Study On The Regulation Mechanism Of Cytokinin Response Regulator OsRR2 In Rice Root Development

Root is one of the most important nutritional organs in plant,In addition to genes,root development is closely related to the plant endogenous hormones.Auxin and cytokinin play important roles in these hormones.OsRR2?Oryza sativa response regulator 2?belongs to type-A response regulators,which has a function as negative regulators of cytokinin signal.The results indicated that OsRR2 was directly regulated by WUSCHEL-Related homeobox11?WOX11?and AP2/ERF transcription factors ERF3 in different stages of crown root development,and affected crown root development.But the regulation mechanism of OsRR2 in crown root development remains unclear.Based on existing research,our study explored regulation mechanism of OsRR1 and OsRR2 in crown root development,our work will further the advance regulation mechanism of WOX11 and cytokinin during crown root formation in rice and provide foundation for understanding root organogenesis and for crop improvement.The main results are as follows:1.Protein sequence alignment of OsRRs showed OsRR1 and OsRR2 have very high sequence similarity?55%?.Expression patterns analysis revealed that OsRR2 had the highest expression level among the OsRRs genes,and is highly expressed in stem,flag leaf,leaf blade,leaf sheath and root tip maturation zone.The expression level of OsRR1 was lower than OsRR2,but the expression level is high in root,flag leaf,leaf blade,leaf sheath and root apical meristem.2.OsRRs may response to exogenous phytohormone auxin and cytokinin in different patterns.The expression level of OsRR1,OsRR5,OsRR7,OsRR9 and OsRR14 can be induced by 6-BA and IAA treatment,but the expression of OsRR2,OsRR3,OsRR4,OsRR6,OsRR10 and OsRR13 were only induced by 6-BA treatment.3.In order to study the function of OsRR1 and OsRR2 in rice crown root development,we created RNA interference lines of OsRR2,and got 24 positive transgenic plants at T₀ generation;we also created OsRR2 over-expression lines and got 26 positive plants at T₀ generation,of which 61.5%are effective.4.7-day-old seedlings of OsRR2 RNAi lines have fewer number of crown roots compared with wild type,while OsRR2 overexpression lines have increased number of crown roots,indicating that OsRR2 may function in rice crown root development.But OsRR1 RNAi lines showed no phenotype.5.In vitro enzymatic activity in rice protoplasts showed that OsRR2 had transcription activation activity,indicating OsRR2 may regulate the expression of downstream genes by activating their transcription.6.Analysis of the RNA-seq data revealed 378 up-regulated genes and 219down-regulated genes in OsRR2 RNAi lines.Expression profoile analysis showed that the down-regulated genes in OsRR2 RNAi lines were specifically expressed in roots.Gene ontology analysis showed the differentially expressed genes were enriched in transferase activity,metabolic process,response to stress,catalytic activity,response to abiotic stimulus and response to endogenous stimulus.These results indicate that OsRR2 might be involved in crown root development by regulating these genes.