Research On The Function Of GIR1 And GIR2 Genes In Arabidopsis Leaf Development

Leaves are the main place for photosynthesis in plants and have an important impact on the growth and development of plants.During leaf development,correct establishment of adaxial-abaxial polarity is the foundation in leaf formation and function-driven.Arabidopsis has a short growth cycle and is an important model plant for studying gene function.GIR1 and GIR2 genes are involved in the development of root hairs,and GIR1 and GIR2 as GL2 adaptors control root hair formation(Wu and Vitaly,2017).Interestingly,we found that gir1-1/gir2-1 mutant leaf was abnormal,but the function of GIR1 and GIR2 genes during leaf development was not clear.Therefore,it is of great significance to study the function of GIR1 and GIR2 genes during Arabidopsis leaf development.In this study,we performed phenotypic analysis of the gir1-1/gir2-1 double mutant,and phenotype annalysis of overexpression transgenic lines of non-mutated GIR1 gene.two phenotypic stable overexpression transgenic lines were obtained using site-directed mutagenesis: gir1 and gir2.We performed phenotypic analysis,leaf roling index analysis,expression analysis of related gene of roling leaf,q RT-PCR analysis,and leaf adaxial–abaxial polarity analysis on gir1 and gir2.Meanwhile,we analysed expression pattern of GIR1 and GIR2 genes,RNAi plants of GIR2 gene,and subcellular localization and interaction protein of GIR1 and GIR2 proteins.The study laid a foundation for further exploring the functional and involved regulatory networks of GIR1 and GIR2 genes during leaf development.The main results are as follows.1 Phenotypic analysis of gir1-1/gir2-1 double mutantCompared to the wild type,one of the first true leaves of the gir1-1 /gir2-1 mutant plants showed abnormal,and a new leave grown on the cracked leaves.The result showed that GIR1 and GIR2 genes are likely to be involved in the development of Arabidopsis leaves.2 Phenotypic observation of overexpression transgenic plants of GIR1 geneTo further investigate how the GIR1 gene affects leaf development,we constructed overexpression transgenic lines of GIR1 gene.The result showed that compared with the wild type,the overexpression transgenic cotyledons showed abnormal,but the phenotype of them was less consistent.Some of them had no cotyledons and directly grew true leaves,some of them one of the two cotyledons had deteriorated,some of them one of the cotyledon had deteriorated and the other cotyledon had a cup shape,and some of them showed a cotyledon fusion.3 Phenotypic and q RT-PCR analysis of gir1 and gir2In order to obtain phenotypic stable overexpresion transgenic lines,we performed site-directed mutagenesis of GIR1 and GIR2 genes,respectively,and over-expressed them after mutation.The results showed that the phenotype of overexpression transgenic lines(gir1)with the sixth Lys locus of the GIR1 gene mutated to Arg and overexpression transgenic lines(gir2)with the mutated second Lys site of the GIR2 gene were relatively stable.Compared with the wild type,the gir1 plant was smaller,the cotyledons were fused,and the rosette leaves were asymmetric leaves that curling downwards,accompanied by the formation of crystals,and curved stems.Rosette leaves of some transgenic plants appeared as needle-shaped leaves.The leaf roling index results showed that compared with the wild type,the transversal curvature index and the longitudinal curvature index of gir1 and gir2 increased significantly.The phenotype of gir2 is very similar to the phenotype of gir1.These results showed that these phenotypes are probably due to the overexpression or ectopic expression of GIR1 and GIR2 genes.Quantitative results showed that the overexpression transgenic plants with a low expression level still had a phenotype,suggesting that it was likely that a relatively stable phenotype was generated due to protein accumulation.4 q RT-PCR analysis of related genes of curling leafThese genes related to curled leaf in Arabidopsis were selected for quantitative analysis.The result showed that compared with the wild type,the expression of KANT1,KANT2,KANT6,and STM in the gir1 showed varying degrees of up-regulation.In the gir2,the expression levels of KANT1,KANT2,and STM showed varying degrees of up-regulation,and the expression of KANT6 was slightly down-regulated.The result showed that GIR1 and GIR2 genes may have positive regulatory effects on KANT1,KANT2 and STM during leaf development.5 Observation of adaxial and abaxial epithelial cells in leaves of gir1 and gir2Scanning electron microscope observations observed that compared with the wild type,the epidermal hair on the leaf surface of gir1 and gir2 was reduced,the adaxial epithelial cells were deformed,and the distance between the adaxial epidermal cells of the leaves became larger.The abaxial epithelial cells were deformed and the distance between the abaxial epidermal cells of the leaves became larger.The result showed that GIR1 and GIR2 genes may participate in leaf development by affecting the polarity of leaf epidermal cells.6 Histological analysis of gir1 mutantThe morphological characteristics of mesophyll cells were observed by tissue section.The result showed that the leaf curling downwards of gir1 mutant was larger than that of the wild type.The polarity of mesophyll cells changed,and the palisade tissue was specialized into sponge tissue.The xylem of main vein of gir1 mutant was more dispersed,and the xylem of microtubule tissue was surrounded by phloem.7 Analysis of expression pattern of GIR1 and GIR2 genesThe p GIRs::GUS and p GIR1::GFP results showed that GIR1 is mainly expressed in leaf primordium,epidermal hair and the root tip of lateral root,and GIR2 is expressed in cotyledon,leaf primordium,SAM,true leaf,epidermal hair,root tip and lateral root.These results showed that GIR1 and GIR2 genes are expressed in leaf primordium.8 Phenotypic and q RT-PCR analysis of RNAi transgenic plant of GIR2 geneCompared with the wild type,the leaf shape of stem leaf of the RNAi transgenic plants of GIR2 gene was changed.The GIR2 expression of the stem leaves of RNAi transgenic plants was detected by q RT-PCR.These results showed that the expression level of RNAi transgenic plant of GIR2 gene were significantly lower than that of wild type.The result showed that GIR2 gene may also be involved in stem leaf development9 Subcellular localization of GIR1 and GIR2 proteinsThe coding sequence of the wild-type GIR1 and GIR2 genes were used to construct the GFP-GIR1 and GFP-GIR2 fusion proteins,and then co-transformed in Arabidopsis protoplasts by the PEG-mediated transformation methods.The GFP-GIR2 fusion proteins detected GFP fluorescence signal in the nucleus,indicating that GIR1 and GIR2 proteins are localized in the nucleus.10 Interaction protein analysis between GIR1 and GIR2 proteinsIn order to further explore the regulatory network involved in GIR1 and GIR2 genes,we screened and verified the interactions between GIRs proteins and other proteins using sequence transformation and yeast two-hybrids.The results of the screening library showed that many of the proteins screened were related to the AS1 and AS2 genes.The results of yeast two-hybrid verification showed that AE7 protein could interact with both GIR1 and GIR2 proteins.