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OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis

Nature volume 514, pages 367–371 (2014)Cite this article

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A Corrigendum to this article was published on 25 February 2015

Abstract

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes1,2,3,4. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca2+]i)5,6. Considering that in bacteria and animals osmosensing Ca2+ channels serve as osmosensors7,8, hyperosmolality-induced [Ca2+]i increases have been widely speculated to be involved in osmosensing in plants1,9. However, the molecular nature of corresponding Ca2+ channels remain unclear6,10,11. Here we describe a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens we isolated Arabidopsis mutants that exhibit low hyperosmolality-induced [Ca2+]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca2+]i increase 1 (osca1), displays impaired osmotic Ca2+ signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca2+]i increases induced by a stimulus in plants, opening up new avenues for studying Ca2+ machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops.

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Figure 1: Isolation of a genetic mutant with reduced hyperosmolality-induced [Ca2+]i increases (OICI) in Arabidopsis.
Figure 2: Impaired guard cell osmotic stress signalling and attenuated plant responses to osmotic stress in osca1 plants.
Figure 3: OSCA1 encodes a novel integral protein in the plasma membrane.
Figure 4: OSCA1 forms hyperosmolality-gated calcium-permeable channels in HEK293 cells.

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Acknowledgements

We thank M. R. Knight for providing an aequorin vector and aequorin expressing Arabidopsis seeds, S. Gilroy for a YC3.6 vector and YC3.6 expressing Arabidopsis seeds, M. R. Knight and S. Gilroy for sharing unpublished data on genetic screening and physical mapping, X. Dong and M. Chen for advice on physical mapping, J. Grandl, G. Chen and Q. Liu for advice concerning electrophysiology, W. G. Zhang and M. H. Zhu for providing HEK293 cells and advice on transfection, Y. Gao and S. Johnson for confocal imaging, and D. R. McClay, T.-p. Sun, J. Grandl and P. N. Benfey for discussions and critical reading of the manuscript. F.Y. and J.Z. were supported in part by grants from Hangzhou Normal University (PanDeng11001008001) and Zhejiang NSF (Z3110433). This work was supported by grants from USDA (CSREES-2005-35304-16196, CSREES-2006-35100-17304) and NSF (MCB-0451072, IOS-0848263) to Z.-M.P.

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Author notes

  1. Huimin Yang and Yan Xue: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Duke University, Durham, 27708, North Carolina, USA

    Fang Yuan, Huimin Yang, Yan Xue, Dongdong Kong, Rui Ye, Chijun Li, Jingyuan Zhang, Lynn Theprungsirikul, Tayler Shrift, Bryan Krichilsky, Yikun He, James N. Siedow & Zhen-Ming Pei

  2. Center on Plant Environmental Sensing, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China,

    Fang Yuan & Jingyuan Zhang

  3. Department of Physics, Duke University, Durham, 27708, North Carolina, USA

    Douglas M. Johnson & Gary B. Swift

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  1. Fang Yuan
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Contributions

F.Y., H.Y., Y.X., D.K., R.Y., C.L., J.Z., T.S., B.K., D.M.J. and G.B.S. conducted aequorin imaging and genetic screen. F.Y., H.Y., Y.X., J.Z., L.T., conducted map-based cloning. F.Y., Y.X., D.K., R.Y. and Z.-M.P. conducted Ca2+ imaging and electrophysiological analyses in HEK293 cells and plant cells. F.Y., H.Y., Y.X., J.Z., L.T., T.S., B.K. and Y.H. carried out physiological analyses. Z.-M.P. designed the overall research with input from J.N.S. Z.-M.P., F.Y. and J.N.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Fang Yuan or Zhen-Ming Pei.

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Extended data figures and tables

Extended Data Figure 1 Events occurring after osmotic stress treatment, and optimized conditions for genetic screens for mutants with low hyperosmolality-induced [Ca2+]i increases.

a, Schematic illustration of events occurring after osmotic stress treatment. It is known that osmotic stress triggers a signalling cascade, in which the earliest detectable event is an increase in [Ca2+]i that lasts ∼5 min (blue)5,55,56. For immediate responses, the signal is funnelled to downstream events, such as the activation of ASK1 protein kinase, ABA accumulation and stomatal closure, leading to the reduction of water loss4. For sustainable responses, the expression profiles for many genes, such as DREB2A and RD29A, are altered57. Collectively, although these events might start as early as the [Ca2+]i increase, they display a dynamic change (phase 1), and take a long time to reach a relative steady state (phase 2; Supplementary Information). Clearly, in contrast to traditional genetic screens, in which the phenotypes scored can take hours or days to reach a steady state58, the entire transient OICI event lasts only ∼5 min, which could be used to genetically dissect osmosensing. Recently, similar screens using pathogen elicitors and ATP have been carried out, while the associated Ca2+ channels have not been identified12,13. The amplitudes of coloured polygons depict the dynamic activities of these events evoked by osmotic stress. b, Optimized conditions for genetic screens for mutants with low hyperosmolality-induced [Ca2+]i increases (OICI). EMS-mutagenized aequorin-expressing Arabidopsis M2 seeds were used to determine the optimum genetic screening conditions. Individual seeds were planted evenly using a template in 150 mm × 15 mm Petri dishes, and grown for 9 days. The sorbitol solution at an indicated concentration was added into the Petri dish, and the aequorin images were acquired. Sorbitol concentrations from 0 to 800 mM were tested and representative aequorin images are shown (bottom). Relative [Ca2+]i in leaves is scaled by a pseudo-colour bar. Corresponding relative [Ca2+]i for each individual seedling was analysed and plotted (top). At 600 mM sorbitol concentration, about 95% of seedlings showed an OICI using an artificial cut-off value (red line), which could be practically used to phenotype/score seedlings. Similar results were seen in more than 10 independent experiments and one representative experiment is shown. c, Isolation of individuals with low OICI in leaves in the first-round screen. The bright-field image was used to identify the position for each seedling (left). Individual seedlings with lower leaf OICI signals in the bioluminescence image (right) were circled via image analyses, and selected seedlings were transferred from the Petri dish to soil. At the first round we picked up seedlings with low leaf OICI signals as putative osca1 candidates.

Extended Data Figure 2 Defect in hyperosmolality-induced [Ca2+]i increases in osca1.

a, b, Similar total amount of aequorin in wild-type (WT) and osca1 seedlings. The same seedlings used in Fig. 1a were treated with a solution containing 0.9 M CaCl2 and 10% (v/v) ethanol to measure the total amount of aequorin, and no difference between wild type and osca1 was observed (a). Similar results were seen in >20 separate experiments. Quantification of total amount of aequorin in wild-type and osca1 plants from experiments as in a is plotted as mean ± s.e.m. (b; n = 6; P > 0.8). c, The osca1 mutant shows reduced OICIs. The aequorin-expressing wild-type and osca1 seedlings grown side-by-side were treated with water or 440 mOsm solutions containing sorbitol, mannitol, sucrose, ribose or N-methyl-d-glucamine (NMDG), and changes in [Ca2+]i in leaves were recorded. Data are mean ± s.e.m. (n = 33 for sorbitol, 29 for mannitol and sucrose, 26 for ribose and 21 for NMDG). The responses to these compounds were significantly reduced in osca1 compared to those in wild type (P < 0.005). d, Averaged increases in [Ca2+]i in wild-type and osca1 roots plotted as a function of applied sorbitol concentrations. Seedlings were grown in a Petri dish that was placed vertically similar to those in Fig. 1a, and aequorin images were acquired and analysed as in Fig. 1. Data for three separate experiments representing 30 seedlings are shown (mean ± s.d.; two-way ANOVA, P < 0.01). e−g, Reduced OICIs in root cells in osca1. FRET imaging of OICIs was carried out in root cells in wild-type and osca1 plants expressing the Ca2+ indicator protein YC3.6. Emission images (F535 and F485) of roots were taken every 3 s, and ratiometric images before and 20 s after addition of 600 mM sorbitol are shown (e). The F535:F485 ratio is scaled by a pseudo-colour bar. The relative [Ca2+]i (F535:F485) in response to sorbitol treatment was quantified from these root cells in e (f; mean ± s.e.m.; n = 10). Peak changes in ratios from experiments similar to e and f are shown (g; boxes represent s.e., error bars are s.d.; n = 26 seedlings; P < 0.001).

Extended Data Figure 3 OSCA1 acts upstream of ABA signalling in stomatal closure and root growth.

a, Comparison of ABA-induced stomatal closing in wild type and osca1. Data shown are mean ± s.e.m. (n = 60; two-way ANOVA, P > 0.5). Stomatal apertures were normalized with respect to the width in the absence of ABA. b, OSCA1 controls transpirational water loss in response to desiccation treatment. Rosette leaves from wild-type and osca1 seedlings were detached, and transpirational water loss was analysed at the indicated time points after leaf detachment. Water loss was calculated as a percentage of the initial fresh weight. Data shown are mean ± s.e.m. (n = 25 leaves; two-way ANOVA, P < 0.001). c, Wild-type and osca1 plants were grown in ½ MS media containing 0–1 μM ABA, and root lengths were analysed similarly as in Fig. 2k, l. Data from three independent experiments are shown (mean ± s.d.; n = 30 seedlings; two-way ANOVA, P > 0.2).

Extended Data Figure 4 Genetic analysis and map-based cloning of OSCA1.

a, All F1 seedlings derived from osca1 × wild-type (WT; ColAQ, Col-0 expressing aequorin) crosses showed wild-type OICI signals. F2 seedlings showed a 3:1 wild-type:osca1 segregation, suggesting that the osca1 phenotype resulted from a recessive mutation in a single nuclear gene. Note that it was not feasible to phenotype the F2 and F3 populations from crosses between osca1 × Landsberg erecta (Ler). The F2 seedlings, which were derived from osca1 × Wassilewskija (Ws) crosses and also identified as aequorin homozygous, showed a 3:1 wild-type:osca1 segregation. The same amount of F2 seeds for each cross were placed in Petri dishes and OICI phenotypes were scored for individual seedlings (mean ± s.e.m.; n = 4 and 6 for osca1 × ColAQ and osca1 × Ws crosses, respectively). b, Physical mapping of OSCA1. OSCA1 was positioned between JV30/31 and MN4.2 markers in the short arm of chromosome 4 close to centromere in a segregating F2 population derived from the osca1 × Ws cross. OSCA1 was fine-mapped to a region between OS114 and OS125 by analysing 1,256 recombinant chromosomes in the F2 population with molecular markers listed in c. We sequenced all open reading frames (ORFs) in this region between these two makers and identified two mutations in an ORF, which corresponded to the gene At4g04340. c, Molecular markers developed for fine mapping. At the time when we were fine-mapping osca1, the whole-genome sequence for Ws was not available. Thus, we used the 250,000 single-nucleotide polymorphism (SNP) data to develop these markers. d, OSCA1 encodes a protein with transmembrane α-helices. The transmembrane α-helices (TM; blue), the putative ion channel pore-forming domain (green), and mutations of glycine 59 to arginine (G59R) and glycine 507 to aspartic acid (G507D) in red in osca1 are shown.

Extended Data Figure 5 Hydropathy and transmembrane probability plots of OSCA1.

a, Hydropathy of OSCA1 was calculated using the Kyte–Doolittle algorithm, with a window size of 19 amino acids. The probabilities of transmembrane helices (TM1–TM9) were predicted using TmHMM 2.0 program59, and the probability plot (red) is superimposed to the hydropathy plot (blue). The region between TM8 and TM9 could be another transmembrane segment (b), or a re-entrant pore loop, a common structure in ion channels. Based on the probability prediction, it is most likely to be a re-entrant pore loop, which needs to be verified in the future. b, Transmembrane α-helical spanners predicted by Aramemnon (http://aramemnon.botanik.uni-koeln.de)60.

Extended Data Figure 6 Verification of the T-DNA insertion osca1-2 mutant and expression patterns and subcellular locations of OSCA1.

a−c, Schematic illustration of the exon–intron structure of OSCA1 with the boxes representing exons (a). The mutations in osca1-1 and T-DNA insertion sites in osca1-2 and osca1-3 are illustrated. Primers for genotyping T-DNA insertion in osca1-2 are shown. BP, T-DNA boarder primer; LP, OSCA1 left primer; RP, OSCA1 right primer. The osca1 refers to osca1-1 in this study. Genotyping of an osca1-2 homozygous line (b). PCR reactions with DNA show a flanking DNA fragment upstream (LP) and downstream of the insertion site (RP) in wild type (WT) but not in osca1-2, and a DNA fragment flanking the T-DNA boarder (BP) and the downstream of the insertion site (RP) in osca1-2 but not wild type, suggesting that osca1-2 is a homozygous T-DNA insertion line. The OSCA1 mRNA level was greatly reduced in osca1-2, but the expression of OSCA1 was not abolished (c), suggesting that osca1-2 is a knock-down mutant rather than a null mutant. d−g, Expression patterns of the pOSCA1::GUS in Arabidopsis leaf (d), flower bud (e), flower (f) and silique (g). The intensity of blue represents the level of GUS activity. h−k, Expression patterns of OSCA1–GFP in Arabidopsis seedlings stably expressing OSCA1 promoter-driven OSCA1–GFP construct (pOSCA1::OSCA1-GFP) (h) or CaMV 35S promoter-driven GFP construct (p35S::GFP) (i). GFP fluorescence was analysed using a Zeiss stereo microscope, and images were merged to generate the whole-seedling images. Insets are enlargements of root tips. Over 10 homozygous single-insertion transgenic lines were generated for each construct, and similar results were observed from these lines. Plasma membrane localization of OSCA1 in Arabidopsis seedlings stably expressing CaMV 35S promoter-driven OSCA1–GFP construct (p35S::OSCA1-GFP) (j) or GFP alone as a control (p35S::GFP) (k). GFP fluorescence was analysed using confocal microscopy. Similar results were seen from over 10 independent homozygous single insertion transgenic lines. In addition, OSCA1 is also predicted to be localized to the plasma membrane by SUBA3 (http://suba.plantenergy.uwa.edu.au/)18. Moreover, OSCA1 has been identified independently by several studies of plasma membrane proteomes19,20,47,61.

Extended Data Figure 7 OSCA1 confers calcium-induced [Ca2+]i increases (CICI) in HEK293 cells.

a, The increases in [Ca2+]i in response to elevated Ca2+ in HEK293 cells expressing empty vector (pcDNA3.2; top), OSCA1 (middle), or mutant OSCA1 (OSCA1(G59R/G507D) (mOSCA1); bottom). HEK293 cells transiently transfected with empty vector pcDNA3.2, OSCA1, or mOSCA1 were incubated in 0.l mM Ca2+ bath solution, and then treated with 2.5 mM Ca2+. The [Ca2+]i increase was analysed by Fura-2 emission ratios (F340 nm:F380 nm) and scaled using a pseudo-colour bar. b−d, Dynamic analysis of CICI in HEK293 cells expressing empty vector (b), OSCA1 (c) or mOSCA1 (d) from experiments as in a. Data are mean ± s.d. (n = 60 cells; r.u., relative unit). Arrows indicate the time of Ca2+ addition. e, Quantitative analysis of the peaks of CICI from 80 to 90 s after addition of Ca2+ from experiments as in b−d. We have also carried out experiments with a range of concentrations of Ca2+, and calculated the Kd as 3.6 ± 0.25 mM. Data for three separate experiments are shown (mean ± s.e.m.). f, g, The [Ca2+]i increases in response to osmotic stress treatment in HEK293 cells expressing pcDNA3.2, which were used as a control for HEK293 cells expressing OSCA1 or mOSCA1 as shown in Fig. 4a. The cells were incubated in the standard bath solution, and then treated with 650 mM sorbitol. The [Ca2+]i increases were analysed by Fura-2 emission ratios (f). OICIs in HEK293 cells expressing empty vector from experiments as in f were quantified (g; mean ± s.d.; n = 60 cells). Sor, sorbitol.

Extended Data Figure 8 OSCA1 is localized to the plasma membrane and forms non-selective cation channels with permeability to Ca2+ in HEK293 cells.

a, b, HEK293 cells were transiently transfected by OSCA1–GFP or GFP constructs, and GFP fluorescence was analysed using the Zeiss Axiovert 200 fluorescence microscope. OSCA1 was localized in the vicinity of the plasma membrane (a); while GFP alone was localized throughout the cells (b). These cells were further analysed by confocal microscopy imaging (Fig. 4e). c, Ca2+ influx across the plasma membrane was analysed using Mn2+ quenching of Fura-2 fluorescence in HEK293 cells. HEK293 cells transfected with pcDNA3.2, OSCA1 or mOSCA1 were loaded with Fura-2 and incubated in the standard bath solution. The bath was perfused with the same solution added with 1 mM Mn2+, and quenching of Fura-2 fluorescence at 358 nm was monitored. Percentages show the relative quenching (F, Fura-2 fluorescence intensity F358; F0, F358 at time zero; mean ± s.e.m.; n = 60 cells). d, Averaged current-–voltage relationships from experiments similar to those in Fig. 4k (mean ± s.e.m.; n = 6 for control and 9 for OSCA1), Ctrl, control. e, Current–voltage relationship from single-channel recordings in experiments as in Fig. 4l (mean ± s.e.m.; n = 4). f, Superimposed whole-cell currents recorded during voltage ramps. OSCA1 currents were first recorded in the standard bath solution and then in solutions containing (in mM) 140 CsCl, 140 KCl, 140 NaCl, 112 CaCl2, or 112 MgCl2. g, Relative ion permeability (PX/PCs) of OSCA1 channels from experiments similar to those in f. Data are mean ± s.e.m. (n = 3 to 9).

Extended Data Figure 9 Phylogeny of OSCA1 family and alignment of eight members from the first clade of Arabidopsis OSCA1 family.

a, The sequences of OSCA1 homologues from Arabidopsis thaliana were retrieved from NCBI GenBank. The phylogeny of OSCA1 homologues was analysed using DNASTAR Lasergene 11 with Clustal Omega(ref. 62), and the phylogenetic tree was illustrated using FigTree 1.4 (http://tree.bio.ed.ac.uk/software/figtree/). b, Eight AtOSCA members from the first clade were aligned using Clustal Omega (ref. 62). The first mutation of G59R and the second mutation of G507D in the osca1 mutant are shown. The mutation of polar glycine 59 to basic arginine (G59R) and especially the mutation of highly conserved glycine 507 to acidic aspartic acid (G507D) might both markedly alter the conformation of OSCA1, leading to changes in OSCA1 activity. Red, small (small and hydrophobic) amino acids; blue, acidic amino acids; magenta, basic amino acids excluding H, R and K; green, hydroxyl, sulphydryl and amine amino acids. (amine amino acids are those with a functional group that contains a basic nitrogen atom, such as asparagine and glutamine). The colour code is identical to the code defined in ref. 62.

Extended Data Figure 10 Phylogeny of OSCA1 family across the taxa.

The sequences of OSCA1 homologues from several species across the taxa were retrieved from NCBI GenBank. The phylogeny of these homologues were analysed using DNASTAR Lasergene 11 using Clustal Omega (ref. 62), and the phylogenetic tree was illustrated using FigTree 1.4. Four clades were classified based on the phylogenetic tree with the clade 3 and 4 uniquely having 1 or 2 genes for vascular plants. At, Arabidopsis thaliana; Bradi, Brachypodium distachyon; Cr, Chlamydomonas reinhardtii; Hs, Homo sapiens; Mm, Mus musculus; Os, Oryza sativa; Pp, Physcomitrella patens; Pt, Populus trichocarpa; Sb, Sorghum bicolor; Sc, Saccharomyces cerevisiae; and Sm, Selaginella moellendorffii. OsOSCA1.1, Os01g0534900; OsOSCA1.2, Os05g0594700; OsOSCA1.3, Os05g0393800; OsOSCA1.4, Os10g0579100; OsOSCA2.1, Os12g0633600; OsOSCA2.2, Os03g0673800; OsOSCA2.3, Os03g0726300; OsOSCA2.4, Os12g0582800; OsOSCA2.5, Os01g0950900; OsOSCA3.1, Os07g0150100; OsOSCA4.1, Os03g0137400.

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Yuan, F., Yang, H., Xue, Y. et al. OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis. Nature 514, 367–371 (2014). https://doi.org/10.1038/nature13593

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