Functional Analysis Of BnA.EPF2 And BnA.EPFL9 Regulating Stomata Development In Brassica Napus

Stomata which locates in the epidermis of stem,leaf or other part of plants is a complex consisting of two guard cells that derived from two specialized epidermis cells.Stomata functions as a key valve that regulates gas exchange in plant,playing an important role in the.transpiration and photosynthesis.Stomata distribution and development models are regulated by the complex and fine internal gene network regulation and simultaneously affected by external environmental stimuli.In Arabidopsis,the whole pathway of the stomatal development has been well described,where EPF2 and EPFL9 in the EPF family function as the upstream signaling factors.EPF2 is highly expressed in the stomatal mother cell,signal from the epidermis cell then negatively regulates the stomata development.While EPFL9 is highly expressed in the mesophyll cell which respond to the photosynthesis and positively regulates the stomata development.Those two regulatory models about the stomata development represent the two opposite approaches that regulate stomata density.However,the stomatal development pathway in Brassica has seldomly been identified.In this study,we focused on the identification of homologous genes BnA.EPF2 and BnA.EPFL9 respectively in Brassica napus in regulation of stomatal development to elucidate the mechanism of plants stomata development.The main findings of this study are as follows:1.BnA.EPF2 and BnA.EPFL9 were expressed in Arabidopsis respectively.Expressing of BnA.EPF2 obviously decreases stomatal density in Arabidopsis,however expressing BnA.EPFL9 clearly increases stomatal density in Arabidopsis.The functions of them are similar to EPF2 and EPFL9 in Arabidopsis.2.When BnA.EPFL9 and BnA.EPF2 were expressed in the corresponding Arabidopsis mutants EPFL9-RNAi and epf2,the expression of BnA.EPFL9 restored EPFL9-RNAi mutant phenotype,and while expression of BnA.EPF2 only partially complemented the corresponding phenotype Arabidopsis epf2 mutants.3.GUS staining analyses are used to identify the expression pattern of BnA.EPF2 and BnA.EPFL9 in Arabidopsis.BnA.EPFL9 has the similar expression pattern as consistent with EPFL9 expression scope;The expression pattern of BnA.EPF2 is not the same as EPF2,BnA.EPF2 expression ismuch wider.4.When two genes are respectively overexpressed in Brassica napus,both type of the transgenic plants show a certain increase of in stomatal density.We find evidence ound that the function of BnA.EPFL9 is conserved in both species,whereas the roles of BnA.EPF2 are somewhat divergent,due to different expression patterns,although they are homologous genes from species with very close genetic relationship.5.This study We also investigated the effects of stomatal density on Plant Physiologyogical traits and the application potential by means of transgenic Arabidopsis over-expressing BnA.EPF2 and BnA.EPFL9 respectively.By analyzing the physiological traits of plants with different stomatal density resulted from expression of BnA.EPF2 and BnA.EPFL9,it is revealed that stomatal density decrease can dramatically enhance drought resistance but simultaneously with side effect,reducing the plant biomass production due to lower photosynthesis from lower CO₂ uptake through stomata.