Molecular Mechanism Of OsNAC2 Regulating Early Root Length Development In Rice

The NAC family is one of the largest families of specific transcription factors in plant,which played an important role in plant development,biological and abiotic stress and hormone signaling.Previous study showed that the root length of OsNAC2 overexpression lines(ON11,ON7)are shorter than WT,but RNAi transgenic line(RNAi31)has longer root length compared with WT.In order to further elucidate the molecular mechanism of OsNAC2 regulation on the early development of rice root length,taking gene microarray,quantitative PCR,ChIP-seq,one yeast hybrid assays and ChIP-qPCR,to study OsNAC2 and its downstream genes how to regulate auxin and cytokinin metabolic signaling pathway,the contents of auxin and cytokinin,and the molecular mechanism of early root growth in rice.The main results were as follows:1.OsNAC2 is involved in the regulation of auxin and cytokinin metabolic pathways.The expression of auxin synthesis genes YUCCA family,the family of auxin response factor ARF and auxin transport carrier PIN were decreased in ON11,but indole acetic acid amination synthetase GH3 family was increased.While the cytokinin receptor gene OsHK and synthetic gene OsLOGL expression was increased in ON 11.The gene expression of cytokinin degradation enzyme OsCKX and response factor OsRR were decreased.2.OsNAC2 regulates the content of IAA and cytokinin in the roots by regulating the metabolism of auxin and cytokinin,and controls the length of early root of rice.The endogenous IAA content in ON 11 was decreased,but the endogenous cytokinin content in ON 11 was increased.In conclusion that OsNAC2 leading to early rice root length shortening may be the reason for the endogenous IAA content was reduced,the endogenous cytokinin content was increased.3.OsNAC2 regulates the synthesis,transport and degradation of auxins and cytokinins by regulating the promoter of OsGH3-6,OsGH3-8,OsARF25 and OsCKX4,and regulates the hormone levels in the early apical meristem of rice.4.Through the tilling technology obtained osgh3.6 and osckx4 mutants from Liu Lab at Key Laboratory of Plant Molecular Physiology.By combined with sequence alignment get homozygous mutants that osgh3.6-19/20 and osckx4-2/12.5.Through to the osgh3-6 mutant phenotype found that the root length was longer and the gene expression of related auxin metabolic pathway is match expectations.The root length of osckx4 mutant was shorter and the gene expression of cytokinins metabolism have also changed,it is consistent with expectation.In conclusion that OsNAC2 is through combined with auxin response factor OsGH3-6 and cytokinin degradation gene OsCKX4 promoter,which is mediated auxin and cytokinin signaling pathways to regulate the rice root length growth.6.According to the crown root phenotypes in ON11,WT and RNAi31,microarray and the expression of crown root development related genes,meanwhile OsNAC2 binds to OsCKX4 promoter,which is related to Oswoxll,crl-4 and crl-5 were significantly down-regulated and the number of crowns was significantly decreased.It was verified that OsNAC2 could involve in the development of rice crowns by binding the promoter of OsCKX4.7.The pathway of OsNAC2 regulats the length of early rice root was constructed.OsNAC2 can bind to the promoter of OsGH3-6,OsGH3-8,OsARF25 and OsCKX4,can inhibit the cytokinin degrading enzyme OsCKX4 and auxin response factor OsARF25 expression,promote indole acetic acid amination synthase OsGH3-6 and OsGH3-8 expression,which mediate the signaling pathways and synthetic metabolic pathway of auxin and cytokinin,and regulate the development of early rice root length.OsGH3-6/OsGH3-8 and auxin form a feedback loop,which can maintain the dynamic balance of auxin in rice roots.