Entanglement of Arabidopsis Seedlings to a Mesh Substrate under Microgravity Conditions in KIBO on the ISS.

The International Space Station (ISS) provides a precious opportunity to study plant growth and development under microgravity (micro-G) conditions. In this study, four lines of Arabidopsis seeds (wild type, wild-type MCA1-GFP, mca1-knockout, and MCA1-overexpressed) were cultured on a nylon lace mesh placed on Gelrite-solidified MS-medium in the Japanese experiment module KIBO on the ISS, and the entanglement of roots with the mesh was examined under micro-G and 1-G conditions. We found that root entanglement with the mesh was enhanced, and root coiling was induced under the micro-G condition. This behavior was less pronounced in mca1-knockout seedlings, although MCA1-GFP distribution at the root tip of the seedlings was nearly the same in micro-G-grown seedlings and the ground control seedlings. Possible involvement of MCA1 in the root entanglement is discussed.

MCAs in Arabidopsis are Ca2+-permeable mechanosensitive channels inherently sensitive to membrane tension.

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.

Mix and match: Patchwork domain evolution of the land plant-specific Ca2+-permeable mechanosensitive channel MCA.

Multidomain proteins can have a complex evolutionary history that may involve de novo domain evolution, recruitment and / or recombination of existing domains and domain losses. Here, the domain evolution of the plant-specific Ca2+-permeable mechanosensitive channel protein, MID1-COMPLEMENTING ACTIVITY (MCA), was investigated. MCA, a multidomain protein, possesses a Ca2+-influx-MCAfunc domain and a PLAC8 domain. Profile Hidden Markov Models (HMMs) of domains were assessed in 25 viridiplantae proteomes. While PLAC8 was detected in plants, animals, and fungi, MCAfunc was found in streptophytes but not in chlorophytes. Full MCA proteins were only found in embryophytes. We identified the MCAfunc domain in all streptophytes including charophytes where it appeared in E3 ubiquitin ligase-like proteins. Our Maximum Likelihood (ML) analyses suggested that the MCAfunc domain evolved early in the history of streptophytes. The PLAC8 domain showed similarity to Plant Cadmium Resistance (PCR) genes, and the coupling of MCAfunc and PLAC8 seemed to represent a single evolutionary event. This combination is unique in MCA, and does not exist in other plant mechanosensitive channels. Within angiosperms, gene duplications increased the number of MCAs. Considering their role in mechanosensing in roots, MCA might be instrumental for the rise of land plants. This study provides a textbook example of de novo domain emergence, recombination, duplication, and losses, leading to the convergence of function of proteins in plants.

A Method Enabling Comprehensive Isolation of Arabidopsis Mutants Exhibiting Unusual Root Mechanical Behavior.

Root penetration into soils is fundamental for land plants to support their own aboveground parts and forage water and nutrients. To elucidate the molecular mechanisms underlying root mechanical penetration, mutants defective in this behavior need to be comprehensively isolated; however, established methods are currently scarce. We herein report a method to screen for these mutants of Arabidopsis thaliana and present their phenotypes. We isolated five mutants using this method, tentatively named creep1 to creep5, the primary roots of which crept over the surface of horizontal hard medium that hampered penetration by the primary root of the wild type, thereby forcing it to spring up on the surface and die. By examining root skewing, which is induced by a touch stimulation that is generated as the primary roots grow along a vertical impenetrable surface, the five creep mutants were subdivided into three groups, namely mutants with the primary root skewing leftward, those skewing rightward, and that growing dispersedly. While the majority of wild type primary roots skewed slightly leftward, nearly half of the primary roots of creep1 and creep5 skewed rightward as viewed from above. The primary roots of creep4 displayed scattered growth, while those of creep2 and creep3 showed a similar phenotype to the wild type primary roots. These results demonstrate the potential of the method developed herein to isolate various mutants that will be useful for investigating root mechanical behavior regulation not only in Arabidopsis, but also in major crops with economical value.

The gravistimulation-induced very slow Ca2+ increase in Arabidopsis seedlings requires MCA1, a Ca2+-permeable mechanosensitive channel.

Gravity is a critical environmental factor affecting the morphology and function of plants on Earth. Gravistimulation triggered by changes in the gravity vector induces an increase in the cytoplasmic free calcium ion concentration ([Ca2+]c) as an early process of gravity sensing; however, its role and molecular mechanism are still unclear. When seedlings of Arabidopsis thaliana expressing apoaequorin were rotated from the upright position to the upside-down position, a biphasic [Ca2+]c-increase composed of a fast-transient [Ca2+]c-increase followed by a slow [Ca2+]c-increase was observed. We find here a novel type [Ca2+]c-increase, designated a very slow [Ca2+]c-increase that is observed when the seedlings were rotated back to the upright position from the upside-down position. The very slow [Ca2+]c-increase was strongly attenuated in knockout seedlings defective in MCA1, a mechanosensitive Ca2+-permeable channel (MSCC), and was partially restored in MCA1-complemented seedlings. The mechanosensitive ion channel blocker, gadolinium, blocked the very slow [Ca2+]c-increase. This is the first report suggesting the possible involvement of MCA1 in an early event related to gravity sensing in Arabidopsis seedlings.

The root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and transmembrane receptor-mediated signal transduction in Arabidopsis.

KEY MESSAGE: We found that mutations in a Ca2+-permeable mechanosensitive channel MCA1, an ethylene-regulated microtubule-associated protein WDL5, and a versatile co-receptor BAK1 affect root growth response to mechanical stress. Plant root tips exposed to mechanical impedance show a temporal reduction in the elongation growth. The process involves a transient Ca2+ increase in the cytoplasm followed by ethylene signaling. To dissect the molecular mechanisms underlying this response, we examined the root growth of a series of Arabidopsis mutants with potentially altered response to mechanical stress after transfer from vertical to horizontal plates that were covered by dialysis membrane as an impedance. Among the plant hormone-response mutants tested, the ethylene-insensitive mutant ein3 was confirmed to show no growth reduction after the transfer. The root growth reduction was attenuated in a mutant of MCA1 encoding a Ca2+-permeable mechanosensitive channel and that of WDL5 encoding an ethylene-regulated microtubule-associated protein. We also found that the growth reduction was enhanced in a mutant of BAK1 encoding a co-receptor that pairs with numerous leucine-rich repeat receptor kinases to modulate growth and immunity. These results suggest the root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and also transmembrane receptor-mediated signal transduction.

Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca2+ channel related to the animal VGCC/NALCN family.

Yeasts and fungi generate Ca2+ signals in response to environmental stresses through Ca2+ channels essentially composed of Cch1 and Mid1. Cch1 is homologous to the pore-forming α1 subunit of animal voltage-gated Ca2+ channels (VGCCs) and sodium leak channels nonselective (NALCNs), whereas Mid1 is a membrane-associated protein similar to the regulatory α2/δ subunit of VGCCs and the regulatory subunit of NALCNs. Although the physiological roles of Cch1/Mid1 channels are known, their molecular regulation remains elusive, including subunit interactions regulating channel functionality. Herein, we identify amino acid residues involved in interactions between the pore-forming Cch1 subunit and the essential regulatory Mid1 subunit of Saccharomyces cerevisiaeIn vitro mutagenesis followed by functional assays and co-immunoprecipitation experiments reveal that three residues present in a specific extracellular loop in the repeat III region of Cch1 are required for interaction with Mid1, and that one essential Mid1 residue is required for interaction with Cch1. Importantly, these residues are necessary for Ca2+ channel activity and are highly conserved in fungal and animal counterparts. We discuss that this unique subunit interaction-based regulatory mechanism for Cch1 differs from that of VGCCs/NALCNs.

The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in Saccharomyces cerevisiae.

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae, a few proteins devoid of a signal peptide are still translocated into the ER and then N-glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain (ste24Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N-glycosylated in the ER in the ste24Δ strain, we identified the transcription factor Rme1 as a protein that is partially N-glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process.

MCA1 and MCA2 Are Involved in the Response to Hypergravity in Arabidopsis Hypocotyls.

Plants respond to and resist gravitational acceleration, but the mechanism of signal perception in the response is unknown. We studied the role of MCA (mid1-complementing activity) proteins in gravity perception by analyzing the expression of the MCA1 and MCA2 genes, and the growth of hypocotyls of mca mutants, under hypergravity conditions in the dark. An MCA1 promoter::GUS fusion reporter gene construct (MCA1p::GUS) and MCA2p::GUS were expressed almost universally in etiolated seedlings. Under hypergravity conditions, the expression levels of both genes increased compared with that under the 1 g condition, and remained higher, especially in the basal supporting region. On the other hand, mca-null and MCA-overexpressing seedlings showed normal growth under the 1 g condition. Hypergravity suppressed elongation growth of hypocotyls, but this effect was reduced in hypocotyls of mca-null mutants compared with the wild type. In contrast, MCA-overexpressing seedlings were hypersensitive to increased gravity; suppression of elongation growth was detected at a lower gravity level than that in the wild type. These results suggest that MCAs are involved in the perception of gravity signals in plants, and may be responsible for resistance to hypergravity.

Ca2+-permeable mechanosensitive channels MCA1 and MCA2 mediate cold-induced cytosolic Ca2+ increase and cold tolerance in Arabidopsis.

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.

Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family.

Saccharomyces cerevisiae Mid1 is composed of 548 amino acids and a regulatory subunit of Cch1, a member of the eukaryotic pore-forming, four-domain cation channel family. The amino acid sequence and voltage insensitivity of Cch1 are more similar to those of Na+ leak channel non-selective (NALCN) than to the α1 subunit of voltage-gated Ca2+ channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC α2/δ regulatory subunits and NALCN-associated proteins. Unlike animal α2/δ subunits, Mid1 and NALCN-associated proteins are essential for the function of the pore-forming subunit. We herein investigated the processing and membrane translocation of Mid1. Mid1 was found to have a 20-amino-acid-long N-terminal signal peptide and appeared to be entirely localized extracellularly. A signal peptide-deleted Mid1 protein, Mid1ΔN23, was N-glycosylated and retained Ca2+ influx activity through Cch1. Moreover, an N-terminal truncation analysis revealed that even truncated Mid1 lacking 209 N-terminal amino acid residues was N-glycosylated and maintained Ca2+ influx activity. A 219-amino-acid-truncated Mid1 protein lost this activity but was still N-glycosylated. In the sec71Δ and sec72Δ single mutants defective in the post-translational protein transport into the endoplasmic reticulum (ER), Mid1ΔN23 could not mediate Ca2+ influx and did not undergo N-glycosylation, whereas wild-type Mid1 exhibited normal Ca2+ influx activity and N-glycosylation in these mutants. Therefore, the signal peptide-lacking Mid1ΔN23 protein may be translocated to the ER exclusively through the post-translational protein translocation, which typically requires an N-terminal signal peptide. Mid1 may provide a tool for studying mechanisms of protein translocation into the ER.

Genetic analysis of the regulation of the voltage-gated calcium channel homolog Cch1 by the γ subunit homolog Ecm7 and cortical ER protein Scs2 in yeast.

The yeast Cch1/Mid1 Ca2+ channel is equivalent to animal voltage-gated Ca2+ channels and activated in cells incubated in low Ca2+ medium. We herein investigated the third subunit, Ecm7, under the same cell culture conditions. The deletion of ECM7 slightly lowered Ca2+ influx activity in the CNB1+ background, in which calcineurin potentially dephosphorylates Cch1, but markedly lowered this activity in the cnb1Δ background. The deletion of the C-terminal cytoplasmic region of Ecm7 also reduced Ca2+ influx activity. We identified a novel Cch1-interacting protein, Scs2, which is known as a cortical endoplasmic reticulum membrane protein. The deletion of SCS2 did not affect Ca2+ influx activity when calcineurin was inhibited by FK506, but enhanced this activity by 35% when the enzyme was not inhibited. However, this enhancement was canceled by the deletion of ECM7. These results suggest that Cch1/Mid1 is regulated differentially by Ecm7 and Scs2 in a manner that is dependent on the phosphorylation status of Cch1.

Coupling of a voltage-gated Ca2+ channel homologue with a plasma membrane H+ -ATPase in yeast.

Yeast has a homologue of mammalian voltage-gated Ca2+ channels (VGCCs), enabling the efficient uptake of Ca2+ . It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore-forming α1 and auxiliary α2 /δ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear. Therefore, we screened candidate proteins that interact with Mid1 by an unbiased proteomic approach and identified a plasma membrane H+ -ATPase, Pma1, as a candidate. Mid1 coimmunoprecipitated with Pma1, and Mid1-EGFP colocalized with Pma1-mCherry at the plasma membrane. The physiological relevance of their interaction was determined using the temperature-sensitive mutant, pma1-10. At the nonpermissive temperature, the membrane potential was less negative and Ca2+ uptake was lower in pma1-10 than in wild-type cells. Increased extracellular H+ increased the rate of Ca2+ uptake. Therefore, H+ extrusion by Pma1 may be important for Ca2+ influx through Cch1/Mid1. These results suggest that Pma1 interacts physically with Cch1/Mid1 Ca2+ channels to enhance their activity via its H+ -pumping activity.

Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana.

Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca(2+)-permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca(2+) in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca(2+)-permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca(2+) and protein interactions.

Involvement of Ca2+ in Vacuole Degradation Caused by a Rapid Temperature Decrease in Saintpaulia Palisade Cells: A Case of Gene Expression Analysis in a Specialized Small Tissue.

Saintpaulia (African violet) leaves are known to be damaged by a rapid temperature decrease when cold water is applied to the leaf surface; the injury is ascribed to the chloroplast damage caused by the cytosolic pH decrease following the degradation of the vacuolar membrane in the palisade cells. In this report, we present evidence for the involvement of Ca(2+) in facilitating the collapse of the vacuolar membrane and in turn in the temperature sensitivity of Saintpaulia leaves. In the presence of a Ca(2+) chelator (EGTA) or certain Ca(2+) channel inhibitors (Gd(3+) or La(3+)) but not others (verapamil or nifedipine), the pH of the vacuole, monitored through BCECF (2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein) fluorescence, did not increase in response to a rapid temperature drop. These pharmacological observations are consistent with the involvement of mechanosensitive Ca(2+) channels in the collapse of the vacuolar membrane. The high level of expression of an MCA- (Arabidopsis mechanosensitive Ca(2+) channel) like gene, a likely candidate for a mechanosensitive Ca(2+) channel(s) in plant cells, was confirmed in the palisade tissue in Saintpaulia leaves by using a newly developed method of gene expression analysis for the specialized small tissues.

Organellar mechanosensitive channels involved in hypo-osmoregulation in fission yeast.

MscS and MscL, bacterial mechanosensitive channels, play crucial roles in the hypo-osmotic shock response. However, only MscS has homologs in eukaryotes. These homologs are called MscS-like proteins or MSL proteins. MSL proteins have changed both structurally and functionally during evolution and are now localized not only to the membrane of the chloroplast, which is thought to be a descendant of an ancient, free-living bacterium, but also the cell membrane and the endoplasmic reticulum (ER) membrane, suggesting that the role of MSL proteins has diverged. In this brief review, we mainly focus on two MSL proteins in the fission yeast Schizosaccharomyces pombe that are localized in the ER membrane and protect cells from hypo-osmotic shock-induced death by regulating intracellular Ca(2+) concentrations. We also discuss Arabidopsis thaliana MSL proteins and other yeast ion channels in terms of osmoregulation in eukaryotes.

Mechanosensitive channel candidate MCA2 is involved in touch-induced root responses in Arabidopsis.

The Ca(2+)-permeable mechanosensitive (MS) channel is a mechanical stress sensor. We previously reported that Arabidopsis MCA1 and its paralog MCA2 functioned individually as Ca(2+)-permeable MS channels. In the present study, we showed that the primary roots of the mca2-null mutant behaved abnormally on the surface of hard medium. First, primary roots are known to exhibit a skewing growth pattern on the surface of vertically placed agar medium. On such surface, the primary roots of mca2-null skewed more than those of the wild type. Second, when seedlings were grown on a tilted agar surface, the primary root of mca2-null showed abnormal waving patterns. Third, wild-type seedlings eventually died when grown on horizontally placed 3.2% gelrite medium, which was too hard to allow the primary roots of the wild type to penetrate, because their primary roots sprang from the surface of the medium and may have been unable to absorb water and nutrients. In contrast, the primary roots of mca2-null, but not those of mca1-null, were able to creep over the surface of the medium and grow. Fourth, when grown on the surface of 3.2% agar medium supplemented with 30 mM CaCl2, only mca2-null grew with a root that coiled in a clockwise direction. Lastly, on the surface of vertically placed rectangular plates that allowed primary roots to grow vertically down to the frame of the plate, wild-type primary roots grew horizontally after touching the frame at an angle of 90(∘). During the horizontal growth, only the extreme root tips maintained contact with the frame. In contrast, the primary roots of mca2-null allowed not only the extreme root tips, but also the meristem and elongation zones to maintain contact with the frame during horizontal growth. These results suggest that MCA2 is involved in touch-related root responses.

Mechanosensitive channels Msy1 and Msy2 are required for maintaining organelle integrity upon hypoosmotic shock in Schizosaccharomyces pombe.

The mechanosensitive channels, Mys1 and Msy2, in fission yeast are localized in the endoplasmic reticulum membrane and control cytoplasmic Ca(2+) levels in the hypoosmotic response. We here investigated changes in organellar structures with hypoosmotic shock using transmission electron microscopy. While msy1(-) and msy2(-) single mutant cells developed a number of swollen vacuoles following hypoosmotic shock, similar to wild-type cells, msy1(-) msy2(-) double mutant cells only had two abnormally large vacuoles and cracks between the inner and outer nuclear membranes. These results suggest that Msy1 and Msy2 may be involved in maintaining vacuole integrity and protecting the nuclear envelope upon hypoosmotic shock and also that these two channels are functionally complementary.