Glutamate has dual roles in metabolism and signaling; thus, signaling functions must be isolatable and distinct from metabolic fluctuations, as seen in low-glutamate domains at synapses. In plants, wounding triggers electrical and calcium (Ca 2+ ) signaling, which involve homologs of mammalian glutamate receptors. The hydraulic dispersal and squeeze-cell hypotheses implicate pressure as a key component of systemic signaling. Here, we identify the stretch-activated anion channel MSL10 as necessary for proper wound-induced electrical and Ca 2+ signaling. Wound gene induction, genetics, and Ca 2+ imaging indicate that MSL10 acts in the same pathway as the glutamate receptor-like proteins (GLRs). Analogous to mammalian NMDA glutamate receptors, GLRs may serve as coincidence detectors gated by the combined requirement for ligand binding and membrane depolarization, here mediated by stretch activation of MSL10. This study provides a molecular genetic basis for a role of mechanical signal perception and the transmission of long-distance electrical and Ca 2+ signals in plants.

ABSTRACTAlthough a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to GFP triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag and could be also induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus are transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants.HIGHLIGHTCell death is triggered by mutations in either the cytoplasmic N- or C-terminus of AìMSLlü. Our proposed model explains how membrane tension may activate signaling through the interaction of these two domains.

+1 314 935-9223 14 15 16 Key words: Programmed cell death, cell swelling, hypo-osmotic, mechanosensitive, reactive 17 oxygen species 18osmotic shock, where they are thought to up-regulate rehydration-responsive genes, including 68 several enzymes that reduce levels of the plant hormone ABA (Tsugama et al., 2012; Tsugama et 69 al., 2014; Tsugama et al., 2016). Despite these many observations, it is still not clear how these 70 downstream signaling components are connected, nor how they might lead to adaptive responses. 71 72It is also poorly understood how cell swelling is initially perceived in plants. The many potential 73 signals include molecular crowding, membrane tension, osmotic gradient, turgor, membrane 74 curvature, cell wall damage, and the disruption of the plasma membrane-cell wall connections; 75 (Haswell and Verslues, 2015; Cuevas-Velazquez and Dinneny, 2018; Hýskova and Ryslava, 2018; 76 Gigli-Bisceglia et al., 2019; Le Roux et al., 2019). Several families of receptor-like kinases, 77including the WALL-ASSOCATED KINASES, Catharanthus roseus RLK family, have been 78 implicated in sensing cell wall integrity (Nissen et al., 2016; Gigli-Bisceglia et al., 2019). In 79 particular, the CrRLK FERONIA has been implicated in the perception of mechanical signals, 80including hypo-osmotic shock ((Shih et al., 2014). Here we investigate the role of another class of 81 candidate cell swelling sensors, mechanosensitive ion channels (MS) (Cazalé et al., 1998; Haswell 82 and Verslues, 2015; Kobayashi et al., 2018). 83 84 MS ion channels are multimeric proteins embedded in the membrane that mediate ion flux across 85 the membrane in response to lateral membrane tension, and are found in all kingdoms of life 86 (Martinac, 2012;Ranade et al., 2015; Basu and Haswell, 2017). Rapid cell swelling is likely to 87 lead to an immediate increase in membrane tension, leading to the opening of MS ion channels 88 and thereby to the exit of osmolytes and/or entry of calcium signals that regulate downstream 89 adaptive events. In Escherichia coli, the MS ion channels MscS and MscL serve as osmotic safety 90 valves, opening in response to severe hypo-osmotic shock and mediating the release of osmolytes 91 (Levina et al., 1999;Sukharev, 2002; Belyy et al., 2010). The subsequent departure of water from 92 the cell presumably reduces swelling and preserves cell integrity. It is also possible that EcMscS 93 mediates the swelling-induced entry of calcium, leading to intracellular signaling events (Cox et 94 al., 2013). In plants, the overexpression of members of the MCA family leads to increased Ca 2+ 95 uptake in response to hypoosmotic shock in Arabidopsis, tobacco, and rice; but are not strictly 96 required for swelling-induced Ca 2+ uptake (Nakagawa et al., 2007; Kurusu et al., 2012b; Kurusu 97 et al., 2012a). Members of the MscS-Like (MSL) family of MS ion channels have been implicated 98 in osmotic homeostasis in chloroplasts (Haswell and Meyerowitz, 2006; Veley et al., 2012), and 99 pollen (Hamilton et al., 2015), bu...

Population growth and climate change will impact food security and potentially exacerbate the environmental toll that agriculture has taken on our planet. These existential concerns demand that a passionate, interdisciplinary, and diverse community of plant science professionals is trained during the 21st century. Furthermore, societal trends that question the importance of science and expert knowledge highlight the need to better communicate the value of rigorous fundamental scientific exploration. Engaging students and the general public in the wonder of plants, and science in general, requires renewed efforts that take advantage of advances in technology and new models of funding and knowledge dissemination. In November 2018, funded by the National Science Foundation through the Arabidopsis Research and Training for the 21st century (ART 21) research coordination network, a symposium and workshop were held that included a diverse panel of students, scientists, educators, and administrators from across the US. The purpose of the workshop was to re‐envision how outreach programs are funded, evaluated, acknowledged, and shared within the plant science community. One key objective was to generate a roadmap for future efforts. We hope that this document will serve as such, by providing a comprehensive resource for students and young faculty interested in developing effective outreach programs. We also anticipate that this document will guide the formation of community partnerships to scale up currently successful outreach programs, and lead to the design of future programs that effectively engage with a more diverse student body and citizenry.