shifts. Interestingly, AGPase is rate-limiting solely under short- but not long-day conditions. These findings suggest a model of enhanced AGPase activity in guard cells under short days such that starch biosynthesis becomes rate-limiting for CO2 -induced stomatal closing.

Quantification of Starch in Guard Cells of Arabidopsis thaliana.

In this protocol, we describe how to quantify starch in guard cells of Arabidopsis thaliana using the fluorophore propidium iodide and confocal laser scanning microscopy. This simple method enables monitoring, with unprecedented resolution, the dynamics of starch in guard cells.

Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening

Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3)-enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H(+)-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light.

Guard cell metabolism and CO2 sensing

In this review we concentrate on guard cell metabolism and CO2 sensing. Although a matter of some controversy, it is generally accepted that the Calvin cycle plays a minor role in stomatal movements. Recent data emphasise the importance of guard cell starch degradation and of carbon import from the guard cell apoplast in promoting and maintaining stomatal opening. Chloroplast maltose and glucose transporters appear to be crucial to the export of carbon from both guard and mesophyll cells. The way guard cells sense CO2 remains an unresolved question. However, a better understanding of the cellular events downstream from CO2 sensing is emerging. We now recognise that there are common as well as unique steps in abscisic acid (ABA) and CO2 signalling pathways. For example, while ABA and CO2 both trigger increases in cytoplasmic free calcium, unlike ABA, CO2 does not promote a cytoplasmic pH change. Future advances in this area are likely to result from the increased use of techniques and resources, such as, reverse genetics, novel mutants, confocal imaging, and microarray analyses of the guard cell transcriptome.

Guard Cell Starch Biosynthesis Regulated by Effectors of ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase catalyzes the regulated step of starch bioynthesis in mesophyll chloroplasts. This enzyme is activated by a high ratio of the concentrations of 3-P-glycerate to inorganic phosphate (Pi) in light. In contrast, starch in guard cell chloroplasts is degraded when stomata open, which usually occurs in light. We have investigated the biochemical causes for this contrasting phenomenon.Vicia faba L. leaflets were sampled in darkness and after various periods of illumination. The samples were quick-frozen and freeze-dried. Guard cells and other cells were dissected out, weighed, and assayed for ADP-glucose pyrophosphorylase activity, 3-P-glycerate, and Pi. In the pyrophosphorolytic direction, ADP-glucose pyrophosphorylase specific activity in guard cells was 2.7 moles per kilogram protein per hour, which was comparable to the values obtained for palisade and spongy cells. The specific activity in epidermal cells was 4-fold lower. Under our assay conditions, the guard cell enzyme activity was 5-fold higher in the presence of 3-P-glycerate and 5-fold lower with Pi (i.e. similar to the results obtained with extracts of fresh leaflet). During three minutes of illumination, 3-P-glycerate concentration in palisade cells increased 2.5-fold to 10 millimoles per kilogram dry mass. The concentration of 3-P-glycerate in guard cells was 20-fold lower and unaffected by illumination. The concentration of Pi was approximately 17 millimoles per kilogram dry mass in palisade cells, but was 10-fold higher in guard cells. These overall cellular Pi concentrations were unaffected by illumination. We conclude that starch biosynthesis in guard cells is not activated by light because of the low and constant 3-P-glycerate concentration there. We interpret this last to be a consequence of the absence of the photosynthetic carbon reduction pathway in chloroplasts of these cells.

Blue light response of stomata with starch-containing ( Vicia faba) and starch-deficient ( Allium cepa) guard cells under background illumination with red light

Two plant species, Vicia faba with starch-containing guard cells and Allium cepa with starch-deficient guard cells, were compared for stomatal response to blue light in order to clarify whether hydrolysis of starch in guard cells is involved in opening of stomata by blue light. The rates of transpiration of Vicia and Allium leaves increased upon illumination with strong red light and very weak blue light remarkably enhanced the rates of these species when applied simultaneously with the red light. The same blue light applied without the background was much less effective for opening of stomata. No significant difference was found between these two species in the rates of transpiration induced by blue light with background red light. It is concluded that blue light effect is not brought about via starch hydrolysis and is observable only when photosynthetic mechanism is operating with red light.

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K(+) and Cl(-) contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl(-) (or Br(-)) was necessary for stomatal opening.Previous nutrient supply of the plants determined whether the charges of K(+) in guard cells were completely balanced by Cl(-) or only partially. Addition of K(2)SO(4) to the nutrient solution reduced the participation of Cl(-) in stomatal ion transfer, even if epidermal strips of these plants were later exposed to KCl solution. The anion supplying the charge complement in these cases is not known.Although malate appeared not to participate in stomatal ion transfer in onion, epidermal samples of this species did contain malate. Malate accumulated in the epidermis of leaves put into the light but disappeared from illuminated epidermal strips floating on solutions. In whole leaves, epidermal malate content was positively correlated with stomatal opening; in epidermal strips floating on solutions, the correlation was negative or absent.

Guard Cell Starch Concentration Quantitatively Related to Stomatal Aperture

Using quantitative histochemical techniques, the carbohydrate levels of guard cells from open and closed stomatal apparatus of Vicia faba L. were compared. To minimize experimental error, all comparisons were between leaflets of the same pair. Stomata on one leaflet were caused to open by light and reduced CO(2). The other leaflet, which was in darkness, had closed stomata. In one experiment, data were also collected on palisade parenchyma, spongy parenchyma, and epidermal cells.Guard cell starch concentration was higher in the leaflets with closed stomata than in open stomata by 72 +/- 16 millimoles per kilogram dry weight (anhydroglucosyl equivalents) (N = 117, P < 0.02). Variation in guard cell starch concentration from one part of a leaflet to another was small. The data are consistent with the hypothesis that starch degradation provides the carbon skeletons for anion synthesis in guard cells during stomatal opening.Sucrose concentration was higher in guard cells when stomata were open than when they were closed in all three experiments (average difference = 45 +/- 7 millimoles per kilogram dry weight [N = 59, P < 0.01]). The variability of sucrose concentration within test leaflets prevented an unequivocal interpretation of these results. When all data are considered, it appears that soluble sugars increase in guard cells when stomata of Vicia faba open.

Structural and functional aspects of stomata

Differential cell wall thickening in developing guard cells of Polypodium vulgare L. has been studied with particular reference to guard cell protoplast deformation and the eventual formation of the stomatal pore. Concomitant studies on the development of guard cell chloroplasts and their starch inclusions during ontogeny of the stomatal complex have provided data which have been incorporated into a model to account for the formation of the pore. Guard cell starch inclusions reach a maximum density per unit volume at the same time as the guard cell walls achieve maximum differential thickening. These events coincide with the development of the pore. It is suggested that, whilst pore formation is initiated enzymatically, the mechanical forces required to bring about the separation of the two guard cells are of an osmotic nature derived from starch hydrolysis. The development of the mesophyll in relation to the epidermis is examined in respect of the formation of substomatal chambers.

Further Observations of Cation-stimulated Stomatal Opening in Isolated Epidermal Peelings of Asphodelus tenuifolius Cav. and Allium cepa L.

Summary Some observations on cation-stimulated opening in isolated epidermal peelings have been made. Water incubation of the peelings caused only turgidity of the guard cells. The incubation of peelings in SnCl 2 and FeCl 3 for a short duration caused the opening of stomata, with disappearance of starch in guard cells. The stomatal width achieved with monovalent, divalent and trivalent cations was nearly similar. It appeared that a quick stomatal opening was a consequence of these cations being actively accumulating in the guard cells.

Role of Monovalent, Divalent Cations and Phenylmercuric Acetate in the Regulation of Stomatal Aperture in Euphorbia neriifolia L.

Normally stomatal apertures in Euphorbia neriifolia remain closed in the day time. Immersion of epidermal strips in water led to an appreciable swelling of guard cells with a slight disappearance of starch grains in them but without any effect on the aperture size. The influx of K + ions caused a tremendous change in the opening of the aperture, with consequent disappearance of starch in guard cells. The influx of K + appeared to cause hydrolysis of starch, leading to an increase of osmotic amounts of guard cells, their uptake of water and finally the opening of the stomata. The K + uptake was only light-stimulated for opening of the apertures. Ca ++ did not cause any effect on the opening of stomata, either in light or darkness. The apertures in KCl-induced open stomata nearly closed down when transfered to phenylmercuric acetate (PMA) in light, associated with simultaneous appearance of starch in guard cells, as if condensation phenomenon had taken place together with the withdrawl of K ions from the guard cells with this treatment. It was also observed that uptake of PMA in light was more than in dark. More uptake of PMA result ed in complete efflux of K + , and the guard cells with lesser uptake of PMA still contained some K + , thus lowering its closing effect.

Role of epidermal cell turgor on stomatal regulation Lin isolated epidermal peelings of Citrullus colocynthis inn. (Schrad.)

The role of epidermal cell turgor appeared to play a definite role in the regulation of stomatal pore in isolated epidermal peelings of C. colocynthis . Guard cells and their surrounding epidermal cells have different osmotic potentials and absorbing capacities. Plasmolysis in epidermal cells caused by incubating peelings in suitable sucrose solutions, led to the lowering of their turgor potentials, which resulted in opening of the stomata. Any increase in turgor potential of epidermal cells caused by incubation of such peelings in water resulted in closure of stomata. Water from plasmolysed epidermal cells was taken up by guard cells due to greater difference of osmotic potentials between epidermal cells and guard cells than between epidermal cells and the incubation medium. When peelings with plasmolysed epidermal and guard cells were incubated in water, water moved into the guard cells from the medium because of its non-availability in plasmolysed epidermal cells. As the pore opened, the starch in guard cells disappeared, but the cause of this could not be ascertained