The Molecular Mechanism Of Cytokinin And Auxin Regulating Poplar Meristem Activities

Shoot regeneration is the prerequisite of plant genetic transformation in most plant species. The activity of vascular cambium leads to the secondary growth and wood formation. The phytohormone cytokinin plays important roles in regulating both shoot regeneration and cambium activity. However, the mechanisms underlying the regulation, especially in woody species, remain elusive.In the present study, we established a stable system for poplar genetic transformation. On the basis, we analyzed the distribution pattern of auxin response signals during shoot regeneration using DR5::GUS reporter line. The results showed that DR5::GUS signals distributed evenly at the edge of the callus at the beginning of shoot induction, and then progressively restricted into specific regions. At the initiation stage of shoot apical meristem (SAM), GUS signals were visualized at the apical and peripheral regions of the SAM. After the formation of the SAM, auxin response signals were detected at the LI cell layer. Further, the patterns of cytokinin response in shoot regeneration were examined. Sequnence analysis revealed that RR12 and RR13 of popular are homologs of the B-type cytokinin response regulater ARR1 of Arabidopsis. The results of in situ hybridization showed that the transcripts of RR12 and RR13 accumulated evenly at the edge of the callus at the beginning of shoot induction, and then restricted into specific regions. After the formation of the SAM, RR12 and RR13 were expressed throught the SAM except for the LI cell layer. These distribution patterns were in accordance with that of ARR1 in Arabidopsis thaliana during shoot regeneration process. These results indicated the similar roles of auxin and cytokinin in regulating shoot regeneration between poplar and Arabidopsis.Knock-down of RR12 and RR13 using artificial microRNA strategy resulted in the decrease of the frequency of shoot regeneration and the number of regenerated shoots on each explant. The results indicate that RR12 and RR13 are involved in the regulation of shoot regeneration. Furthermore, RR12 and RR13 were found to be preferentially expressed in the cambium zone. Decreased expression levels of RR12 and RR13 in amiR-RR12,13 lines led to the reduction of the growth rate and the diameter of the 20th internode. Cytological results showed that the formation of xylem and phloem were inhibited in amiR-RR12,13 lines, implying a reduced secondary growth in the stem. The relative transcriptional levels of RR12 and RR13 decreased significantly in the amiR-RR12,13 stem compared to those of wild-type plant, while those of other B-type RRs did not exhibit obvious changes, suggesting that the phenotype of the amiR-RR12,13 stem were resulted from decreased expression levels of RR12 and RR13.Our results suggest that cytokinin regulates shoot regeneration and cambium activity through RR12 and RR13, which act as positive regulators in these processes.