The Role Of Extracellular ATP In The Stomatal Closure Of Arabidopsis Thaliana And Its Relationship With H <2>, 2 2, NO

ATP, as an extracellular signal molecule, has been known to regulate virious growth and development processes in plant. However, the effect of extracellular ATP (eATP) on stomatal movement and the potential mechanism by which eATP regulates stomatal movement remains incomplete clear. The present study provided evidence that eATP mediates darkness-induced stomatal closure, and the relationship between eATP and hydrogen dioxide (H2O2) or nitric oxide (NO) during darkness-induced stomatal closure was investigated. The main results were as follows:1. Apyrase, an enzyme that can hydrolyze nucleoside triphosphates and/or diphosphates, but not nucleoside monophosphates or nonnucleoside phosphates, PPADS, an inhibitor of purine receptor, and Brefeldin A, an inhibitor of vesicle transport, significantly inhibited darkness-induced stomatal closure. ATP, ATPyS, a weakly hydrolyzable ATP analogs, and ADP obviously induced stomatal closure. The data indicate that eATP is involved in darkness-induced stomatal closure. In addition, Brefeldin A and PPADS inhibition of darkness-induced stomatal closure suggest that eATP is derived from its exocytotic release, and purine receptor mediates its perception.2. Apyrase, PPADS and Brefeldin A inhibited darkness-induced H2O2 production in guard cells, ATP, ATPyS and ADP increased H2O2 production in guard cells in light. H2O2 reversed the inhibitory effect of Apyrase, PPADS and Brefeldin A on darkness-induced stomatal closure, but ATPyS did not reverse the inhibitory effect of ASA and DPI on darkness-induced stomatal closure. The results show that H2O2 mediates the effect of eATP on stomatal closure. In addition, ATPyS induced H2O2 generation and stomatal closure in the wild type and AtrbohD mutant, but did not in the wild type treated with ASA or DPI and in AtrbohF and AtrbohD/F mutants. The data not only provide evidence that H2O2 is involved in eATP-led stomatal closure, and show that AtrbohF catalyzes eATP-induced H2O2 synthesis.3. Apyrase, PPADS and Brefeldin A prevented darkness-induced NO production in guard cells, ATP, ATPyS and ADP elevated NO production in guard cells in light. SNP reversed the inhibitory effect of Apyrase, PPADS and Brefeldin A on darkness-induced stomatal closure, but ATPyS did not prevent the effect of c-PTIO and Na2WO4 on darkness-induced stomatal closure. The results show that NO participates in eATP- induced stomatal closure. Furthermore, ATPyS triggered NO generation and stomatal closure in the wild type and Nia2-1 mutant, but did not in the wild type treated with c-PTIO or Na2WO4 and in Nia1-2 and Nia2-5/Nia1-2 mutants. The data support that NO is involved in eATP-induced stomatal closure, also show that eATP-induced NO synthesis is Nial-dependent.4. H2O2 did not close stomata of wild type treated with c-PTIO or Na2WO4 and Nial-2 and Nial-2/Nia2-5 mutants, but SNP closed stomata of the wild type treated with ASA or DPI and AtrbohF and AtrbohD/F mutants. H2O2 failed to rescue the defects of wild type treated with c-PTIO or Na2WO4 and Nial-2 and Nial-2/Nia2-5 mutants, but SNP restored the lesions of wild type treated with ASA or DPI and AtrbohF and AtrbohD/F mutants, in ATPyS-induced stomatal closure. H2O2 and NO levels in guard cells were monitored, data show that, ATPyS induced H2O2 generation in wild type treated with c-PTIO or Na2WO4 and Nial-2 and Nial-2/Nia2-5 mutants, but failed to induce NO generation in wild type treated with ASA or DPI and AtrbohF and AtrbohD/F mutants. The results clearly indicate that H2O2, as an intermediate signal molecular, acts upstream of NO.To sum up, the data presented here show that eATP mediates darkness-led stomatal closure via inducing AtrbohF-dependent H2O2 production and Nia1-catalyzed NO synthesis, and NO acts downstream of H2O2.