Origin Of AMI7 In Amidase Family Of Plants And Functional In Vascular Bundle Development

Vascular bundles are responsible for the translocation of water and nutrients,and provide mechanical support of the whole plant body,which is of great significance for the adaptive evolution of terrestrial plants.Furthermore,vascular bundles connect the source and sink,and also play a key role in the transport of photosynthetic products and crop yield.In this study,we identified an amide enzyme family gene OsAMI7,which was originated in green plants through horizontal gene transfer,and is specifically expressed in the vascular tissue.The function of OsAMI7 gene in vascular development and rice yield was further explored through the T-DNA insertion mutant characterization,construction of transgenic materials,phenotypic observation,and physiological mechanism analysis.Moreover,the favorable haplotype of OsAMI7 was identified through natural population genetic analysis,which revealed the potential application value of this gene in rice breeding.The main results are as follows:（1）Through phylogenetic analysis of plant amidase genes,a special subfamily AMI7 was identified,and the homologous genes of this subfamily in eukaryotes are only present in green plants,and the homologous genes of green plants and CFB bacteria form a monophyly with a high bootstrap support.Sequence consistency and conservative motif analysis showed that the green plant AMI7 protein had high sequence consistency with the CFB bacterial AMI7 protein,and they shared a specific conserved motif,suggesting that the ancestors of the green plant acquired the AMI7 gene from CFB bacteria through a single horizontal gene transfer event.Adaptive evolutionary analysis found that AMI7 gene experienced positive selection for a short period of time in green plants after acquisition from CFB bacteria through horizontal gene transfer.Purification selection dominated over a longer period of time,which facilitated the AMI7 gene in maintaining its optimal function.（2）A T-DNA insertion mutant of the rice OsAMI7 gene was acquired and identified,and the T-DNA was inserted into the first intron of OsAMI7,leading to a significant decrease in the transcription level of the OsAMI7 gene.Phenotypic observation at the mature stage revealed that the plant height,stem length,flag leaf length and panicle length of osami7 plants were significantly reduced compared with the wild type,and the number of effective tillers,panicle grains and yields were significantly decreased.Co-segregation analysis revealed that the above phenotype of the mutant was co-segregated with the T-DNA insertion of the OsAMI7 gene.The complementary line showed no significant difference with wild type.These results suggest that OsAMI7 had a significant effect on the growth and development and grain yield of rice.（3）The expression pattern of OsAM17 gene was analyzed by qRT-PCR,and the results showed that OsAMI7 gene was expressed in root,leaves,stem,sheath and panicle of rice,while the expression levels in leaves,sheath and stem were higher than that of root and panicle.Through construction of the POsAMI7:GUS transgenic line and GUS activity assay,it was revealed that OsAMI7 was specifically expressed in the phloem of vascular bundles of mature leaves,stem and sheath,indicating that OsAMI7 may have important functions in vascular bundle development.In addition,the subcellular localization results showed that the OsAMI7GFP fusion protein was localized in the cytoplasm.（4）Vascular bundle morphology was examined by paraffin section,and the results showed that the number of large and small vascular bundles and the area of large vascular bundles in the internodes and leaves of osami7 were significantly reduced compared with that of wild type,while there was no significant difference between the complementary strains and the wild type.These results indicate that OsAMI7 may affect rice growth and grain yield through promoting vascular development.（5）The overexpression and CRISPR/Cas9 knock-out lines of OsAMI7 were constructed,and their phenotype was observed.Results revealed that the plant height,stem length and flag leaf length were significantly decreased in OsAMI7-Cas9 lines,while significantly increased in OsAMI7-OE lines,compared with wild type.The effective tiller number,panicle length,grain number per panicle,and grain yield per plant of OsAMI7-Cas9 were all significantly lower than those of the wild type,while the OsAMI7-OE lines increased them.Furthermore,the number and area of large vascular bundles of internodes and leaves were significantly increased in OsAMI7-OE lines,while reduced in OsAMI7-Cas9 lines.The above results confirmed that OsAMI7 possess the function in promoting the development of vascular bundles,and positively regulating the grain number per panicle and grain yield per plant.（6）The expression levels of vascular tissue development-associated genes in OsAMI7 transgenic lines were detected by qRT-PCR.Two essential transcriptional factors in the maintenance of meristem activity,WOX4 and FON2,were significantly upregulated in OsAMI7-OE lines,while decreased in OsAIM7-Cas9 lines and T-DNA insertion mutants.Cytokinin content in young panicles was determined by high performance liquid chromatography-mass spectrometer（HPLC-MS/MS）,and the results showed that endogenous cytokinin levels in the OsAMI7-OE line were significantly higher than those in the wild type,while the OsAMI7-Cas9 line and T-DNA insertion mutants were significantly reduced.These results indicate that OsAMI7 promotes the development of vascular tissue by regulating the biosynthesis of cytokinin and maintaining the activity of meristem.（7）By analyzing the haplotype of OsAMI7 gene and yield traits in natural populations,a favorable haplotype of OsAMI7 gene was identified,which significantly improved the panicle number and yield per plant of rice.The gene expression analysis showed that the expression level of OsAMI7 in the favorable haplotype was significantly higher than the other haplotypes.These results suggest that natural variations of OsAMI7 regulated the grain number per panicle and yield per plant through affecting its expression level.Moreover,the favorable haplotype of OsAMI7 have important application values in rice breeding.