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WUSCHEL Mediated Bifunctional Transcriptional Regulation of CLAVATA3 Levels and Spatial Pattern in Arabidopsis thaliana

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The balance between self-renewing stem cell divisions and differentiation of stem cell progeny in Shoot Apical Meristems (SAMs) is crucial for the development of all the above-ground organs. This is regulated by a self-sustaining feedback loop between WUSCHEL(WUS) and CLAVATA (CLV) genes. WUS, a homeodomain transcription factor expressed in the rib-meristem (RM), migrates into overlying stem cell domain, also referred to as the central zone (CZ). In the CZ, WUS represses a set of differentiation-promoting transcription factors to prevent premature differentiation of stem cell progeny and also activates its own negative regulator CLV3. CLV3, a secreted peptide, negatively regulates WUS expression through CLV1 and related transmembrane receptors. This process is critical for the homeostasis of the stem cell population. The transcriptional mechanisms underlying the CLV3 activation and repression of differentiation factors in stem cells are not known. It is also not known how WUS protein levels are regulated. The structure-function analysis of the WUS protein has led to the identification of the last 63 amino acid region that is both sufficient and necessary to maintain higher WUS in the RM and lower WUS in the CZ. This region contains information for subcellular partitioning, stability and transcriptional activity of WUS. Analysis of the CLV3 promoter uncovered 5 closely spaced cis-elements that bind WUS, referred to as the cis-regulatory module (CRM). The in vivo cis element deletion analysis shows that the collective activity of all 5 cis elements is required for activation of CLV3 in the CZ and repression in the RM. Biochemical analysis of the WUS-binding behavior to CLV3 elements uncovered a concentration dependent switch from monomeric to dimeric/higher molecular weight complex. Taken together, it led to a model where WUS activates and represses CLV3 at lower and higher WUS, respectively. The manipulation of WUS levels and the affinity of cis elements largely agreed with the concentration dependent switch in CLV3 regulation. While determining the contribution of the number of cis elements and affinity of cis elements to CLV3 regulation, we found that the expression data from mutant CLV3 promoters could only be explained by a model based on cooperativity between elements. These new findings show that the cis-element grammar (number, affinity and possibly spacing) may be crucial in interpreting local differences in WUS levels in regulating CLV3 levels and spatial pattern.

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