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 Ca 2؉ -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 Ca 2؉ influx with mechanosensing in Arabidopsis thaliana. MCA1 cDNA was cloned by the functional complementation of lethality of a yeast mid1 mutant lacking a putative Ca 2؉ -permeable SA channel component. Mca1 was localized to the yeast plasma membrane as an integral membrane protein and mediated Ca 2؉ influx. Mca1 also increased [Ca 2؉ ]cyt upon plasma membrane distortion in Arabidopsis. The growth of MCA1-overexpressing plants was impaired in a high-calcium but not a low-calcium medium. The primary roots of mca1-null plants failed to penetrate a harder agar medium from a softer one. These observations demonstrate that Mca1 plays a crucial role in a Ca 2؉ -permeable SA channel system that leads to mechanosensing in Arabidopsis. We anticipate our findings to be a starting point for a deeper understanding of the molecular mechanisms of mechanotransduction in plants.calcium ͉ calcium channel ͉ calcium uptake ͉ mechanosensing

By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling. The mutants were classified into five complementation groups, one of which we studied extensively. This mutation defines a new gene, designated MIDI, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues. The mid)-) mutant has low Ca21 uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2. The MID) gene is nonessential for vegetative growth. The efficiency of mating between AL4Ta mid)-) and MATa mid)-) cells is low. These results demonstrate that MID) is required for Ca2+ influx and mating.

Ca2+ is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca 2+ -permeable channels or transporters involved in Ca 2+ uptake in roots are largely unknown. We recently identified a candidate for the Ca 2+ -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 Ca 2+ uptake deficiency in yeast cells lacking a Ca 2+ 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. Ca 2+ 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 Mg 2+ 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 Ca 2+ uptake in roots and an overlapping role with MCA1 in plant growth.Calcium ion (Ca 2+

Tryptophan uptake appears to be the Achilles' heel in yeast physiology, since under a variety of seemingly diverse toxic conditions, it becomes the limiting factor for cell growth. When growing cells of Saccharomyces cerevisiae are subjected to high hydrostatic pressure, tryptophan uptake is down-regulated, leading to cell cycle arrest in the G 1 phase. Here we present evidence that the two tryptophan permeases Tat1 and Tat2 are differentially regulated by Rsp5 ubiquitin ligase in response to high hydrostatic pressure. Analysis of highpressure growth mutants revealed that the HPG1 gene was allelic to RSP5. The HPG1 mutation or the bul1⌬ bul2⌬ double mutation caused a marked increase in the steady-state level of Tat2 but not of Tat1, although both permeases were degraded at high pressure in an Rsp5-dependent manner. There were marked differences in subcellular localization. Tat1 localized predominantly in the plasma membrane, whereas Tat2 was abundant in the internal membranes. Moreover, Tat1 was associated with lipid rafts, whereas Tat2 localized in bulk lipids. Surprisingly, Tat2 became associated with lipid rafts upon the occurrence of a ubiquitination defect. These results suggest that ubiquitination is an important determinant of the localization and regulation of these tryptophan permeases. Determination of the activation volume (⌬V ) for Tat1-and Tat2-mediated tryptophan uptake (89.3 and 50.8 ml/mol, respectively) revealed that both permeases are highly sensitive to membrane perturbation and that Tat1 rather than Tat2 is likely to undergo a dramatic conformational change during tryptophan import. We suggest that hydrostatic pressure is a unique tool for elucidating the dynamics of integral membrane protein functions as well as for probing lipid microenvironments where they localize.

Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli. However, genes encoding such channels have not been identified in eukaryotes. The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae. Functional expression of Mid1 in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium. These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels. Single-channel analyses with cell-attached patches revealed that Mid1 acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette. Thus, Mid1 appears to be a eukaryotic, SA Cat channel.

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Ciliates and flagellates temporarily swim backwards on collision by generating a mechanoreceptor potential. Although this potential has been shown to be associated with cilia in Paramecium, the molecular entity of the mechanoreceptor has remained unknown. Here we show that Chlamydomonas cells express TRP11, a member of the TRP (transient receptor potential) subfamily V, in the proximal region of the flagella, and that suppression of TRP11 expression results in loss of the avoiding reaction. The results indicate that Chlamydomonas flagella exhibit mechanosensitivity, despite constant motility, by localizing the mechanoreceptor in the proximal region, where active bending is restricted.