Cytokinin stabilizes WUSCHEL by acting on the protein domains required for nuclear enrichment and transcription

Concentration-dependent transcriptional regulation and the spatial regulation of transcription factor levels are poorly studied in plant development. WUSCHEL, a stem cell-promoting homeodomain transcription factor, accumulates at a higher level in the rib meristem than in the overlying central zone, which harbors stem cells in the shoot apical meristems of Arabidopsis thaliana. The differential accumulation of WUSCHEL in adjacent cells is critical for the spatial regulation and levels of CLAVATA3, a negative regulator of WUSCHEL transcription. Earlier studies have revealed that DNA-dependent dimerization, subcellular partitioning and protein destabilization control WUSCHEL protein levels and spatial accumulation. Moreover, the destabilization of WUSCHEL may also depend on the protein concentration. However, the roles of extrinsic spatial cues in maintaining differential accumulation of WUS are not understood. Through transient manipulation of hormone levels, hormone response patterns and analysis of the receptor mutants, we show that cytokinin signaling in the rib meristem acts through the transcriptional regulatory domains, the acidic domain and the WUSCHEL-box, to stabilize the WUS protein. Furthermore, we show that the same WUSCHEL-box functions as a degron sequence in cytokinin deficient regions in the central zone, leading to the destabilization of WUSCHEL. The coupled functions of the WUSCHEL-box in nuclear retention as described earlier, together with cytokinin sensing, reinforce higher nuclear accumulation of WUSCHEL in the rib meristem. In contrast a sub-threshold level may expose the WUSCHEL-box to destabilizing signals in the central zone. Thus, the cytokinin signaling acts as an asymmetric spatial cue in stabilizing the WUSCHEL protein to lead to its differential accumulation in neighboring cells, which is critical for concentration-dependent spatial regulation of CLAVATA3 transcription and meristem maintenance. Furthermore, our work shows that cytokinin response is regulated independently of the WUSCHEL function which may provide robustness to the regulation of WUSCHEL concentration.

浓度依赖性转录调控与转录因子水平的空间调控机制，在植物发育过程中尚未得到充分研究。WUSCHEL，一种可促进干细胞的同源域转录因子（homeodomain transcription factor），在拟南芥（Arabidopsis thaliana）的茎尖分生组织（shoot apical meristem）中，其在肋状分生组织（rib meristem）中的积累水平高于上方的中央区（central zone）——该区域包含干细胞。WUSCHEL在相邻细胞中的差异积累，对于CLAVATA3的空间调控及其水平至关重要，而CLAVATA3是WUSCHEL转录的负调控因子。此前的研究表明，依赖DNA的二聚化、亚细胞分区以及蛋白质去稳定化过程，共同调控WUSCHEL蛋白的水平与空间积累模式。此外，WUSCHEL的去稳定化可能同样依赖于其自身的蛋白浓度。然而，外源性空间信号在维持WUSCHEL差异积累过程中的作用仍未阐明。本研究通过对激素水平、激素响应模式的瞬时操控，以及对受体突变体的分析，证实肋状分生组织中的细胞分裂素（cytokinin）信号通路可通过转录调控结构域、酸性结构域与WUSCHEL-box，实现WUSCHEL蛋白的稳定化。进一步研究发现，同一WUSCHEL-box结构域在中央区的细胞分裂素缺失区域中，可作为降解子序列（degron sequence）发挥作用，进而介导WUSCHEL蛋白的去稳定化。正如此前研究所报道的，WUSCHEL-box在核滞留过程中的协同功能，与细胞分裂素感知共同作用，强化了WUSCHEL在肋状分生组织中的核内高积累水平。与之相反，亚阈值浓度的WUSCHEL蛋白可能会使中央区中的WUSCHEL-box暴露于去稳定化信号之下。因此，细胞分裂素信号通路作为一种非对称性空间信号，通过稳定WUSCHEL蛋白使其在相邻细胞间产生差异积累，这一过程对于CLAVATA3转录的浓度依赖性空间调控以及分生组织维持均具有关键作用。此外，本研究发现细胞分裂素响应的调控并不依赖于WUSCHEL的功能，这一机制可为WUSCHEL浓度的调控提供鲁棒性。