Functional Analysis Of STOMAGEN-Like Gene In Regulating Stomatal Development In Maize

Stoma is a key channel for gas exchange between plant and environment.The efficiency of plant photosynthesis and transpiration rate is regulated by stomatal density in leaf epidermis.Therefore,the study of stomatal development has been a hotspot and frontier in the field of plant developmental biology and crop breeding.In view of the particularity of the composition and distribution of stomata in the leaves of Grass,the study of stomatal development is lagging behind the study of stomata in the model plant Arabidopsis thaliana.Recently some bHLH transcription factors in grass have been identified and which function was revealed.However,the signaling molecules and mechanisms that regulate transcription factor activity have not been reported.STOMAGEN positively regulates stomatal density in Arabidopsis.ZmSTOMAGENl and ZmSTOAMGEN2 is two STOMAGEN-like genes in maize genome.In our laboratory,STOMAGEN homologous gene ZmSTOMAGEN1 has been cloned from maize genome.The sequence and three-dimensional structure of the protein were analyzed,and the expression of the gene in maize was studied,suggests that it may be key gene to promote maize stomatal development.This study uses CRISPR/Cas9 genome editing,plant genetic transformation,microscopic imaging,gene expression of quantitative detection technology,analysis the function of maize STOMAGEN-Like gene in stomatal development.Got some meaningful results as follows:1?ZmSTOMAGEN1 and eGFP fusion protein expression vector(pCAMBIA2300-35S::ZmSTOMAGEN 1-eGFP)were successfully constructed.The vector was introduced into Agrobacterium tumefaciens GV3101,and transient transformation of tobacco leaves.Confocal imaging showed that the fluorescence signal was concentrated near the cell membrane.These results suggest that ZmSTOMAGEN1 may function on the cell membrane.2?Agrobacterium carrying pCAMBIA2300-35S::ZmSTOMAGEN1-GFP vector was transformed into Arabidopsis thaliana by floral dip method.T1 Arabidopsis plants overexpressing the ZmSTOMAGEN1 gene were obtained.The stomatal analysis of the mature epidermis of transgenic lines showed that the stomatal density and index increased by 4.01 and 1.75 times,respectively.The stomatal distribution also breaks the "One-cell spacing" rule,with clusters of stomatal distribution.3?In order to clarify the function of ZmSTOMAGEN-like gene in maize stomatal development,this study constructed a CRISPR/Cas9 system for editing ZmSTOMAGEN1 and ZmSTOMA GEN2.The mutant plants of ZmSTOMA GEN-like gene were obtained by Agrobacterium tumefaciens-mediated transformation of maize immature embryos.The stomatal density of the leaves epidermis was found to be 63.26%lower than that of the wild type and the stomatal index was reduced by 57.2%.4?The stomatal morphological structure and distribution pattern of the maize leaves were observed by optical microscopy and scanning electron microscopy.It was found that the stomata of the lower epidermis of wild type maize were arranged at intervals between the stomatal complex and epidermal cells.In addition to the normal stomatal complex between the two epidermal cells in the mutant,there are cases where small cells similar to stomatal precursors and two epidermal cells are directly adjacent to each other.The frequency of these two cases was 24.7%and 32.2%,respectively,while the frequency in the wild was only 1.9%and 2.9%.The results showed that the deletion of ZmSTOMAGEN-like gene in maize T1 mutant caused the initial defects of stomatal development,resulting in the presence of two epidermal cells.At the same time,it may also lead to stomatal precursor cell development is suspended,permanently stay in the early development of stomatal.5?The results showed that the stomatal conductance,photosynthetic efficiency and transpiration rate of the mutant decreased by 45.24%,30.32%and 41.92%,respectively,compared with the wild type.The maximum net photosynthetic rate,dark respiration rate,light compensation point,light saturation point and apparent quantum efficiency of the mutant were lower than wild type.These results indicate that the deletion of ZmSTOMAGEN-like gene in the mutant causes the defects of maize stomatal development,and also significantly affects the photosynthetic characteristics.6.qRT-PCR analysis showed that the expression of ZmSTOMAGEN-like gene was inhibited in maize mutant.The genes encoding transcription factors of regulatory stomatal development ZmICE1,ZmSCRM2,ZmSPCH1,ZmSPCH2,ZmMUTE,ZmFAMA and stomatal development signal transduction related genes ZnSDD1,ZmTMM1,ZmERL,ZmERL,ZmERL1/2,ZmYODA were significantly down-regulated,indicating that ZmSTOMAGEN acts as a signal molecule and TMM/ERf on the cell membrane to form a ligand-receptor pair,which regulates the activity of key transcription factors in the development of stomata through the MAPK signaling pathway development of maize stomata.In summery,this study showed that stomatal protein ZmSTOMAGEN played an important role in originating stomatal development and promoting cell differentiation of stomatal precursor in maize,which suggested a divergent function of ZmSTOMAGEN-like gene in regulating stomatal development.