Analysis Of The Cytokinins Response Regulator PtRR Gene Family In Populus

Cytokinin is one of the important hormones,which regulates plant growth and development through its signaling transduction.RR family members are the key signaling factors in cytokinin pathway,based on the studies in annual plants like Arabidopsis and rice.Similar to many important regulatory factors in woody plants,RR family in poplar genome has enlarged family members than that in Arabidopsis thus their functions in woody plants need further investigation.Poplar is a model for woody plants and systematic characterization of poplar PtRR members,their expression pattern and functions can help to uncover their roles in woody plant development,which may have potential applications in tree breeding.Through the investigation of Populus genome,we identified thirty-three PtRR protein coding sequences.Phylogenetic,gene and protein structure analysis showed that 33 PtRRs could be divided into three families,class A,class B and PtPRR.There were 9 members in class A and 13 in class B in poplar.According to the corresponding relationship with Arabidopsis thaliana,we renamed the poplar ARRs to reflect the evolutionary relationships to Arabidopsis,thus the functions of related members between different plants could be easily compared.The expansion of the poplar PtRR family,as positioned on the chromosomes,may mainly come from the duplication of chromosomes,with some members of Arabidopsis homologs lost.Tissue-specific expression by RNA-seq analysis showed obvious differences among the three families in Populus,and a number of PtRR members presented high expression in leaf and cambium respectively,which gave the hints for their functions in the growth and development in poplar.PtRRI from class A and PtRR10/12 d from class B with high expression specific in xylem and cambium were further studied.By cloning the upstream ca2200 bp promoters of PtRR ? and Pt RR10/12 d and fused to GUS,we analyzed the PtRR ? and PtRR10/12 d gene expression pattern through the transgenic plants,and also PtRR ? in the process of adventitious rooting.High expression of PtRR?and PtRR10/12 d was detected incambium and xylem regions,in accordance with that by RNA-seq,semi-quantitative PCR,this suggested that Pt RR ? and PtRR10/12 d gene might participate in the secondary growth of poplar and PtRR might play an important role in adventitious rooting.By fused with 35s::PtRR:: YFP,the two PtRR proteins were confirmed to be nuclear localized.The Pt RR ?transcription was induced by 6-BA,GA3 and IBA while 6-BA,GA3,ABA and IBA failed to change the abundance of PtRR10/12 d transcripts,suggests PtRR10/12 d gene could not be induced by hormones.The 35s:: Pt RR10/12 d construct was transformed into poplar clone 84k(Populus alba × P.glandulosa)in order to elevate PtRR10/12 d expression in poplar thus the PtRR10/12 d function in the development of poplars could be investigated.Over-expression of PtRR10/12 d inhibited the adventitious root regeneration but promoted the root development in the transgenic plants.This indicated that PtRR10/12 d played an important role in the regulation of the development of adventitious roots.We have systematically analyzed PtRR family in Populus and revealed that the PtRR exhibit diversified expression patterns in different tissues.In addition,dynamic changes of expression of PtRR10/12 d in the process of adventitious rooting and the possible mechanism involved in the regulation was preliminarily investigated.The results suggest that PtRR10/12 d play a significant role in regulation of adventitious rooting down cytokinin signaling.This study provides important information of PtRR family and should be useful for future research on their roles in growth and development in poplar.