OsbHlLH98 Regulates Leaf Angle Through Transcription Repression Of OsBUL1

Plant architecture is an important factor in determining rice yield,and leaf angle is one of the important agronomic traits.Since the leaves of erect-leaf rice varieties do not shade each other,they are more advantageous in high-density planting and can increase the unit area yield.In rice,researches on the characteristics of leaf angle have been ongoing,and many bHLH family transcription factors related to the occurrence and regulation of leaf angle have been reported.In this study,we identified a novel bHLH transcription factor that involved in leaf angle regulation.We constructed osbhlh98 mutants using CRISPR-Cas9 technology and developed OsbHLH98-overexpression lines.Combining cytology,genetics,molecular biology and other methods,the function and molecular mechanism of OsbHLH98 in regulating rice leaf angle were studied in detail,and the following main research conclusions were obtained:1.Compared with the wild type,the osbhlh98 mutants showed a larger leaf angle,a looser plant architecture at the mature stage,and a larger flag leaf angle.However,there was no significant difference in the grain traits including grain length,grain width,and thousand-seed weight between the mutants and wild type.Sections of the lamina joint region showed that the main reason for the increased leaf angle of the osbhlh98 mutant was the increase in the size and number of parenchyma cells at the adaxial side of the lamina joint,indicating that OsbHLH98 mainly inhibits the elongation and proliferation of parenchyma cells at the adaxial side of the lamina joint and regulate leaf angle;OsbHLH98 was specifically expressed in adaxial parenchyma cells of midvein,which was consistent with its function of inhibiting the elongation and proliferation of adaxial parenchyma cells of lamina joint;2.The BR sensitivity assay showed that the osbhlh98 mutants were very sensitive to exogenous BR treatment.On the contrary,the OsbHLH98 overexpression lines were not sensitive to exogenous BR treatment,indicating that OsbHLH98 plays negative role on leaf angle enlargement by BR treatment.3.ChIP-seq,CUT & RUN experiments and luciferase reporter gene experiments proved that OsbHLH98 can directly binds to E-Box and G-Box in the promoter region of Os BUL1,and inhibit the expression of Os BUL1;RT-q PCR experiments also proved that the induction of Os BUL1 expression by BR treatment increased in osbhlh98 mutants,compared with that of wild type,while in the OsbHLH98 overexpression lines,the induction of Os BUL1 was lower than that of the wild type.We introduced the osbul1 mutation into the osbhlh98 mutant,and the resulting double mutant showed reduced leaf angle,suggesting that mutation of Os BUL1 restores the increased leaf angle caused by the osbhlh98 mutant,which also indicates that Os BUL1 is the target gene of OsbHLH98.4.Sequence evolution analysis showed that the OsbHLH98 gene locus was selective in the domestication breeding of japonica rice and indica rice,indicating that OsbHLH98 may be selected as an agronomic trait in domestication.The transgenic lines overexpressing OsbHLH98 have a more upright leaf angle phenotype without affecting tiller numbers,grain size,seed setting rate and other agronomic traits.This indicates that by optimizing the expression level of OsbHLH98,the angle of rice leaves can be adjusted to fit high-density planting.Therefore,OsbHLH98 can be used as a selection target in breeding to help increase yield through high-density planting.