Cytokinin Type-B ARRs Negatively Regulate Auxin Biosynthetic Genes YUCCAs Expression Involved In Formation Of Shoot Apical Meristem In Arabidopsis

Auxin and cytokinin play antagonistically important roles in many aspects of plant growth and development. The interaction between auxin and cytokinin is particularly important to control the formation and maintenance of meristems. Cytokinin and auxin reveal the regional distribution during shoot regeneration. The local biosynthesis of cytokinin in this regeneration system is required for the regional distribution of cytokinin and WUS expression. The pioneering work has shown that auxin suppresses the expression of STM to disturb the cytokinin biosynthesis. So far, the molecular mechanism of cytokinin regulating the regional distribution of auxin remains mostly unknown.In this study we used root explants of Arabidopsis to investigate cytokinin regulating the expression level of auxin biosynthesis-related enzyme gene. We aim to provide new datas for clarifying the molecular mechanism on cytokinin and auxin regulating shoot regeneration. Shoot regeneration system were used to study the interaction between auxin and cytokinin during shoot regeneration in order to clarify the relationship between cytokinin and auxin.We used the roots of type-B arr mutants to induce shoot regeneration. The results show that arr1,12, arr10,12, arr1,10double mutants and arr1,10,12triple mutant could not induce the regeneration of shoot. They could regenerate the analogy of root. But arrl, arr10and arr12single mutants could induce the regeneration of shoot. We propose that type-B ARR redundantly play pivotal roles in the regeneration of shoot.The development of zygote embryo of arrl,10,12and yuccal gain function mutation delayed. Moreover, the both seedlings were smaller than Col and the size of SAM for both seedlings was also smaller than the found in the Col seedlings. We analyzed the patterns of glucuronidase (GUS) staining of YUCCA4promoter and ARR10promoter in seedlings. The results show that YUCCA4slightly expressed in L1-L3layer of SAM. But ARR10mainly-expressed in organizing center (OC) and peripheral cell. This suggested that ARR10may inhibit the expression of YUCCA in OC.We further analyzed the patterns of glucuronidase (GUS) staining of YUCCA4promoter and ARR10promoter during the shoot regeneration These results show that YUCCA4predominantly expressed in epidermal cell. The regional expression of ARR10was observed under the epidermal cell. The results of qRT-PCR show that YUCCA genes (YUCCA1, YUCCA2, YUCCA4) up-regulated in arrl,12double mutant, comparing with the wild type.Above all, these results show that cytokinin regulation factor type-B ARRs negatively regulate the transcription of auxin biosynthesis-related enzyme gene YUCCAs in inducing the shoot regeneration. We analyzed the promoter sequence of the auxin biosynthesis-related enzyme gene YUCCA1, YUCCA2, YUCCA4and YUCCA6. Indeed, the promoter contains the key binding motif (G/A)GAT(T/C) for the ARR protein. So we speculated that YUCCAs may be the target genes for ARR protein. Then the results of ChIP suggested that ARR1can directly bind to the promoter of YUCCA6, and ARR10can directly bind to the promoter of YUCCA6as well as YUCCA4. ARR12can directly bind to the promoter of YUCCA4. So we argued that cytokinin regulation factor type-B ARRs may regulate the transcription of auxin biosynthesis-related enzyme genes YUCCA through direct binding with the promoter of YUCCA.In summary, when we transferred calli from auxin-rich callus-inducing medium (CIM) to cytokinin-rich shoot-inducing medium (SIM), type-B ARR combined with the promoter motif of YUCCAs as the result of down-regulating the expression level of YUCCAs to make auxin accumulate in specific region. Finally, the specific distribution pattern of cytokinin and auxin can induce shoot regeneration.