Dark Treatment Activates The Signal Transduction Mechanism Of AtMPK3 And AtMPK6 And The Mechanism Of AtMPK12 In Dark-induced Stomatal Closure

Previous studies have shown that phyto-sphingosine-1-phosphate?Phyto-S1P?,cytoplasmic alkalization,hydrogen peroxide?H₂O₂?and nitric oxide?NO?were involved in the darkness-induced stomatal closure,and our previous genetic analysis also showed that MEK1 and MPK6 were involved in darkness-induced stomatal closure in Arabidopsis.However,which MPKs are activated by the dark-stimulation,and the relationships among MPKs,dark-stimulation,MEK1,Phyto-S1P,cytoplasmic alkalization,H₂O₂ and NO are largely unknown.Also,whether other MPKs also participate in the darkness-induced stomatal closure is not clear.Based on preliminary researches,we want to know which MPKs are activiated by darkness,and whether the activation of these MPKs by darkness depend on MEK1,Phyto-S1P,cytoplasmic alkalization,H₂O₂,NO.Meanwhile,we want to find whether MPK3,MPK9 MPK12 are involved in the darkness-induced stomatal closure in Arabidopsis,and their relationship with H₂O₂ and NO.In general,we got the following main results and conclusions:1.Compared with visible light,darkness could obviously induce the actication ofMPK3 and MPK6,especially MPK6,in wild-type Arabidopsis leaves.Also we found that the appropriate processing time for the activation of Arabidopsis MPKs by darkness was 2 hours.2.Compared with the wild-type,the homozygous T-DNA insertion mutation of MEK1 significantly inhibited the darkness-induced activation of MPK3 and MPK6,suggesting that MEK1-MPK6 signal cascade pathway was involved in this process.3.The inhibitor of phyto-S1P biosynthesis,DMS?N,N-dimethyl sphingosine?,can significantly inhibit the darkness-induced activation of MPK3 and MPK6 in wild-type Arabidopsis,suggesting the darkness-induced activation of MPKs depends on Phyto-S1P generation.4.Methylamine could enhance the activation of MPK3 and MPK6 by darkness in wild-type Arabidopsis leaves,while butyric acid could significantly inhibit the darkness-induced activation of MPK3 and MPK6,indicating that the darkness-induced activation of MPK3 and MPK6 depend on cytosolic alkalization.5.Compared to wild type,the mutation of one NADPH oxidase,AtrbohF,partially inhibited the activation of MPK3 and MPK6 by darkness in leaves of Arabidopsis,while the mutation of another NADPH oxidase,AtrbohD,had no significant effect on darkness-induced MPK3 and MPK6 activation.But the activation of MPK3 and MPK6 by darkness was siginificantly inhibited in the double mutant AtrbohD/F,showing that the functions of AtrbohD and AtrbohF are redundant,and AtrbohF may play a major role.Meanwhile the exogenous H₂O₂ treatment can significantly induced the activiation of MPK3 and MPK6 in visible light.The results indicated that H₂O₂ from NADPH-oxidase AtrbohF and AtrbohD mediated the darkness-induced activation of Arabidopsis MPK3 and MPK6.6.Compared to wild type,the activation of MPK3 and MPK6 by darkness in Arabidopsis leaves was not affected by the single mutation or double mutatiaon of nitrate reductase gene NIA1 and NIA2,suggesting that NO from nitrate reductase pathway was not involved in the MPK3 and MPK6 activation by darkness.Maybe NO from nitrate reductase pathway was downstream of MPK3 and MPK6.7.Compared to wild type,the homozygous T-DNA insertion mutation of MPK12 partially inhibited darkness-induced Aabidopsis stomatal closure,but the homozygous T-DNA insertion mutation of MPK3 and MPK9 had no effects on darkness-induced stomatal closure,showing that MPK12 was involved in darkness-induced stomatal closure,but MPK9 and MPK3 were not involved.8.Exogenous H₂O₂ and the NO donor SNP?sodium nitroprusside?treatment could significantly induce Wild-type Arabidopsis stomatal closure in visible light,but this effect could be partially suppressed by the mutation of mpkl2.At the same time,exogenous H₂O₂ and SNP treatment could not rescue the inhibition of dark-induced stomatal closure by the mutation of mpkl2.Also in the mpk12 mutant darkness could still induce the generation of H₂O₂ and NO in guard cells,and activate MPK6 as well as MPK3.These results indicated that MPK12 are downstream of MPK6,H₂O₂ and NO in dark-induced stomatal closure process.In summary,our research firstly showed that darkness majorly induced the activation of MPK3 and MPK6,not other MPKs,in leaves of Arabidopsis,and the activation depend on Phyto-S1P,cytoplasmic alkalization,H₂O₂ and MEK1,instead of NO.The results not only supported our previous genetic analysis which showed that the role of MEK1?MPK6 cascades in darkness-induced stomatal closure and their relationship with H₂O₂ and NO,but also further rationalized the interaction of MEK1?MPK6 signaling cascade pathway with Phyto-S1P and cytoplasmic alkalization in darkness-induced stomatal closure.Also,our research showed that MPK12 was involved in the darkness-induced stomatal closure in Arabidopsis,and MPK12 was downstream of MPK6,H₂O₂ and NO.Besides,we found that MPK3 and MPK9 may play no roles in the darkness-induced stomatal closure.These results will help us to understand the MPK cascade pathway in darkness-induced stomatal closure and their relationships with other signaling molecules.