Extracellular Nucleotides and Apyrases Regulate Stomatal Aperture in Arabidopsis    

Abstract

This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5#-[g-thio]triphosphate (ATPgS) or adenosine 5#-[b-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5#-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPgS-and adenosine 5#-[b-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPgS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions.

Extracellular ATP signaling and homeostasis in plant cells

“…In Arabidopsis, low concentrations of eATP (15-35 mM) triggered the opening of stomata while high concentrations (greater than 250 mM) caused stomatal closure. 15 The application of 100 to 200 mM ATP increased hypocotyl elongation by 10-20% in etiolated seedlings but higher doses led to a 10-55% reduction of hypocotyl elongation. 32 In addition, low doses of eATP (3 to 100 mM) induced an obvious Ca 2+ influx in elongation zone of Arabidopsis roots while higher doses of eATP (1,000 mM) caused a significant increase of Ca 2+ efflux.…”

“…150-250 mM). 15 Moreover, intracellular Ca 2+ mobilizations (including Ca 2+ influx, Ca 2+ elevation in the cytosol, Ca 2+ release from the vacuole and mitochondrial Ca 2+ uptake), and mitochondrial H 2 O 2 burst were implicated in the signaling pathway of eATP-induced PCD in P. euphratica cells. 13 Under salt shock, the release of intracellular ATP from P. euphratica cells and the activation of putative plasma membrane ATP receptors were found able to trigger the downstream signaling components, H 2 O 2 and Ca 2+ (Sun J., Deng S. and Chen S., unpublished data).…”

“…In Arabidopsis, the ABA-induced stomatal closure was mediated by eATP and apyrase. 15 Chemical inhibition of apyrase activities led to stomatal closure and ABA treatment induced an obvious elevation of eATP in guard cells. 15 These results suggest that ABA-induced stomatal closure is partially caused by an inhibition of apyrase activity and a higher accumulation of eATP around guard cells.…”

“…16 Exogenous application of ATP to plant cells caused numerous physiological responses, including alteration of membrane potential, 17 14,15 and modulation of ion fluxes across the plasma membrane and tonoplast. 13,19,24 In addition to artificially increasing eATP concentrations in the ECM, a few studies focused on the effects of eATP depletion on plant and cellular responses.…”

“…13 Another two reports disclosed the regulation of eATP on stomatal movement. 14,15 Several excellent reviews have comprehensively summarized recent progress in plant eATP signaling (see refs. [2][3][4][5].…”