The Role Of COP1 In Dark-induced Stomatal Closure And Its Relationship With H₂O₂, NO, FT, TSF And SOC1

As an E3 ligase,COP1 degrades HY5 in darkness by direct interaction with HY5,which is a positive regulator in photomorphogenesis.Thus under darkness,the photomorphogenesis is inhibited but skotomorphogenesis is initiated.Under light,COP1 is inactive,and thus HY5 is accumulated and initiates the photomorphogenesis.In the floral induction of photoperiod pathway,previous studies have shown that COP1 degrades CONSTANS(CO)by direct interaction with it and thus inhibits the expression of flowering integrons FLOWERING LOCUS(FT),TWIN SISTER OF FT(TSF)and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1(SOC1)to regulate flowering time.In the stomatal movement,studies have shown that COP1 and HY5 mediate the UV-B-induced stomatal closure by inducing the formation of H2O2 and NO in guard cells.The blue light promotes the expression of flowering regulators FT,TSF,SOC1 and APETALA1(AP1),and thus induces the stomatal opening.At the same time,previous studies also show that COP1 mediates the dark-induced stomatal closure,but the mechanism is not clear.In this study,Arabidopsis thalinaa was used as a material to study the role of COP1 in dark-induced stomatal closure and its interrelationship with H2O2,NO and flowering regulators of photoperiod pathway with methods of pharmacology,genetics,molecular biology and cell biology.The results showed that:1.Dark-induced stomatal closure was remarkably inhibited by the inhibitors of 26S proteasome degradation pathway and COP1 mutation,whereas COP1 overexpression plants showed a stomatal closure phenotype under light.However,the stomata of hy5 mutant could open normally under light and closed as wild type in darkness,indicating that COP1 acts as a positive regulator to mediate dark-induced stomatal closure and HY5 does not participate in this signal transduction process.2.Darkness significantly induced the formation of H2O2 in guard cells of wild-type and copl mutant of Arabidopsis and exogenous H2O2 could not induce the stomatal closure of cop1 mutant under darkness.The results showed that COP1 acts downstream of H2O2 in the dark-induced stomatal closure.3.Darkness could also significantly induce the formation of NO in guard cells of wild-type of Arabidopsis,but this response was inhibited in cop1 mutant,and exogenous NO could notably induce stomatal closure of cop1 mutant under either light or darkness.The results showed that COP1 plays a role in the upstream of NO during dark-induced stomatal closure.4.Darkness significantly inhibited the expression of FT,TSF and SOC1 in guard cells of wild-type Arabidopsis;ft,tsf and soc1 mutants showed partly stomatal closure phenotype under light,and darkness could further induce their stomatal closure,it is indicated that one of the reasons for dark-induced stomatal closure is that darkness inhibits the gene expression of FT,TSF,and SOC1 in guard cell.5.The inhibitors of 26S proteasome degradation pathway and COP1 mutation not only inhibited dark-induced stomatal closure,but also markedly inhibited dark-induced reduction of FT,TSF,,and SOC1 gene expression,suggesting that COP1 mediates dark-induced stomatal closure by inhibiting the gene expression of the flowering factors in photoperiod pathway.6.The endogenous level of H2O2 in guard cells of ft,tsf and socl mutants was low in light,as well as wild-type,but their endogenous levels of NO were higher than that of wild-type,indicating that the flowering integron FT,TSF and SOC1 are involved in the regulation of stomatal movement by inhibiting the production of the guard cell NO.Combining with that darkness could not induce the production of NO or stomatal closure in cop1 mutant,and ft,tsf,socl mutants showed partly stomatal closure under light,it is concluded that COP1 mediates dark-induced stomatal closure via inhibiting gene expressions of FT,TSF,and SOC1 to promote the NO production in guard cells.In summary,this study demonstrated that COP1 acts as a positive regulator to mediate dark-induced stomatal closure and its role is not related to the transcription factor HY5,but because COP1 inhibits gene expressions of flowering factors FT,TSF and SOC1,and thus promotes the production of NO in guard cell,finally results in the dark-induced stomatal closure.At the same time,the results also suggest that the role of H2O2 is in the upstream of COP1 in the signal transduction pathway of dark-induced stomatal closure,and that COP 1 mediates dark-induced stomatal closure also via FT,TSF and SOC1 independent-pathways.These results preliminarily demonstrate the role of COP1 in dark-induced stomatal closure,which will help us to further understand the signal transduction mechanisms of dark-induced stomatal closure.