Transcriptional Regulation Of Initiation And Developmental Process Of Lamina Joints In Rice(Oryza Sativa)

The LJs is a mechanical structure between blade and sheath,including LJ,auricle and ligule,in rice.The presence or absence and the development degree of LJs directly affect leaf angle,and thus affect the plant architecture and yield.However,the initiation mechanism and the regulation of the developmental process of LJs are not clear.In this paper,we explored the mechanism of LJ initiation through the identification and analysis of genes and mutants associated with LJ initiation.Simultaneously,we explored the cytological structure and the expression dynamics of differentially expressed genes during the LJ development as well;and we identified the specific genes involved in the development of LJs,and verified the functions of some key transcription factors.The results are as follows:First,OsLG2 and OsLG2L were identified in rice by sequences alignment with ZmLG2.Observing the phenotypes of mutants through stereoscope and scanning electron microscope,we comfirmed the functions of OsLG1 and OsLG2/OsLG2L in the process of LJ initiation.The results of fluorescence quantitative PCR and GUS staining suggested that OsLG1,OsLG2,OsLG2L specifically expressed in LJs,compared with blades;and the expression of OsLG2/OsLG2L preceded that of OsLG1.The results of subcellular localization and BIFC revealed that OsLG1,OsLG2 and OsLG2L can form homodimers in the nucleus,and OsLG2 and OsLG2L can form heterodimers,OsLG1 and OsLG2 can interact.The analysis of transcription factor binding sites on promoters of OsLG1,OsLG2 and OsLG2L revealed the binding sites of many HD-ZIP,KANADI and RAMOS A1 transcription factors.The analysis of RNA-seq data provides a specific set of genes participating in LJ initiation,especially the expression danamics of transcription factor families and genes involved in organ initiation and boundary formaiton.We proposed the mechanism of transcription regulation of LJ initiation through LJ genes and downstream genes of OsLG1 and OsLG2/OsLG2L.The cytological structure and mechanical strength of LJs determine the leaf angle.According to the morphological features and cytological structures of the first complete leaf of rice,we identified five successive and complete development stages during the development of LJ,and conducted RNA-seq and small RNA(sRNA)-seq of LJ and connected leaves.For the time-course profiles of LJs,we obtained 23 mRNAs and 20 miRNAs expression patterns with the significant stage specificity and found key biological processes responsible for the cytological changes.In view of the differences in tissue development between LJs and blades,we identified specific genes and miRNAs sets,which could be used as marker genes for LJs and blades.Through the prediction of overrepresented TF binding sites(TFBS)on the promotors of specific genes and miRNAs,we identified 60 key TFs involved in a certain stage or biological process of LJ development.We constructed the mutants of some key TFs using CRISPR/CAS9 technology;through phenotypic observation,paraffin section,freehand section,qrt-pcr and chip-qpcr,we confirmed that these key TFs are involved in LJ development,laying foundation for establishment of the network for LJ development and leaf angle formation.