Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

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 ß-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.

The Thioredoxin-Regulated α-Amylase 3 of Arabidopsis thaliana Is a Target of S-Glutathionylation.

Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, AtAMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that AtAMY3 activity was sensitive to ROS, such as H2O2. Treatments with H2O2 inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized AtAMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated AtAMY3 and performing oxidation experiments of Cys-to-Ser variants of AtAMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the pK a values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (pK a1 = 5.70 ± 0.28; pK a2 = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of AtAMY3 is proposed.

New insights into redox control of starch degradation.

Starch is one of the major sinks of fixed carbon in photosynthetic tissues of higher plants. Carbon fixation and the synthesis of primary starch occur during the day in the chloroplast stroma, whereas starch degradation typically occurs during the following night to fuel the whole plant with energy and carbon in the absence of photosynthesis. Redox-based regulatory systems play a central role in the modulation of several chloroplastic pathways. Reversible oxidations of cysteine residues are post-translational modifications that orchestrate the precise functioning of chloroplast pathways together with changes in pH, Mg(2+) and concentrations of metabolic intermediates. Leaf starch metabolism has been intensively studied. The enzymes involved in starch synthesis and degradation have been identified and characterized. However, the redox control of the enzymes responsible for starch degradation at night remains elusive, and their response to redox transitions conflicts with the timing of the physiological events. Most of the enzymes of starch degradation are activated by reducing conditions, characteristic of daytime. Thus, redox control may have only a minor role during starch degradation at night, but could become relevant for daily stomatal opening in guard cells or in the re-allocation of fixed carbon in mesophyll cells in response to stress conditions.

Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism.

Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.