P-type Ca2 Research Articles

MiR1432 negatively regulates cold tolerance by targeting OsACAs.

miRNAs function as negative regulators that significantly influence plant growth and stress responses. Within rice and other monocotyledonous plants, miR1432 plays a conserved role in seed development and disease resistance. However, its involvement in the response to abiotic stresses remains unclear. Our study aimed to elucidate this mechanism by predicting the targeting of the rice P-type IIB Ca2+ ATPase gene OsACAs by miR1432 and identifying its cleavage sites via 5'RACE. We observed induced expression of miR1432 and its target gene, OsACA6, under abiotic stresses. Overexpression (OX) of miR1432 and suppression of OsACA6 resulted in reduced cold, salt, and drought tolerance, while OsACA6 suppression/knockout and OX had opposite effects on cold tolerance. Additionally, miR1432 may target other OsACA6 homologs. RNA-sequencing data highlighted the differential expression of stress-related genes in miR1432-overexpressing rice. Furthermore, miR1432-overexpressing rice exhibited weakened vigor, dwarfism, yellowing leaves and reduced fertility. Collectively, our results strongly suggest that miR1432 not only negatively modulates abiotic stress tolerance by suppressing Ca2+ ATPase gene(s) but also influences plant growth and development.

P-type calcium ATPases play important roles in biotic and abiotic stress signaling.

Knowledge of Ca2+-ATPases is imperative for improving crop quality/ food security, highly threatened due to global warming. Ca2+-ATPases modulates calcium, essential for stress signaling and modulating growth, development, and immune activities. Calcium is considered a versatile secondary messenger and essential for short- and long-term responses to biotic and abiotic stresses in plants. Coordinated transport activities from both calcium influx and efflux channels are required to generate cellular calcium signals. Various extracellular stimuli cause an induction in cytosolic calcium levels. To cope with such stresses, it is important to maintain intracellular Ca2+ levels. Plants need to evolve efficient efflux mechanisms to maintain Ca2+ ion homeostasis. Plant Ca2+-ATPases are members of the P-type ATPase superfamily and localized in the plasma membrane and endoplasmic reticulum (ER). They are required for various cellular processes, including plant growth, development, calcium signaling, and even retorts to environmental stress. These ATPases play an essential role in Ca2+ homeostasis and are actively involved in Ca2+ transport. Plant Ca2+-ATPases are categorized into two major classes: type IIA and type IIB. Although these two classes of ATPases share similarities in protein sequence, they differ in their structure, cellular localization, and sensitivity to inhibitors. Due to the emerging role of Ca2+-ATPase in abiotic and biotic plant stress, members of this family may help promote agricultural improvement under stress conditions. This review provides a comprehensive overview of P-type Ca2+-ATPase, and their role in Ca2+ transport, stress signaling, and cellular homeostasis focusing on their classification, evolution, ion specificities, and catalytic mechanisms. It also describes the main aspects of the role of Ca2+-ATPase in transducing signals during plant biotic and abiotic stress responses and its role in plant development and physiology.

Dietary Clostridium butyricum and 25-Hydroxyvitamin D3 modulate bone metabolism of broilers through the gut—brain axis

Leg disorders have become increasingly common in broilers, leading to lower meat quality and major economic losses. This study evaluated the effects of dietary supplementation with Clostridium butyricum (C. butyricum) and 25-hydroxyvitamin D3 (25-OH-D3) on bone development by comparing growth performance, tibial parameters, Ca and P contents of tibial ash, bone development-related indicators' level, and cecal short-chain fatty acids in Cobb broilers. All birds were divided into four treatment groups, which birds fed either a basal diet (Con), basal diet + 75 mg chlortetracycline/kg (Anti), basal diet + C. butyricum at 109 CFU/kg (Cb), basal diet + C. butyricum at 109 CFU/kg and 25-OH-D3 at 25 μg/kg (CbD), or basal diet + 25-OH-D3 at 25 μg/kg (CD). Our results suggest that the dietary supplementation in Cb, CbD, and CD significantly increased the body weight (BW) and average daily gain (ADG), and reduced the feed-to-weight ratio (F/G) at different stages of growth (P < 0.05). Dietary supplementation in Cb, CbD, and CD prolonged (P < 0.05) the behavioral responses latency-to-lie (LTL) time, reduced (P < 0.05) the levels of osteocalcin (BGP) and peptide tyrosine (PYY), and increased (P < 0.05) serotonin (5-HT) and dopamine (DA). Treatment with Cb increased (P < 0.05) the levels of acetic acid, isobutyric acid, butyric acid, and isovaleric acid compared with those in Con group. The cecal metagenome showed that Alistipes spp. were significantly more abundant in Cb, CbD, and CD groups (P < 0.05). A total of 12 metabolic pathways were significantly affected by supplementation, including the signaling pathways of glucagon, insulin, and PI3K-AKT; primary and secondary bile acid biosynthesis; and P-type Ca 2+ transporters (P < 0.05). Hence, the CbD supplementation modulates bone metabolism by regulating the mediators of gut-brain axis, which may inform strategies to prevent leg diseases and improve meat quality in broilers.

Astragaloside depresses compound action potential in sciatic nerve of frogs involved in L-type Ca2+-channel dependent mechanism

The sciatic nerve is the largest sensorimotor nerve within the peripheral nervous system (PNS), possessing the ability to produce endogenous neurotrophins. Compound nerve action potentials (CNAPs) are regarded as a physiological/pathological indicator to identify nerve activity in signal transduction of the PNS. Astragaloside (AST), a small-molecule saponin purified from Astragalus membranaceus, is widely used to treat chronic disease. Nonetheless, the regulatory effects of AST on the sciatic nerve remain unknown. Therefore, the present investigation was undertaken to study the effect of AST on CNAPs of frog sciatic nerves. Here, AST depressed the conduction velocity and amplitude of CNAPs. Importantly, the AST-induced responses could be blocked by a Ca2+-free medium, or by applying all Ca2+ channel antagonists (CdCl2/LaCl3) or L-type Ca2+ channel blockers (nifedipine/diltiazem), but not the T-type and P-type Ca2+ channel antagonist (NiCl2). Altogether, these findings suggested that AST may attenuate the CNAPs of frog sciatic nerves in vitro via the L-type Ca2+-channel dependent mechanisms.

Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [3 H]choline and subsequent synthesis and release of [3 H]ACh were investigated in rat striatal segments. Incubation with [3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [3 H]choline and [3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [3 H]choline led predominantly to the accumulation of [3 H]ACh. Electrical stimulation and KCl depolarization selectively released [3 H]ACh but not [3 H]choline. [3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

A Transient Receptor Potential-like Calcium Ion Channel in the Filamentous Fungus Aspergillus nidulans.

Transient Receptor Potential (TRP) proteins constitute a superfamily that encodes transmembrane ion channels with highly diverse permeation and gating properties. Filamentous fungi possess putative TRP channel-encoded genes, but their functions remain elusive. Here, we report that a putative TRP-like calcium channel, trpR, in the filamentous fungus Aspergillus nidulans, performs important roles in conidiation and in adapting to cell wall disruption reagents in a high temperature-induced defect-dependent manner, especially under a calcium-limited culture condition. The genetic and functional relationship between TrpR and the previously identified high-affinity calcium channels CchA/MidA indicates that TrpR has an opposite response to CchA/MidA when reacting to cell wall disruption reagents and in regulating calcium transients. However, a considerable addition of calcium can rescue all the defects that occur in TrpR and CchA/MidA, meaning that calcium is able to bypass the necessary requirement. Nevertheless, the colocalization at the membrane of the Golgi for TrpR and the P-type Golgi Ca2+ ATPase PmrA suggests two channels that may work as ion transporters, transferring Ca2+ from the cytosol into the Golgi apparatus and maintaining cellular calcium homeostasis. Therefore, combined with data for the trpR deletion mutant revealing abnormal cell wall structures, TrpR works as a Golgi membrane calcium ion channel that involves cell wall integration.

Relationship Between Tensile Strength and Durability of Oxide Thermoelectric Modules

The durability of thermoelectric modules is investigated by conducting material property tests over a range of module preparation pressures. Tensile strength and bending strength tests are carried out at room temperature for devices comprising one-piece oxide thermoelectric elements (p-type Ca2.7Bi0.3Co4O9 and n-type CaMn0.98Mo0.02O3), silver paste, and silver sheet between two sheets of alumina substrate. Tensile strength was found to increase with device preparation pressure, reaching approximately 20 MPa for p-type devices at a preparation pressure of 6.36 MPa. The tensile strength of the n-type devices was less than 10 MPa. Module performance was tested under heat cycling and vibration by measuring thermoelectric generation properties including internal resistance, open-circuit voltage, and output power of the modules. Modules prepared at pressures above 3.19 MPa were found to have no degradation of thermoelectric properties during the vibration tests. Furthermore, no degradation during heat cycling was observed for modules prepared at 6.36 MPa. Continuous power generation tests at 973 K in air were performed for the modules prepared at 0.116 MPa and 6.36 MPa and no degradation was observed over 2400 h. For the module prepared at 6.36 MPa, no degradation was observed over 10000 h.

Genome-wide analysis of manganese homeostasis in Saccharomyces cerevisiae.

Manganese is a crucial cofactor for a wide range of enzymes in many living cells. However, excessive manganese can induce cellular toxicity by affecting a number of metabolic reactions and even cause severe neurological diseases in humans. To understand manganese homeostasis fully, a genome-scale screen was performed using the homozygous diploid yeast deletion mutant library. We identified 152 manganese-sensitive and 13 manganese-tolerant gene deletion mutations. We found that 62 of the manganese-sensitive mutants (40% of the total) accumulated higher intracellular manganese compared to wild type. Our results also reinforced the genetic functional link between manganese and calcium, and the addition of 100 mM CaCl2 confirmed that the manganese sensitivities of 103 (67.8 % of the total) strains could be inhibited by calcium. Finally, this study demonstrated that there might be some significant interactions between manganese and calcium regulated by the calcium/calcineurin signaling pathway through the P-type Ca2+- and Mn2+-transporting ATPase, Pmr1. Taken together, our current findings would provide new insights into the molecular causes of manganese toxicity in yeast cells.

The role of P-type IIA and P-type IIB Ca2+-ATPases in plant development and growth.

As sessile organisms, plants have evolved mechanisms to adapt to variable and rapidly fluctuating environmental conditions. Calcium (Ca2+) in plant cells is a versatile intracellular second messenger that is essential for stimulating short- and long-term responses to environmental stresses through changes in its concentration in the cytosol ([Ca2+]cyt). Increases in [Ca2+]cyt direct the strength and length of these stimuli. In order to terminate them, the cells must then remove the cytosolic Ca2+ against a concentration gradient, either taking it away from the cell or storing it in organelles such as the endoplasmic reticulum (ER) and/or vacuoles. Here, we review current knowledge about the biological roles of plant P-type Ca2+-ATPases as potential actors in the regulation of this cytosolic Ca2+ efflux, with a focus the IIA ER-type Ca2+-ATPases (ECAs) and the IIB autoinhibited Ca2+-ATPases (ACAs). While ECAs are analogous proteins to animal sarcoplasmic-endoplasmic reticulum Ca2+-ATPases (SERCAs), ACAs are equivalent to animal plasma membrane-type ATPases (PMCAs). We examine their expression patterns in cells exhibiting polar growth and consider their appearance during the evolution of the plant lineage. Full details of the functions and coordination of ECAs and ACAs during plant growth and development have not yet been elucidated. Our current understanding of the regulation of fluctuations in Ca2+ gradients in the cytoplasm and organelles during growth is in its infancy, but recent technological advances in Ca2+ imaging are expected to shed light on this subject.

Coordinated Roles of the Putative Ceramide-Conjugation Protein, Cwh43, and a Mn2+-Transporting, P-Type ATPase, Pmr1, in Fission Yeast

Genetically controlled mechanisms of cell division and quiescence are vital for responding to changes in the nutritional environment and for cell survival. Previously, we have characterized temperature-sensitive (ts) mutants of the cwh43 gene in fission yeast, Schizosaccharomyces pombe, which is required for both cell proliferation and nitrogen starvation-induced G0 quiescence. Cwh43 encodes an evolutionarily conserved transmembrane protein that localizes in endoplasmic reticulum (ER). Defects in this protein fail to divide in low glucose and lose mitotic competence under nitrogen starvation, and also affect lipid metabolism. Here, we identified mutations of the pmr1 gene, which encodes an evolutionarily conserved Ca2+/Mn2+-transporting P-type ATPase, as potent extragenic suppressors of ts mutants of the cwh43 gene. Intriguingly, these pmr1 mutations specifically suppressed the ts phenotype of cwh43 mutants, among five P-type Ca2+- and/or Mn2+-ATPases reported in this organism. Cwh43 and Pmr1 co-localized in the ER. In cwh43 mutant cells, addition of excessive manganese to culture media enhanced the severe defect in cell morphology, and caused abnormal accumulation of a cell wall component, 1, 3-β-glucan. In contrast, these abnormal phenotypes were abolished by deletion of the pmr1+ gene, as well as by removal of Mn2+ from the culture medium. Furthermore, nutrition-related phenotypes of cwh43 mutant cells were rescued in the absence of Pmr1. Our findings indicate that the cellular processes regulated by Cwh43 are appropriately balanced with Pmr1-mediated Mn2+ transport into the ER.

Expression of the Tobacco Non-symbiotic Class 1 Hemoglobin Gene Hb1 Reduces Cadmium Levels by Modulating Cd Transporter Expression Through Decreasing Nitric Oxide and ROS Level in Arabidopsis.

Hemoglobin (Hb) proteins are ubiquitous in plants, and non-symbiotic class 1 hemoglobin (Hb1) is involved in various biotic and abiotic stress responses. Here, the expression of the tobacco (Nicotiana tabacum) hemoglobin gene NtHb1 in Arabidopsis (Arabidopsis thaliana) showed higher cadmium (Cd) tolerance and lower accumulations of Cd, nitric oxide (NO), and reactive oxygen species (ROS) like hydrogen peroxide (H2O2). NtHb1-expressing Arabidopsis exhibited a reduced induction of NO levels in response to Cd, suggesting scavenging of NO by Hb1. In addition, transgenic plants had reduced accumulation of ROS and increased activities of antioxidative enzymes (catalase, superoxide dismutase, and glutathione reductase) in response to Cd. While the expression of the Cd exporters ABC transporter (PDR8) and Ca2+/H+ exchangers (CAXs) was increased, that of the Cd importers iron responsive transporter 1 (IRT1) and P-type 2B Ca2+ ATPase (ACA10) was reduced in response to Cd. When Col-0 plants were treated with the NO donor sodium nitroprusside (SNP) and H2O2, the expression pattern of Cd transporters (PDR8, CAX3, IRT1, and ACA10) was reversed, suggesting that NtHb1 expression decreased the Cd level by regulating the expression of Cd transporters via decreased NO and ROS. Correspondingly, NtHb1-expressing Arabidopsis showed increased Cd export. In summary, the expression of NtHb1 reduces Cd levels by regulating Cd transporter expression via decreased NO and ROS levels in Arabidopsis.

Alcohol withdrawal upregulates mRNA encoding for CaV2.1-α1 subunit in the rat inferior colliculus.

We previously reported increased current density through P-type voltage-gated Ca2+ channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased P-type channel current is currently unknown. Here, we probe changes in mRNA and protein expression of the pore-forming CaV2.1-α1 (P/Q-type) subunits in IC neurons during the course of alcohol withdrawal-induced seizures (AWSs). Rats received three daily doses of ethanol or the vehicle every 8h for 4 consecutive days. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV2.1-α1 subunits were measured. In separate experiments, rats were tested for acoustically evoked seizure susceptibility 3, 24, and 48h after alcohol withdrawal. AWSs were observed 24h after withdrawal; no seizures were observed at 3 or 48h or in the control-treated rats. Compared to control-treated rats, the mRNA levels of the CaV2.1-α1 subunit were increased 1.9-fold and 2.1-fold at 3 and 24h, respectively; change in mRNA expression was nonsignificant at 48h following alcohol withdrawal. Western blot analyses revealed that protein levels of the CaV2.1-α1 subunits were not altered in IC neurons following alcohol withdrawal. We conclude that expression of the Cacna1a mRNA increased before the onset of AWS susceptibility, suggesting that altered CaV2.1 channel expression may play a role in AWS pathogenesis.

(Invited) Durability of Thermoelectric Modules and Development of Generation Systems

The durability against high temperature and heat cycling, and vibration of thermoelectric modules composed of p-type Ca2.7Bi0.3Co4O9 and n-type CaMn0.98Mo0.02O3 devices was investigated quantitatively. Long life time tests under power generation have been carried out for the oxide modules up to 1073 K of the heat source temperature by water circulation at 293 K under the air atmosphere. No degradation in the continuous power generation is confirmed up to 1073 K (T H). The durability against heat cycling between 373 and 873 K of T H was investigated in air. The maximum out-put power is kept constant during 700 times of the heat cycling.

Melatonin-mediated inhibition of Purkinje neuron P-type Ca²⁺ channels in vitro induces neuronal hyperexcitability through the phosphatidylinositol 3-kinase-dependent protein kinase C delta pathway.

Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P-type Ca(2+) channels and action-potential firing in rat Purkinje neurons. Melatonin at 0.1 μm reversibly decreased peak currents (I(Ba)) by 32.9%. This effect was melatonin receptor 1 (MT(R1)) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the Gβ subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors abolished the melatonin-induced decrease in I(Ba). Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1-fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin-induced decrease in I(Ba). Melatonin application increased the membrane abundance of PKCδ, and PKCδ inhibition (either pharmacologically or genetically) abolished the melatonin-induced IBa response. Functionally, melatonin increased spontaneous action-potential firing by 53.0%; knockdown of MT(R1) and blockade of P-type channels abolished this effect. Thus, our results suggest that melatonin inhibits P-type channels through MT(R1) activation, which is coupled sequentially to the βγ subunits of G(i/o)-protein and to downstream PI3K-dependent PKCδ signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.

Low pH-induced changes of antioxidant enzyme and ATPase activities in the roots of rice (Oryza sativa L.) seedlings.

Soil acidification is the main problem in the current rice production. Here, the effects of low pH on the root growth, reactive oxygen species metabolism, plasma membrane functions, and the transcript levels of the related genes were investigated in rice seedlings (Oryza sativa L.) in a hydroponic system at pH 3.5, 4.5, and 5.5. There were two hybrid rice cultivars in this trial, including Yongyou 12 (YY12, a japonica hybrid) and Zhongzheyou 1 (ZZY1, an indica hybrid). Higher H+ activity markedly decreased root length, the proportion of fine roots, and dry matter production, but induced a significant accumulation of hydrogen peroxide (H2O2), and led to serious lipid peroxidation in the roots of the two varieties. The transcript levels of copper/zinc superoxide dismutase 1 (Cu/Zn SOD1), copper/zinc superoxide dismutase 2 (Cu/Zn SOD2), catalase A (CATA) and catalase B (CATB) genes in YY12 and ZZY1 roots were significantly down-regulated after low pH exposure for two weeks. Meanwhile, a significant decrease was observed in the expression of the P-type Ca2+-ATPases in roots at pH 3.5. The activities of antioxidant enzymes (SOD, CAT) and plasma membrane (PM) Ca2+-ATPase in the two varieties were dramatically inhibited by strong rhizosphere acidification. However, the expression levels of ascorbate peroxidase 1 (APX1) and PM H+-ATPase isoform 7 were up-regulated under H+ stress compared with the control. Significantly higher activities of APX and PM H+-ATPase could contribute to the adaptation of rice roots to low pH.

Bcpmr1 encodes a P-type Ca2+/Mn2+-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea

The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn2+ provided by the Ca2+/Mn2+-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca2+/Mn2+-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.

Cordycepin Decreases Compound Action Potential Conduction of Frog Sciatic Nerve In Vitro Involving Ca (2+) -Dependent Mechanisms.

Cordycepin has been widely used in oriental countries to maintain health and improve physical performance. Compound nerve action potential (CNAP), which is critical in signal conduction in the peripheral nervous system, is necessary to regulate physical performance, including motor system physiological and pathological processes. Therefore, regulatory effects of cordycepin on CNAP conduction should be elucidated. In this study, the conduction ability of CNAP in isolated frog sciatic nerves was investigated. Results revealed that cordycepin significantly decreased CNAP amplitude and conductive velocity in a reversible and concentration-dependent manner. At 50 mg/L cordycepin, CNAP amplitude and conductive velocity decreased by 62.18 ± 8.06% and 57.34% ± 6.14% compared with the control amplitude and conductive velocity, respectively. However, the depressive action of cordycepin on amplitude and conductive velocity was not observed in Ca2+-free medium or in the presence of Ca2+ channel blockers (CdCl2/LaCl3). Pretreatment with L-type Ca2+ channel antagonist (nifedipine/deltiazem) also blocked cordycepin-induced responses; by contrast, T-type and P-type Ca2+ channel antagonists (Ni2+) failed to block such responses. Therefore, cordycepin decreased the conduction ability of CNAP in isolated frog sciatic nerves via L-type Ca2+ channel-dependent mechanism.

Five vacuolar Ca2+ exchangers play different roles in calcineurin-dependent Ca2+/Mn2+ tolerance, multistress responses and virulence of a filamentous entomopathogen

Multiple Vcx1 (vacuolar calcium exchanger) paralogues exist in many filamentous fungi but are functionally unexplored unlike a single Vcx1 ortholog well characterized in yeasts. Here we show that five Vcx1 paralogues (Vcx1A–E) in Beauveria bassiana are conditionally functional for intracellular Ca2+ homeostasis and contribute differentially to multistress tolerance and virulence in the filamentous entomopathogen. Each vcx1 deletion drastically upregulated transcriptional expressions of four other partners and six P-type Ca2+-ATPases, resulting in elevated or lowered intracellular Ca2+ concentration in some deletion mutants treated with Ca2+ stress or untreated at 25 and 30°C. When calcineurin was inactivated by cyclosporine A, Ca2+ tolerance decreased by 11–17% in five Δvcx1 mutants, but Mn2+ sensitivity increased only in Δvcx1A and Δvcx1D, at optimal 25°C. These two mutants were also more sensitive to Ca2+ stress at 30°C when calcineurin was active, and showed minor growth defect at 25 and 30°C when calcineurin was inactive. Moreover, all the Δvcx1 mutants were more sensitive to dithiothreitol (stress-response trigger to endoplasmic reticulum) and Congo red (cell wall stressor); three of them were consistently less tolerant to the oxidants menadione and H2O2. The fungal virulence to Galleria mellonella larvae decreased by 15–40% in four Δvcx1 mutants excluding Δvcx1E, which was uniquely defective in conidial thermotolerance. All the changes were restored by each vcx1 complementation. Our findings indicate that the five Vcx1 paralogues in B. bassiana contribute differentially to calcineurin-dependent Ca2+/Mn2+ tolerance, multistress responses and virulence, and recall attention to multifunctional Vcx1 paralogues in filamentous fungi.

Putative PmrA and PmcA are important for normal growth, morphogenesis and cell wall integrity, but not for viability in Aspergillus nidulans.

P-type Ca(2+)-transporting ATPases are Ca(2+) pumps, extruding cytosolic Ca(2+) to the extracellular environment or the intracellular Ca(2+) store lumens. In budding yeast, Pmr1 (plasma membrane ATPase related), and Pmc1 (plasma membrane calcium-ATPase) cannot be deleted simultaneously for it to survive in standard medium. Here, we deleted two putative Ca(2+) pumps, designated AnPmrA and AnPmcA, from Aspergillus nidulans, and obtained the mutants ΔanpmrA and ΔanpmcA, respectively. Then, using ΔanpmrA as the starting strain, the promoter of its anpmcA was replaced with the alcA promoter to secure the mutant ΔanpmrAalcApmcA or its anpmcA was deleted completely to produce the mutant ΔanpmrAΔpmcA. Different from the case in Saccharomyces cerevisiae, double deletion of anpmrA and anpmcA was not lethal in A. nidulans. In addition, deletion of anpmrA and/or anpmcA had produced growth defects, although overexpression of AnPmc1 in ΔanpmrAalcApmcA could not restore the growth defects that resulted from the loss of AnPmrA. Moreover, we found AnPmrA was indispensable for maintenance of normal morphogenesis, especially in low-Ca(2+)/Mn(2+) environments. Thus, our findings suggest AnPmrA and AnPmcA might play important roles in growth, morphogenesis and cell wall integrity in A. nidulans in a different way from that in yeasts.

Erratum to: OsACA6, a P-type 2B Ca2+ ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load

Erratum to: OsACA6, a P-type 2B Ca2+ ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load