STM, As An Interacting Cofactor Of WUS, Regulates Transcription Of CLV3 For The Control Of Stem Cell Function In Arabidopsis

Plant stem cells in the shoot apical meristem (SAM) are necessary for postembryonic development of above-ground tissues. WUSCHEL (WUS), a homeodomain transcription factor expressed in the organizing center of the Arabidopsis SAM, is a key regulatory factor controlling SAM stem cell population. Although some genes have important functions in downstream of WUS, they cannot fully explain the molecular links underlying WUS function in stem cell niches.In this study, we have identified some WUS-interacting cofactors, including SHOOT MERISTEMLESS (STM). We first confirmed that STM can directly interact with WUS. Then we found that WUS and STM can coordinately bind to the promoter of CLV3 for the activation of its expression, and therefore regulate stem cell activity of SAM. The main results and conclusions are as follows:(1) STM physically interact with WUSIn an attempt to bring insights into the cofactors of WUS, we performed mass spectrometry experiments by taking advantage of pWUS::WUS-3GFP transgenic plants. Here, we demonstrate that another homeobox protein, SHOOT MERISTEMLESS (STM), acts as a direct interacting protein with WUS in SAM regulation. Both WUS and STM are required for the formation and maintenance of stem cell populations. To verify the reliability of the interaction, in vitro pull-down assays and yeast two-hybrid were employed, and both genes are involved in SAM regulation by the direct interaction of both proteins.(2) Expression patterns of STM and WUS genesConsistently with physical and genetic interactions between STM and WUS, visualization of STM-VENUS and WUS-GFP fluorescent protein reporters revealed that STM and WUS expression overlapped in the SAM of planta. WUS is first detected in the inner cells at the apical region of the 16-cell stage embryos, but STM is not expressed at this stage. At transition-stage and heart-stage embryo, the expression regions of STM and WUS are overlapped. Whereas in vegetative and inflorescence meristems of seedlings, STM expression is markedly expanded in the whole SAM, while WUS is still expressed in the organize centre of SAM.(3) STM protein binds to CLV3 regulatory regions to activate its transcriptionWe determined the expression levels of CLV3 in stm mutants and 35S::STM-GR transgenics. The expression of CLV3 gene is enhanced in 35S::STM-GR transgenics treated with DEX, whereas its expression is reduced in stm mutants.Because WUS can directly activate CLV3 expression through binding to its promoter, we determine whether STM can bind to the promoter of CLV3 as WUS. We performed chromatin immunoprecipitation (ChIP) coupled to qPCR analysis (ChlP-qPCR). The results showed that STM directly binds to the promoter regions of CLV3 in vivo. We next performed electrophoretic mobility shift assays (EMSA) to confirm the presence of the STM-binding elements in the upstream regions of CLV3 gene.(4) WUS and STM have synergistic effects on regulating CLV3 expressionCLV3 coding region driven by its promoter can fully rescued the phenotypes of clv3 mutant. However, CLV3 coding region driven by the CLV3 promoter with mutant binding sites of STM or WUS can partially rescued the clv3 mutant phenotypes. Then mutations of both STM and WUS binding sites in the promoter can hardly rescued the clv3 mutant phenotypes. Moreover, downregulation of STM expression can reduce the binding ability of WUS on the promoter of CLV3 gene, and vice versa.(5) The feedback loop between CLV3 and STMqRT-PCR analysis showed that the expression of STM gene in clv3 mutants is much higher than that in wild type. A series of in situ RNA hybridization experiments indicate that the expression of STM gene is enhanced in clv3 mutants. These findings suggest a model in which the stem cell population is partially maintained by a regulatory feedback loop between CLV3 and STM.In a summary, STM and WUS proteins regulate the Arabidopsis SAM development through interaction. Furthermore, STM can activate CLV3 transcription through binding to its promoter. Therefore, STM and WUS share common targets CLV3 and produces synergistic effects on CLV3 transcription in promoting stem cell proliferation. These results provide a new framework for the control of stem cell production during SAM development.