Abstract:
Mitogen-activated protein kinase (MAPK) pathways are highly conserved signaling cascades in eukaryotes and play a key role in plant growth, development, reproduction and stress responses. In Arabidopsis, its role in cell differentiation has drawn close attention in the past two decades, with a body of research focusing on the ERECTA-YODA (ER-YDA) pathway in embryogenesis and stomata development. ER-YDA signaling essentially comprises three transduction steps: 1) extracellular ligands are recognized by the upstream ER family (ERf) receptor complex which contains two receptor-like cytoplasmic kinases BRASSINOSTEROID-SIGNALING KINASE 1/2 (BSK1/2) at the membrane; 2) signals from the ERf receptor complex through BSK1/2 activate the YODAMKK4/5-MPK3/6 phosphorylation cascade; 3) the MAPK phosphorylation activates the downstream transcription factor WRKY2 in the embryo or represses the heterodimer complex of two basic helix-loop-helix (bHLH) transcription factors SPEECHLESS (SPCH) and SCREAM (SCRM) in stomata. Previous publications revealed details of the ER-YDA pathway in these two developmental contexts and indicated conserved upstream receptor components and distinct downstream targets. However, former genetic analysis is yet to demonstrate any functional difference in BSK1 and BSK2 as vital upstream signal relays connecting the ER receptor complex and the YDA cascade, and little is known about the conserved mechanism in the regulation of downstream regulators during cell proliferation.
This project aims to fill the gaps mentioned above and to gain a better understanding of the ER-YDA pathway from a comparative point of view. To accomplish this, we generated the new loss-of-function mutants of BSK1 and BSK2 using CRISPR/Cas9 and conducted detailed phenotypic analyses which showed that BSK1 contributes mainly to the cell polarity in the embryo and stomata, while BSK2 plays a prominent role in the growth of rosette leaves. Confocal microscopy and co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) assays in YPet-tagged BSK1/2 transgenic lines further identified the differences in expression patterns, new phosphorylation sites and interacting proteins for BSK1 and BSK2 in vivo. These functional differences between BSK1 and BSK2 suggest an evolutionary divergence and support us to propose a collaborative working model. Furthermore, we disclose that SCREAM (SCRM), a well-known target of the ER-YDA pathway in stomata, functions as a regulator of embryo development. However, the regulation of SCRM by the ER-YDA pathway switches from phosphorylation-dependent degradation in stomata to phosphorylation-dependent activation in the embryo. Further studies verified that SCRM works in synergy with WRKY2 to upregulate WUSCHEL RELATED HOMEOBOX 8 (WOX8) in the apical-basal axis formation during embryogenesis. Finally, we conducted a phenotypic screen and found bHLH35, a paralogue of SCRM subfamily, is a potential interacting partner of SCRM in early embryos.
This work provides a comparative insight into the ER-YDA pathway, enabling us to have a more comprehensive understanding of the MAPK cascade. Two aspects of this thesis, the overlapping but differential functions of BSK1/2 and the altered regulation of the conserved target SCRM, together exhibit the conservation and diversity of the ER-YDA pathway, further expanding the knowledge on how the MAPK pathway delicately controls the signal for cell differentiation and proliferation.
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