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

A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 μM, and NH4 + was absent. NH4 + specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4 + had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4 +. Wild-type plants were much more resistant to NH4 +. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4 +, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 μM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the “high-affinity” range of K+ concentrations. In the absence of AKT1 channel activity, an NH4 +-sensitive, Na+/H+-stimulated mechanism can suffice.

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogendefense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep-and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca 2+ -dependent manner.calcium signaling | plant innate immunity | flagellin receptor 2 | brassinosteroid associated kinase 1

The amino acids glutamate (Glu) and glycine (Gly) trigger large, rapid rises in cytosolic Ca 21 concentration and a concomitant rise in membrane potential (depolarization) in plants. The possibility that plant homologs of neuronal ionotropic glutamate receptors mediate these neuron-like ionic responses was tested in Arabidopsis (Arabidopsis thaliana) seedlings using a combination of Ca 21 measurements, electrophysiology, and reverse genetics. The membrane depolarization triggered by Glu was greatly reduced or completely blocked in some conditions by mutations in GLR3.3, one of the 20 GLR genes in Arabidopsis. The same mutations completely blocked the associated rise in cytosolic Ca 21 . These results genetically demonstrate the participation of a glutamate receptor in the rapid ionic responses to an amino acid. The GLR3.3-independent component of the depolarization required Glu concentrations above 25 mM, did not display desensitization, and was strongly suppressed by increasing extracellular pH. It is suggested to result from H 1 -amino acid symport. Six amino acids commonly present in soils (Glu, Gly, alanine, serine, asparagine, and cysteine) as well as the tripeptide glutathione (g-glutamyl-cysteinylGly) were found to be strong agonists of the GLR3.3-mediated responses. All other amino acids induced a small depolarization similar to the non-GLR, putative symporter component and in most cases evoked little or no Ca 21 rise. From these results it may be concluded that sensing of six amino acids in the rhizosphere and perhaps extracellular peptides is coupled to Ca 21 signaling through a GLR-dependent mechanism homologous to a fundamental component of neuronal signaling.

Caesium (Cs(+)) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs(+) is chemically similar to potassium (K(+)), and much is known about K(+) transport mechanisms, it is not clear through which K(+) transport mechanisms Cs(+) is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K(+) and Cs(+) uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K(+) deprivation, when it is expressed. Growth analysis showed that AtHAK5 plays a role during severe K(+) deprivation. Under K(+)-deficient conditions in the presence of Cs(+), Arabidopsis seedlings lacking AtHAK5 had increased inhibition of root growth and lower Cs(+) accumulation, and significantly higher leaf chlorophyll concentrations than wild type. These data indicate that, in addition to transporting K(+) in planta, AtHAK5 also transports Cs(+). Further experiments showed that AtHAK5 mediated Cs(+) uptake into yeast cells and that, although the K(+) deficiency-induced expression of AtHAK5 was inhibited by low concentrations of NH(4)(+) in planta, Cs(+) uptake by yeast was stimulated by low concentrations of NH(4)(+). Interestingly, the growth of the Arabidopsis atakt1-1 mutant was more sensitive to Cs(+) than the wild type. This may be explained, in part, by increased expression of AtHAK5 in the atakt1-1 mutant. It is concluded that AtHAK5 is a root plasma membrane uptake mechanism for K(+) and Cs(+) under conditions of low K(+) availability.

ATP induces Ca(2+) influx across the cell membrane and activates release from intracellular Ca(2+) pools in vascular endothelial cells (ECs). Ca(2+) signaling leads to the modification of a variety of EC functions, including the production of vasoactive substances such as nitric oxide and prostacyclin. However, the molecular mechanisms for ATP-induced Ca(2+) influx in ECs have not been thoroughly clarified. Here we demonstrate evidence that a P2X(4) receptor for an ATP-gated cation channel is predominantly expressed in human ECs and is involved in the ATP-induced Ca(2+) influx. Northern blot analysis distinctly showed the expression of P2X(4) mRNA in human ECs cultured from the umbilical vein, aorta, pulmonary artery, and skin microvessels. Competitive PCR revealed that P2X(4) mRNA expression was much higher in ECs than was the expression of other subtypes, including P2X(1), P2X(3), P2X(5), and P2X(7). Treatment of ECs with antisense oligonucleotides designed to target the P2X(4) receptor decreased the P2X(4) mRNA and protein levels to approximately 25% of control levels and markedly prevented the ATP-induced Ca(2+) influx.

Ca 2+ influx is an early signal initiating cytosolic immune responses to pathogen perception in plant cells; molecular components linking pathogen recognition to Ca 2+ influx are not delineated. Work presented here provides insights into this biological system of non-self recognition and response activation. We have recently identified a cyclic nucleotide-activated ion channel as facilitating the Ca 2+ flux that initiates immune signaling in the plant cell cytosol. Work in this report shows that elevation of cAMP is a key player in this signaling cascade. We show that cytosolic Ca 2+ elevation, nitric oxide (NO) and reactive oxygen species generation, as well as immune signaling, lead to a hypersensitive response upon application of pathogens and/or conserved molecules that are components of microbes and are all dependent on cAMP generation. Exogenous cAMP leads to Ca 2+ channel-dependent cytosolic Ca 2+ elevation, NO generation, and defense response gene expression in the absence of the non-self pathogen signal. Inoculation of leaves with a bacterial pathogen leads to cAMP elevation coordinated with Ca 2+ rise. cAMP acts as a secondary messenger in plants; however, no specific protein has been heretofore identified as activated by cAMP in a manner associated with a signaling cascade in plants, as we report here. Our linkage of cAMP elevation in pathogen-inoculated plant leaves to Ca 2+ channels and immune signaling downstream from cytosolic Ca 2+ elevation provides a model for how non-self detection can be transduced to initiate the cascade of events in the cell cytosol that orchestrate pathogen defense responses.

Physicochemical similarities between K+ and Na+ result in interactions between their homeostatic mechanisms. The physiological interactions between these two ions was investigated by examining aspects of K+ nutrition in the Arabidopsis salt overly sensitive (sos) mutants, and salt sensitivity in the K+ transport mutants akt1 (Arabidopsis K+ transporter) and skor (shaker-like K+ outward-rectifying channel). The K+-uptake ability (membrane permeability) of the sos mutant root cells measured electrophysiologically was normal in control conditions. Also, growth rates of these mutants in Na+-free media displayed wild-type K+ dependence. However, mild salt stress (50 mm NaCl) strongly inhibited root-cell K+ permeability and growth rate in K+-limiting conditions of sos1 but not wild-type plants. Increasing K+ availability partially rescued the sos1 growth phenotype. Therefore, it appears that in the presence of Na+, the SOS1 Na+-H+ antiporter is necessary for protecting the K+ permeability on which growth depends. The hypothesis that the elevated cytoplasmic Na+ levels predicted to result from loss of SOS1 function impaired the K+ permeability was tested by introducing 10 mm NaCl into the cytoplasm of a patch-clamped wild-type root cell. Complete loss of AKT1 K+ channel activity ensued. AKT1 is apparently a target of salt stress in sos1 plants, resulting in poor growth due to impaired K+ uptake. Complementary studies showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal. Thus, the effect of Na+ on K+ transport is probably more important at the uptake stage than at the xylem loading stage.