The Involvement Of OsEPFs In Regulating Plant Stomatal Development

Stomata are openings bordered by a pair of guard cells on the epidermal tissues of the aerial part of plants.Fully functional stomata are crucial for plant growth and survival as they are in charge of gas exchange between the plant and the environment,as well as controlling the entry of specific microbes.EPIDERMAL PATTERNING FACTORs(EPFs)are members of a family of secreted cysteine-rich peptides.The Arabidopsis genome contains 11 members of EPFs which participate in various developmental processes.Existing evidence indicates that three EPF members,EPF1,EPF2 and EPFL9 influence stomatal development.EPF1 and EPF2 specifically express in the stomatal lineage cells and surpress adjacent cells to develop into stomata.EPFL9 expresses in mesophyll cell and serves as a positive regulator in stomatal development.The function of EPFL9 is likely antagonistic to that of EPF2 by competitive binding to ER.In contrast to Arabidopsis,the leaves of rice exhibit a very orderly distribution of stomata with unique morphology,featuring dumbbell-shaped guard cells flanked by subsidiary cells.The function of EPFs in rice stomatal development is yet to be determined.In this study,we investigate the function conserveness of EPF genes.Our main findings are as follows:1.Members of EPF family can only be found in the genomes of higher plants.Interestingly,there is only one homologue to EPF 1/2 whilst no homologue to EPFL9 found in Physcomitrella patens,suggesting that the evolution of EPFL9 is likely later than those of EPF1 and EPF2.Rice and corn plants we examined have two EPFL9 homologues with high amino acid identity particularly in their C-terminal.2.All the OsEPF1-OE,OsEPF2-OE and OsEPFL9-1-RNAi transgenic plants displayed reduced stomatal files and a reduction in stomatal density compared with the wild type rice.Furthermore,it seems that part of stomatal lineage cells escaped their stomatal fate and developed into sinuous pavement cells,indicating that OsEPFs play key roles in the stomatal development in rice.3.OsEPFs mainly express in the aerial parts of plants,including coleoptiles,young leaves,pulvinus,stem,internode and inflorescence,supporting our hypothesis that OsEPFs function in regulating the development and distribution pattern of stomata in rice plants.4.Overexpression of OsEPFl and OsEPF2 in Arabidopsis caused a decrease of stomatal density of both rosette leaves and cotyledons,whereas overexpression of OsEPFL9-l and OsEPF9-2 increased stomata density.Moreover,OsEPFL9-1-OE transgenic plants exhibit stomatal clusters on both leaves and hypocotyl,however,stomatal clusters can only be observed on the hypocotyl of OsEPFL9-2-OE transgenic plants,indicating that two OsEPFL9 function different in modulating stomatal development.