Cytokinin Type-B Response Regulators Regulate Auxin-related Genes During Shoot Induction In Arabidopsis

Cytokinin and auxin have been recognized as crucial signalling molecules controlling plant growth and development for a long time. Cytokinins play important roles in numerous aspects of plant development, including seed germination, de-etiolation, chloroplast differentiation, apical dominance, plant pathogen interactions, flower and fruit development, and leaf senescence. During the past years, more and more physiological, genetic and transcriptomic evidences have been accumulated to confirm the importance of cytokinin-auxin interactions during plant development, but how cytokinin regulates auxin to form their specific spatio-temporal distribution pattern during de novo shoot regeneration is still not understood.The Arabidopsis response regulators (ARRs) are divided into three classes: type A ARRs, type B ARRs and type C ARRs. Type B ARRs act as transcription factors to induce the transcription of cytokinin primary response genes, including type A ARRs. Whereas type B ARRs are positive regulators of the cytokinin response, type A ARRs are negative regulators of cytokinin signalling. Recent studies showed that ARR1 is a positive regulator in cytokinin signaling pathway. In this studiy, we examined the interaction between type B ARRs and auxin-related genes to uncover the role of cytokinin in the regulation of auxin spatio-temporal distribution pattern during de novo shoot regeneration.Type B ARRs mutants arr1,arr10,arr11,arr12,arr18 and arr1,10,11 triple mutant were used to study the role of type B ARRs during shoot regeneration in vitro. We found that all of the single mutant of type B ARRs can regenerate normal shoot. However, in arr1,10,11 triple mutant, the shoot regeneration was inhibited while root-like structure formed on the callus. qRT-PCR analysis indicated that, in arr1,10,11 triple mutant, expression levels of auxin relate genes, such as ARF4, ARF5, ARF12 and YUC1, YUC4, YUC6 were changed substantially. Therefore, we speculate that Type B ARRs directly interact with the promoters of the auxin-related genes. The indeed, type B ARR binding sites were enriched in the promoter of YUC2, YUC4, YUC6, ARF3, ARF5, ARF17, TAA1, PIN1. Furthermore, EMSA analysis indicated that ARR14 protein can directly bind to the promoters of ARF17, YUC4, YUC6 in vivo. In summary, our results showed that type B ARRs positively regulate the expression of some auxin related genes during in vitro shoot regeneration. This is likely the molecular mechanism underlying the spatio-temporal auxin distribution controlled by cytokinin during this process.