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Summary
Summary
The establishment and maintenance of cell polarity is required for asymmetric cell division (ACD) and indispensable for multi-cellular organisms to generate cellular diversity. Through ACD, a single mother cell can produce daughter cells with distinctive identities in developmental differentiation. Our research focuses on the mechanisms by which cell polarity is initiated and maintained in the stomatal lineage cells in Arabidopsis and the mechanisms by which differential cell fates are specified in plant ACD. The identification of the plant-specific protein BASL (Breaking of Asymmetry in the Stomatal Lineage) provided strong evidence that plant cells have the capability to polarize non-transmembrane proteins and utilize such polarized protein distribution to regulate many cellular events during stomatal asymmetric cell division (Fig. 1A).
By using BASL as an anchor for screens to isolate genetic and physical interactors, including the BRXL2 protein (Fig. 1B), and by using the features of the protein itself as a probe for cell’s ability to correctly establish polar cortical localization, our lab is in the process of building a model for plant cell polarization and its regulation during stomatal ACD. In this process, cell polarity orients cell division plane, specifies cell-division potential and asymmetric daughter cell fates.
Fig. 1A Dynamic localization (green) of the BASL protein during stomatal asymmetry cell division (ACD) that produces two distinct cell identities: guard cell and pavement cell, respectively.
Fig. 1B Dynamic localization (green) of YFP-labelled BRXL2, the partner of BASL in a young leaf, in which stomatal ACDs are actively occurring.
Project 1: Dynamic assembly of the polarity complex during stomatal ACD
Prior work showed that, before a stomatal ACD, the polarity complex employs POLAR to recruit the GSK3-like kinase BIN2 that releases the suppression of YODA on stomatal differentiation, so that stomatal ACD is promoted (Houbaert et al., 2018, Nature). Therefore, the stomatal polarity complex by scaffolding different signaling molecules could promote the division potential before an ACD and suppress the division potential after an ACD.
However, how the transition of these two seemingly opposing procedures can be achieved by the same polarity complex remained a major challenge towards understanding stomatal ACD. Here, by using immunoprecipitation combined with mass spectrometry (IP-MS), we identify a family of protein Ser/Thr BSL phosphatases, as BASL partners (Guo et al., 2021, Nature Plants). Genetic analysis places BSL upstream of the YDA MAP kinase cascade and downstream of the plasma membrane receptors. In addition, the founding member BSL1 colocalizes with BASL in a polarized manner at the cell periphery. Interestingly, the recruitment of the BSL phosphatases in the polarity module confers a negative role to BIN2 complex but a positive role to the YDA MAPK module (Figure 2). Thus, our study reveals a crucial function of the BSL phosphatases in bridging the two opposing protein functional modules to control the balance of cell-division potential and cell-fate determination in plant ACDs.
Figure 2. A working model for BSL phosphatases to function in stomatal ACD. The BSL proteins function as the spatiotemporal molecular switch enabling the coordination of cell division and cell-fate differentiation in stomatal ACD. BSL1 join the polarity complex in the ACD mother cells that are committed to cell division. Association of BSL1 with the polarity complex dislodges BIN2 from the cell membrane to prevent subsequent rounds of cell division and activates the YDA MAPK signaling cascade to promote cell-fate differentiation. BIN2, GSK3-like kinase; YODA, MAPK Kinase Kinase; SPCH, bHLH transcription factor; BASL, BRX, and POLAR are scaffold proteins.
Project 2: Spatial cell signaling in determining stomatal production
Figure 3. A working model: BSL phosphatases-based signaling dichotomy controls stomatal development in Arabidopsis. At the cell cortex close to the PM, BSL1 is a predominant regulator, together with the other three BSL phosphatases, activating the MAPKKK YODA to promote MAPK signaling. Activated MPK3/6 molecules phosphorylate the key stomatal fate transcription factors, SPCH and ICE1/SCRMs, for degradation, thereby suppressing stomatal production. In the nucleus, BSU1 plays a primary role, together with BSL2 and BSL3, deactivating MPK3/6, resulting in stabilized SPCH and ICE1/SCRMs, thereby promoting stomatal production.
Stomatal guard cells play a critical biological role by controlling gas exchange and water balance in plants. Located on the epidermal surface of leaves, guard cells regulate the opening and closing of stomatal pores, enabling CO₂ uptake necessary for photosynthesis while simultaneously managing water loss through transpiration. This dynamic function positions guard cells as central players in plant adaptation to fluctuating environmental conditions such as drought, heat, and varying CO₂ concentrations. Moreover, guard cell responses profoundly influence overall plant productivity, growth, and survival. Understanding the molecular and cellular mechanisms governing guard cell development and behavior is therefore crucial for improving agricultural resilience and addressing challenges related to climate change and global food security.
MAPK signaling modules play crucial roles in regulating numerous biological processes in all eukaryotic cells. How MAPK signaling specificity and strength are tightly controlled remains a major challenging question. In Arabidopsis stomatal development, the MAPKK Kinase YODA (YDA) functions at the cell periphery to inhibit stomatal production by activating MAPK 3 and 6 (MPK3/6) that directly phosphorylate stomatal fate-determining transcription factors for degradation in the nucleus. Recently, we demonstrated that BSL1, one of the four BSL protein phosphatases, localizes to the cell cortex to activate YDA, elevating MPK3/6 activity to suppress stomatal formation. In this study, we showed that at the plasma membrane, all four members of BSL proteins contribute to the YDA activation. However, in the nucleus, specific BSL members (BSL2, BSL3, and BSU1) directly deactivate MPK6 to counteract the linear MAPK pathway, thereby promoting stomatal formation (Figure 3). Thus, the pivotal MAPK signaling in stomatal fate determination is spatially modulated by a signaling dichotomy of the BSL protein phosphatases in Arabidopsis, providing a prominent example of how MAPK activities are integrated and specified by signaling compartmentalization at the subcellular level (Guo et al., 2022, Nat Commun).
Project 3: Membrane trafficking required for establishing the polarity site
Figure 4. A working model for PRAF-mediated trafficking in establishing BASL polarity. In wild-type plants, the polarization of BASL protein (green) in the stomatal lineage cell requires the physical partner, four PRAF proteins (orange), as well as the Golgi-localized Arf GEF GNOM (blue). The PRAF8 proteins are predominantly localized to the plasma membrane, where they may polarize together with BASL. The PRAF8 proteins may also partially associate with the Golgi, TGN/EE and a subset of endosomes/vesicles decorated by RabC1 and RabF2b. Furthermore, the PRAF proteins physically interact with GNOM, possibly leading to the association of GNOM to the RabC1- and RabE1d-decorated membrane structures.
Cell polarity is essential for organizing cells into functionally distinct domains, influencing growth direction, differentiation, and developmental patterning. In plants, polarity dictates fundamental processes including asymmetric cell division, directional transport of hormones, nutrient uptake, and establishment of developmental axes. During stomatal development, precise control of polarity ensures correct placement of daughter cells, determination of stomatal lineage fate, and optimal distribution of stomata for efficient gas exchange.
The molecular mechanisms for protein polarization in plants have been extensively studied in two systems: 1) vesicular trafficking-based polarization of PIN auxin efflux carriers (membrane embedded proteins) and 2) cytoskeleton-dependent and –independent positive feedback loop-based ROP polarization (small Rho-like GTPases from plants). Polar trafficking of BASL, a non-membrane novel protein, has not been successfully connected to both pathways and might represent an unknown mechanism. Fluorescence Recovery After Photobleaching (FRAP) was performed on GFP-BASL and the recovery curves suggested that BASL dynamics is more comparable to the membrane-embedded PIN proteins, hinting the possible regulation of membrane trafficking in BASL polarization. Indeed, through the identification of physical interactors of BASL, we recently established that four members of the PRAF protein family interact with BASL and are required for the establishment of the polarity site (Wang et al., 2022 Nat. Commun) (Figure 4). The PRAF proteins are plant specific but contain phospholipid-binding domains and co-localize with small GTPases, both of which are conserved across the kingdoms in regulating membrane trafficking and delivery. Detailed molecular mechanisms for the PRAF-mediate membrane trafficking need to be further investigated.
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