Function And Regulation Of OsRhoGAP2 In Seed Germination And Leaf Development In Rice

Rice is one of the major food crops in the world.Uncovering the function of key genes involved in rice growth and development is of great significance to improve rice yield by the genetic engineering.In our pervious study,we have coloned a rice gene OsRhoGAP2 encoding a Rho GTPase-activating protein from the rice panicle c DNA library.In addation,we found that the promoter activity of the OsRhoGAP2 gene responds to abiotic stress and hormonal signals.However,the exact function of OsRhoGAP2 in rice growth and development is still largely unknown.Here,we constructed OsRhoGAP2 overexpressing rice and OsRhoGAP2 knocked-out plants by the CRISPR / Cas9 technology to futher explore the function of OsRhoGAP2 in of rice growth and development.Observation of the leaf phenotype of OsRhoGAP2 overexpressed rice(T5-1,T5-5,T5-6)revealed that the leaf phenotype was normal at the early stage.However,at the late stage of rice heading,the leaves of transgenic leaves were rolled up.The statistical results showed that the flag leaf rolling index of WT and three transgenic rice lines were 10.86%,27.38%,35.36%,and 31.07%,respectively,while those of WT and three transgenic lines were 95.53%,99.34%,100%,and 100%,respectively.The flag leaf rolling index and the flag leaf erect index of the expressing lines were extremely significantly higher than those of WT.The statistical results of paraffin section showed that the bulliform cells area of transgenic rice flag leaves was e significantly smaller than that of the WT at the heading stage,Toluidine blue staining results showed of leaf bulliform cells of OsRhoGAP2 overexpressed rice were smaller than that of WT,which is consistent with the statistical results of paraffin sections.The distinct physiological parameters of rice flag leaves at heading stage were also measured.Result showed that the chlorophyll contents and net photosynthetic rate of OsRhoGAP2 overexpressed rice were significantly higher than those of WT,and their stomatal conductance and transpiration rate were extremely significantly lower than those of WT.These results indicate that OsRhoGAP2-overexpression not only regulate leaf morphology by affecting the size of leaf bulliform cells,but also increase photosynthetica activities in rice.The seed germination rates of WT and OsRhoGAP2 overexpression rice were 84.5%,62%,39.5% and 28%,respectively,while the α-amylase content was 13.82,10.82,9.85 and 9.66 mg / g · min,respectively.,.The seed germination rate and α-amylase contents of OsRhoGAP2-overexpressed rice were significantly lower than those of WT,indicating that over-accmulation of OsRhoGAP2 inhibite rice seed germination probably by down-regulating the α-amylase activities.After treatment with 1000 mg / L GA in vitro,this inhibition effects can be relieved to a certain extent.Assays of the content of endogenous GA showed that the content of endogenous GA1,GA3 and GA7 of overexpressed rice seeds were significantly lower than those of WT.The q RT-RCR assay showed that the expression levels of GA synthetic genes Os CPS1,Os KS1,and Os KAO in OsRhoGAP2-overexpressed rice seeds were reduced compared to those of WT wherease the expression levels of GA inactivating genes Os GA2ox6 and Os GA2ox8 were higher than those of WT.The expression levels of four kinds of α-amylase synthesis genes was also lower than that of WT.These results suggest that reduce of germination rate of rice seeds in OsRhoGAP2-overexpressed rice may be related to the downregulation of α-amylase activity and GA synthesis genes.The plant height of WT and three OsRhoGAP2-overexpressed rice ines at mature stage were 92.12,86.5,86.4,and 89.22 cm,respectively which were significantly lower than that of WT.Further statistical analysis of internode and panicle length revealed that the panicle length of OsRhoGAP2-overexpressed rice transgenic rice was not changed compared to that of WT,howver,the length of the first,second and third internodes was shorter than that of WT.The paraffin section statistics showed that the length of internodal cells in overexpressed rice is less than that of WT,and the number of internodal cells per unit area is greater than that of WT,both of them reached a extremely significant difference.It suggestes that the dwarfing of OsRhoGAP2 overexpressed rice might result from the reduced internode cell elongation.In order to identify the function of the OsRhoGAP2 by the loss-of-function strategy,this study constructed the OsRhoGAP2 gene-edited rice using CRISPR / Cas9 technology.Two homozygous mutants(lines 1 and 16)were obtained after screening and identification.Sequence alignment showed that they deleted the bases "TC" and "T" at the target site,respectively,and they are expected to produce truncated proteins containing 149 and 60 amino acids,respectively.Both of them had not typical RhoGAP domain.The mutant OsRhoGAP2 in strain 1 had Only 3 amino acids matching the CRIB motif of WT,while the CRIB motif in line 16 was completely deleted.Scanning electron microscopy results showed that both the leaf and glume surface of line 1 lacked epidermal hair,while line 16 had similar phenotypes to WT.The photosynthesis index of flag leaf was tested that the net photosynthetic rate and the stomatal conductance of the two mutants were significantly higher than those of WT.This indicates that the deletion of OsRhoGAP2 gene improved the photosynthesis level of rice,and the light-leaf phenotype in line 1 suggested that the knockout of this gene may also affect the development of epidermal hair.Overall,OsRhoGAP2 overexpressed rrice was used as experimental material to identify the biological function of OsRhoGAP2 gene during plant growth and development in this studay.The functions of this gene involves cell growth,seed germination,photosynthesis and leaf morphology might relateto GA signaling pathway.On the other hand,the OsRhoGAP2 gene knockout rice was constructed in this paper.It was found that OsRhoGAP2 gene knockout not only affected the photosynthesis of rice leaves,but also affected the development of epidermal hair.This laid the foundation for the subsequent exploration of the regulatory network of the OsRhoGAP2 gene,and also provided new insights into the function of the RhoGAP gene in plants.