Direct interaction of ligand-receptor pairs specifying stomatal patterning. Genes Dev

Department of Biology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
Genes & development (Impact Factor: 10.8). 01/2012; 26(2):126-36. DOI: 10.1101/gad.179895.111
Source: PubMed


Valves on the plant epidermis called stomata develop according to positional cues, which likely involve putative ligands (EPIDERMAL PATTERNING FACTORS [EPFs]) and putative receptors (ERECTA family receptor kinases and TOO MANY MOUTHS [TMM]) in Arabidopsis. Here we report the direct, robust, and saturable binding of bioactive EPF peptides to the ERECTA family. In contrast, TMM exhibits negligible binding to EPF1 but binding to EPF2. The ERECTA family forms receptor homomers in vivo. On the other hand, TMM associates with the ERECTA family but not with itself. While ERECTA family receptor kinases exhibit complex redundancy, blocking ERECTA and ERECTA-LIKE1 (ERL1) signaling confers specific insensitivity to EPF2 and EPF1, respectively. Our results place the ERECTA family as the primary receptors for EPFs with TMM as a signal modulator and establish EPF2-ERECTA and EPF1-ERL1 as ligand-receptor pairs specifying two steps of stomatal development: initiation and spacing divisions.

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Available from: Keiko U Torii, Sep 30, 2015
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    • "For example, the kinase-deleted version of the receptor kinase ERECTA (ER: At2g26330) accumulates at much higher levels than the full-length, endogenous ERECTA protein (Shpak et al, 2003), while the removal of the kinase domain in the CLAVA- TA1 (CLV1: At1g75820) receptor improves its expression without affecting the ligand-binding affinity of its ectodomain (Ogawa et al, 2008). These truncated proteins have been successfully used in binding assays such as pull down, gel filtration, fluorescence anisotropy, and surface plasmon resonance (Pollard, 2010, Luoni et al, 2006, Lee et al 2012). However, interpretation of this binding data has to include a thorough analysis of the potential biological effects caused by removing the kinase domains and the changes in stoichiometry caused by the over-accumulation of stabilized receptors. "
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    ABSTRACT: The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane.
    The Arabidopsis Book 06/2015; 13:e0180. DOI:10.1199/tab.0180
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    • "Conversely, CLEL6 and CLEL7 are small peptides that play a role in later asymmetric divisions within lateral root development, although their corresponding LRR-RLK has not been identified (Meng et al., 2012). The ERECTA LRR-RLK influences both ( pro)cambial and stomatal development, demonstrating one case where a single receptor can regulate asymmetric cell division in multiple cell types (Shpak et al., 2005; Lee et al., 2012; Etchells et al., 2013). Perhaps this conservation of function is due to the fact that these are examples of asymmetric amplifying divisions. "
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    • "EPF-LIKE9 (EPFL9/Stomagen) can facilitate stomatal differentiation from internal tissues (Torii, 2012). Very recently, biochemical evidences have indicated that the ERECTA family is a primary receptor for EPF ligands and TMM regulates the activity of ERECTA-family proteins (Lee et al., 2012). Both the genetic and biochemical findings reveal a crucial function of TMM in preventing stomatal precursors from auto-inhibiting themselves from signals they produce (Torii, 2012). "
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    ABSTRACT: One goal of modern agriculture is the improvement of plant drought tolerance and water-use efficiency (WUE). Although stomatal density has been linked to WUE, the causal molecular mechanisms and engineered alternations of this relationship are not yet fully understood. Moreover, YODA (YDA), which is a MAPKK kinase gene, negatively regulates stomatal development. BR-INSENSITIVE 2 interacts with phosphorylates and inhibits YDA. However, whether YDA is modulated in the transcriptional level is still unclear. Plants lacking ANGUSTIFOLIA3 (AN3) activity have high drought stress tolerance because of low stomatal densities and improved root architecture. Such plants also exhibit enhanced WUE through declining transpiration without a demonstrable reduction in biomass accumulation. AN3 negatively regulated YDA expression at the transcriptional level by target-gene analysis. Chromatin immunoprecipitation analysis indicated that AN3 was associated with a region of the YDA promoter in vivo. YDA mutation significantly decreased the stomatal density and root length of an3 mutant, thus proving the participation of YDA in an3 drought tolerance and WUE enhancement. These components form an AN3–YDA complex, which allows the integration of water deficit stress signalling into the production or spacing of stomata and cell proliferation, thus leading to drought tolerance and enhanced WUE.
    Plant Biotechnology Journal 01/2015; 13(7). DOI:10.1111/pbi.12324 · 5.75 Impact Factor
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