Transcriptional mechanisms that underlie the dose-dependent regulation of gene expression in animal development have been studied extensively. However, the mechanisms of dose-dependent transcriptional regulation in plant development have not been understood. In Arabidopsis shoot apical meristems, WUSCHEL (WUS), a stem cell-promoting transcription factor, accumulates at a higher level in the rib meristem and at a lower level in the central zone where it activates its own negative regulator, CLAVATA3 (CLV3). How WUS regulates CLV3 levels has not been understood. Here we show that WUS binds a group of cis-elements, cis-regulatory module, in the CLV3-regulatory region, with different affinities and conformations, consisting of monomers at lower concentration and as dimers at a higher level. By deleting cis elements, manipulating the WUSbinding affinity and the homodimerization threshold of cis elements, and manipulating WUS levels, we show that the same cis elements mediate both the activation and repression of CLV3 at lower and higher WUS levels, respectively. The concentrationdependent transcriptional discrimination provides a mechanistic framework to explain the regulation of CLV3 levels that is critical for stem cell homeostasis.WUSCHEL | cis-regulatory module | CLAVATA3 | shoot apical meristem | gene regulation
The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana. WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not wellunderstood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factorassociated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.CLAVATA3 | feedback | homodimerization | meristem | nuclear-cytoplasmic partitioning
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.
One of the central problems in animal and plant developmental biology is deciphering how chemical and mechanical signals interact within a tissue to produce organs of defined size, shape and function. Cell walls in plants impose a unique constraint on cell expansion since cells are under turgor pressure and do not move relative to one another. Cell wall extensibility and constantly changing distribution of stress on the wall are mechanical properties that vary between individual cells and contribute to rates of expansion and orientation of cell division. How exactly cell wall mechanical properties influence cell behavior is still largely unknown. To address this problem, a novel, subcellular element computational model of growth of stem cells within the multilayered shoot apical meristem (SAM) of Arabidopsis thaliana is developed and calibrated using experimental data. Novel features of the model include separate, detailed descriptions of cell wall extensibility and mechanical stiffness, deformation of the middle lamella and increase in cytoplasmic pressure generating internal turgor pressure. The model is used to test novel hypothesized mechanisms of formation of the shape and structure of the growing, multilayered SAM based on WUS concentration of individual cells controlling cell growth rates and layer dependent anisotropic mechanical properties of subcellular components of individual cells determining anisotropic cell expansion directions. Model simulations also provide a detailed prediction of distribution of stresses in the growing tissue which can be tested in future experiments.
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