S1p, Phyto S1p And Cytoplasmic Ph In The Dark, The Role Of Inducing Stomatal Closure And H < Sub > 2 < / Sub > O < Sub > 2 < / Sub > And No Relationship

In the present study, by means of stomatal bioassays, laser scanning confocal microscopy and high-performance liquid chromatography, whether sphingosine-1-phosphate (SIP), phytosphingosine-1-phosphate (Phyto-S1P) and cytosolic pH are involved in darkness-induced stomatal closure in Vicia faba or Arabidopsis thaliana were studied, and the interrelationships between S1P, Phyto-S1P, cytosolic pH and H2O2, NO during darkness-induced stomatal closure were revealed. The main results are as follows:1. Darkness obviously raised cytosolic pH, hydrogen peroxide (H2O2) and nitric oxide (NO) levels in guard cells while inducing Vicia faba stomatal closure. These darkness effects were prevented by weak acid butyric acid, H2O2modulators ASA, CAT, DPI and NO modulators c-PTIO, L-NAME, respectively. The data suggested that cytosolic alkalization, H2O2and NO all participate in darkness-induced stomatal closure. During darkness treatment, pH rise became noticeable at10min, while H2O2and NO production significantly increased at20min. The H2O2and NO levels were increased by methylamine in light and decreased by butyric acid in darkness. The results showed that cytosolic alkalization induces H2O2and NO production. ASA, CAT and DPI suppressed NO production by methylamine, c-PTIO and L-NAME prevented H2O2generation by methylamine, suggesting that H2O2mediates NO synthesis by alkalization, and vice versa. Extracellar and intracellar calcium chelators BAPTA and BAPTA-AM restricted darkness-induced cytosolic alkalization, H2O2and NO production and stomatal closure, indicating calcium may act upstream of cytosolic alkalization, H2O2and NO production during darkness-induced stomatal closure.2. Darkness substantially raised SIP and H2O2levels and closed stomata. These darkness effects were significantly suppressed by DL-threo-dihydrosphingosine (DL-threo-DHS) and N,N-dimethylsphingosine (DMS), two inhibitors of long-chain base kinases. Exogenous S1P led to stomatal closure and H2O2production, and the effects of S1P were largely suppressed by the H2O2modulators ASA, CAT and DPI. These results indicated that SIP mediates darkness-induced stomatal closure by triggering H2O2production. In addition, DL-threo-DHS and DMS significantly suppressed darkness-induced cytosolic alkalization in guard cells and stomatal closure. Exogenous S1P caused cytosolic alkalization and stomatal closure, which could be largely abolished by butyric acid. These results demonstrated that SIP synthesis is necessary for cytosolic alkalization during stomatal closure by darkness. Furthermore, together with the data described above, inhibition of darkness-induced H2O2production by butyric acid revealed that SIP synthesis-induced cytosolic alkalization is a prerequisite for H2O2production during stomatal closure by darkness, a conclusion supported by the facts that the pH increase caused by exogenous S1P had a shorter lag and peaked faster than H2O2levels and that butyric acid prevented exogenous SIP-induced H2O2production.3. Darkness obviously raised SIP and NO levels and closed stomata. These darkness effects were largely suppressed by DL-threo-DHS and DMS. Exogenous SIP led to stomatal closure and NO production, and the effects of SIP were significantly prevented by cPTIO and L-NAME. These results indicated that S1P mediates darkness-induced stomatal closure by causing NO production. Furthermore, together with the data described above, inhibition of darkness-induced NO production by butyric acid revealed that SIP synthesis-induced cytosolic alkalization is necessary for NO production during stomatal closure by darkness. This conclusion were supported by the facts that the pH increase caused by exogenous SIP had a shorter lag and peaked faster than NO levels and that butyric acid prevented exogenous SI P-induced NO production.4. DL-threo-DHS, DMS and SPHK1mutation significantly inhibited darkness-induced stomatal closure in Arabidopsis thaliana, these effects were substantially reversed by exgenous Phyto-SIP, showing that Phyto-SIP is involved in darkness-induced stomatal closure. DPI and AtRbohF, AtRbohD/F mutation significantly suppressed darkness-induced stomatal closure and H2O2production, but AtRbohD did not, indicating that H2O2generated by AtrbohF is implicated in darkness-induced stomatal closure. In addition, L-NAME, tungstate and AtNOAl, Nial, NialNia2mutation evidently prevented darkness-induced stomatal closure and NO production, but Nia2mutation did not. The results showed that NO production is dependent on Nial and AtNOAl pathways during darkness-induced stomatal closure.5. Exogenous H2O2and SNP significantly reversed the inhibitory effects of DL-threo-DHS, DMS and SPHK1mutation on darkness-induced stomatal closure. However, the effects of ASA, CAT and cPTIO on this process couldn’t be reversed by exogenous Phyto-SIP. In addition, DL-threo-DHS, DMS and SPHK1mutation largely depressed darkness-induced H2O2and NO production. These results indicated that Phyto-S1P mediates darkness-induced stomatal closure by triggering H2O2and NO production in Arabidopsis thaliana. The further results showed that ASA, CAT, DPI and AtRbohF, AtRbohD/F mutation significantly inhibited Phyto-SIP-induced stomatal closure and H2O2production, but AtRbohD mutation did not. cPTIO, L-NAME, Na2WO4and AtNOAl, Nial, NialNia2mutation could inhibit exogenous Phyto-S IP-induced stomatal closure and NO production, but Nia2mutation could not. The results showed that Phyto-S1P mediates darkness-induced stomatal closure via triggering AtrbohF-dependent H2O2production and AtNOAl-and Nial-dependent NO synthesis in Arabidopsis thaliana. In conclusion, our results suggested that darkness raises S1P content and intracellular calcium level, hence causes cytosolic alkalization, and finally induces H2O2, NO production and stomatal closure in Vicia faba. Additionally, H2O2production is dependent on AtrbohF and NO synthesis is associated with AtNOA1-and Nial-dependent pathways during Phyto-SIP-mediated darkness-induced stomatal closure in Arabidopsis thaliana.