| Stomata consist of two guard cells around a pore in the epidermis of plants,allow gas exchange between plant and the external atmosphere.The quantity and morphology of stoma profoundly affect the efficiency of transpiration and photosynthesis.SnRK1(SNF1-related kinase 1)protein complex,the key plant energy sensor,can regulate the metabolism and energy homeostasis in plants.Our previous work has shown that KIN10 kinase,the catalytic subunit of SnRK1,is specially expressed in the nucleus of the meristemoid cells in the epidermal cells of Arabidopsis leaves to phosphorylate and stabilize SPCH,a key regulator of stomatal development,which ultimately promotes stomatal development.However,the molecular mechanism of the specific nuclear localization of KIN10 is not clear.To elucidate the molecular mechanism of the specific spatial localization of KIN10 in leaf epidermal cells,we conducted the following studies.1.H2O2 regulates the subcellular localization of KIN10.Treatment with DCMU,the inhibitor of photosynthesis,significantly increased the nuclear-to-cytoplasmic ratio of pKIN10:KIN10-YFP in the epidermal cells,but such effects of DCMU were counteracted by the cotreatments with H2O2 scavenger KI,suggesting that H2O2 may play an important role for DCMU-induced KIN10 nuclear localization in plants.H2O2 treatment significantly increased the nuclear-to-cytoplasmic ratio of KIN10-YFP,while the subcellular localization of histone H3 was not significantly affected.In addition,treatment with cycloheximide(CHX),a protein translation inhibitor,did not affect the H2O2-mediated nuclear localization of KIN10,indicating that the H2O2-induced KIN10 translocation into the nucleus was not dependent on the synthesis of KIN10 protein.These results suggest H2O2 promotes the KIN10 nuclear localization in plants.2.H2O2 exhibits specific spatial distribution in leaf epidermal cells of Arabidopsis.Considering the strong nuclear localization signal of KIN10 in meristemoids,and the important role of H2O2 in KIN10 nuclear localization in plants,we speculated that the meristemoid cells may contain high levels of H2O2.Then we examined the H2O2 distribution pattern in cotyledon epidermal cells of wild-type plants by H2DCFDA,BES-H2O2-Ac,DAB,pHyPer methods.The results showed that H2O2 is highly enriched in the abaxial meristemoids and guard cells in the epidermal cells of Arabidopsis leaves,while less in pavement cells.Further analysis of plants showed that the spatial pattern of H2O2 in the epidermal cells is widespread under the conditions of different photoperiod,different light intensity and with or without exogenous sucrose.In addition,the specific spatial distribution of H2O2 is highly consistent with the specific nuclear localization of KIN10,which further verifies the essential role of H2O2 in maintaining the nuclear localization of KIN10.3.The specific expression of CAT2 and APX1 result in the specific spatial distribution of H2O2 in leaf epidermal cells.Through analyzing the published cell typespecific transcriptomic data of Arabidopsis leaves,we found that the expression levels of ROS-scavenging genes,CAT2 and APX1 were lower in the meristemoid cells than in other types of cells.The analysis of the expression pattern of CAT2 and APX1 using pCAT2::GFP and pAPX1::GFP transgenic plants showed that the expression of CAT2 and APX1 were strongest in pavement cells,but weak in the meristemoid and guard cell.Further studies revealed that SPCH,a key factor in stomatal development,could directly bind to CAT2 and APX1 promoters and inhibit their expression,thereby causing H2O2 accumulation in stomatal meristem cells.4.H2O2 that exhibits specific spatial distribution in leaf epidermal cells regulates stomatal development.We then analyzed the ratio of meristemoid and stomata in H2O2related mutants.Compared to the wild-type Col-0,cat2,cat3,cat2 cat3 and cat1 cat2 cat3 mutants with higher H2O2 level had significantly higher proportion of meristem cells and stomata.While in the p35S::CAT2-Myc overexpression plants with lower H2O2 level,the proportion of meristemoids and stomata was lower than Col-0.Furthermore,we manipulated CAT2 activity in the stomatal lineage cells by expressing CAT2 under SPCH promoter.The results showed that the expression of CAT2 in stomatal lineage cells significantly inhibited stomatal initiation and reduced the stomatal index,and H2O2 treatment also partially suppressed the decreased stomatal index of pSPCH::CAT2-RFP transgenic plants.These results suggest that the specific spatial distribution of H2O2 is critical for stomatal development.5.H2O2 weakens the interaction between KIN10 and KINβ2 and promotes the nuclear localization of KIN10.Previous studies have shown that the subcellular localization of KIN10 is regulated by the membrane-associated SnRK1 regulatory subunit KINβ,which restricts KIN10 nuclear localization.Pull-down and Co-IP assays showed that H2O2 significantly inhibited the interaction between KIN10 and KINβ2 both in vivo and in vitro.The transient tobacco co-injection experiment showed that KIN10-YFP distributed in the nucleus and cytoplasm,co-expression with KINP2 resulted in the exclusion of KIN10-YFP from the nucleus,and H2O2 treatment promoted the nuclear localization of KIN10.Moreover,the ratio of nuclear-tocytoplasmic KIN 10 protein was significantly decreased in p35S::KINβ2-RFP transgenic plants,but increased in kinβ2 mutant,and H2O2 treatment significantly increased the nuclear-to-cytoplasmic ratio of KIN10 in plants.These results indicate that H2O2 could promote the nuclear localization of KIN 10 by inhibiting the interaction between KIN 10 and KINβ2.In conclusion,SPCH,the core transcription factor of stomatal development,directly binds to the promoters of CAT2 and APX1 to suppress their expression and increase H2O2 levels in meristemoid cells.Then H2O2 reduces the interaction between KIN10 and KINP2,thereby promoting the nuclear localization of KIN10.Finally,nuclear-localized KIN 10 phosphorylates and stabilizes SPCH,and subsequently promotes stomatal development. |
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