Plants can sense and respond to mechanical stimuli, like animals. An early mechanism of mechanosensing and response is speculated to be governed by as-yet-unidentified sensory complexes containing a Ca²⁺-permeable, stretch-activated (SA) channel. However, the components or regulators of such complexes are poorly understood at the molecular level in plants. Here, we report the molecular identification of a plasma membrane protein (designated Mca1) that correlates Ca²⁺influx with mechanosensing inArabidopsis thaliana.MCA1cDNA was cloned by the functional complementation of lethality of a yeastmid1mutant lacking a putative Ca²⁺-permeable SA channel component. Mca1 was localized to the yeast plasma membrane as an integral membrane protein and mediated Ca²⁺influx. Mca1 also increased [Ca²⁺]_cytupon plasma membrane distortion inArabidopsis. The growth ofMCA1-overexpressing plants was impaired in a high-calcium but not a low-calcium medium. The primary roots ofmca1-null plants failed to penetrate a harder agar medium from a softer one. These observations demonstrate that Mca1 plays a crucial role in a Ca²⁺-permeable SA channel system that leads to mechanosensing inArabidopsis. We anticipate our findings to be a starting point for a deeper understanding of the molecular mechanisms of mechanotransduction in plants.

Ca²⁺ is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca²⁺-permeable channels or transporters involved in Ca²⁺ uptake in roots are largely unknown. We recently identified a candidate for the Ca²⁺-permeable mechanosensitive channel in Arabidopsis (Arabidopsis thaliana), named MCA1. Here, we investigated the only paralog of MCA1 in Arabidopsis, MCA2. cDNA of MCA2 complemented a Ca²⁺ uptake deficiency in yeast cells lacking a Ca²⁺ channel composed of Mid1 and Cch1. Reverse transcription polymerase chain reaction analysis indicated that MCA2 was expressed in leaves, flowers, roots, siliques, and stems, and histochemical observation showed that an MCA2 promoter::GUS fusion reporter gene was universally expressed in 10-d-old seedlings with some exceptions: it was relatively highly expressed in vascular tissues and undetectable in the cap and the elongation zone of the primary root. mca2-null plants were normal in growth and morphology. In addition, the primary root of mca2-null seedlings was able to normally sense the hardness of agar medium, unlike that of mca1-null or mca1-null mca2-null seedlings, as revealed by the two-phase agar method. Ca²⁺ uptake activity was lower in the roots of mca2-null plants than those of wild-type plants. Finally, growth of mca1-null mca2-null plants was more retarded at a high concentration of Mg²⁺ added to medium compared with that of mca1-null and mca2-null single mutants and wild-type plants. These results suggest that the MCA2 protein has a distinct role in Ca²⁺ uptake in roots and an overlapping role with MCA1 in plant growth.

Cold shock triggers an immediate rise in the cytosolic free calcium concentration ([Ca²⁺]_cyt) in Arabidopsis thaliana and this cold-induced elevation of [Ca²⁺]_cyt is inhibited by lanthanum or EGTA. It is suggested that intracellular calcium mainly contributes to the cold-induced [Ca²⁺]_cyt response by entering into the cytosol. Two calcium-permeable mechanosensitive channels, MCA1 and MCA2 (mid1-complementing activity), have been identified in Arabidopsis. Here, we demonstrate that MCA1 and MCA2 are involved in a cold-induced increase in [Ca²⁺]_cyt. The cold-induced [Ca²⁺]_cyt increase in mca1 and mca2 mutants was markedly lower than that in wild types. The mca1 mca2 double mutant exhibited chilling and freezing sensitivity, compared to wild-type plants. Expression of At5g61820, At3g51660, and At4g15490, which are not regulated by the CBF/DREB1s transcription factor, was down-regulated in mca1 mca2. These results suggest that MCA1 and MCA2 are involved in the cold-induced elevation of [Ca²⁺]_cyt, cold tolerance, and CBF/DREB1-independent cold signaling.

Background: Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca²⁺-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca²⁺ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca²⁺ influx mediated by mechanosensitive Ca²⁺ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated. Results: We here identified and investigated OsMCA1, the sole rice homolog of putative Ca²⁺-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca²⁺ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca²⁺ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca²⁺-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels. Conclusions: We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca²⁺-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca²⁺ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca²⁺-permeable MS channel that is directly activated by membrane tension. The N-terminal 173 residues of MCA1 and MCA2 were synthesized in vitro, purified, and reconstituted into artificial liposomal membranes. Liposomes reconstituted with MCA1(1-173) or MCA2(1-173) mediate Ca²⁺ influx and the application of pressure to the membrane reconstituted with MCA2(1-173) elicits channel currents. This channel is also activated by voltage. Blockers for MS channels inhibit activation by stretch, but not by voltage. Since MCA proteins are found exclusively in plants, these results suggest that MCA represent plant-specific MS channels that open directly with membrane tension.

A key molecule of sensing machineries essential for survival upon hypo-osmotic shock is the mechanosensitive channel. The bacterial mechanosensitive channel MscS functions directly for this purpose by releasing cytoplasmic solutes out of the cell, whereas plant MscS homologues are found to function in chloroplast organization. Here we show that the fission yeast MscS homologues, designated Msy1 and Msy2, participate in the hypo-osmotic shock response by a mechanism different from that operated by the bacterial MscS. Upon hypo-osmotic shock, msy2^– and msy1^– msy2^– cells display greater cell swelling than wild-type cells and undergo cell death. Cell swelling precedes an intracellular Ca²⁺ increase, which was greater in msy1^– and msy1^– msy2^– cells than in wild-type cells. Fluorescent microscopy showed that Msy1 and Msy2 localize mainly to the endoplasmic reticulum. These observations suggest that organellar Msy1 and Msy2 regulate intracellular Ca²⁺ and cell volume for survival upon hypo-osmotic shock.