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1.
Plant Biotechnol J ; 10(8): 945-55, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22762155

ABSTRACT

While various signalling networks regulate plant responses to heat stress, the mechanisms regulating and unifying these diverse biological processes are largely unknown. Our previous studies indicate that the Arabidopsis monothiol glutaredoxin, AtGRXS17, is crucial for temperature-dependent postembryonic growth in Arabidopsis. In the present study, we further demonstrate that AtGRXS17 has conserved functions in anti-oxidative stress and thermotolerance in both yeast and plants. In yeast, AtGRXS17 co-localized with yeast ScGrx3 in the nucleus and suppressed the sensitivity of yeast grx3grx4 double-mutant cells to oxidative stress and heat shock. In plants, GFP-AtGRXS17 fusion proteins initially localized in the cytoplasm and the nuclear envelope but migrated to the nucleus during heat stress. Ectopic expression of AtGRXS17 in tomato plants minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane systems under heat stress. This enhanced thermotolerance correlated with increased catalase (CAT) enzyme activity and reduced H2O2 accumulation in AtGRXS17-expressing tomatoes. Furthermore, during heat stress, expression of the heat shock transcription factor (HSF) and heat shock protein (HSP) genes was up-regulated in AtGRXS17-expressing transgenic plants compared with wild-type controls. Thus, these findings suggest a specific protective role of a redox protein against temperature stress and provide a genetic engineering strategy to improve crop thermotolerance.


Subject(s)
Acclimatization/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Glutaredoxins/metabolism , Oxidative Stress/physiology , Solanum lycopersicum/genetics , Solanum lycopersicum/physiology , Crops, Agricultural/genetics , Crops, Agricultural/physiology , Gene Expression Regulation, Plant , Genes, Plant , Genetic Engineering , Genetic Variation , Genotype , Hot Temperature , Plants, Genetically Modified , Up-Regulation , Yeasts/genetics , Yeasts/physiology
2.
FEBS J ; 278(14): 2525-2539, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21575136

ABSTRACT

Glutaredoxins (Grxs) have been shown to be critical in maintaining redox homeostasis in living cells. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified. However, the biological and physiological functions of this group of proteins have not been well characterized. Here, we characterize a mammalian monothiol Grx (Grx3, also termed TXNL2/PICOT) with high similarity to yeast ScGrx3/ScGrx4. In yeast expression assays, mammalian Grx3s were localized to the nuclei and able to rescue growth defects of grx3grx4 cells. Furthermore, Grx3 inhibited iron accumulation in yeast grx3gxr4 cells and suppressed the sensitivity of mutant cells to exogenous oxidants. In mice, Grx3 mRNA was ubiquitously expressed in developing embryos, adult tissues and organs, and was induced during oxidative stress. Mouse embryos absent of Grx3 grew smaller with morphological defects and eventually died at 12.5 days of gestation. Analysis in mouse embryonic fibroblasts revealed that Grx3(-/-) cells had impaired growth and cell cycle progression at the G(2) /M phase, whereas the DNA replication during the S phase was not affected by Grx3 deletion. Furthermore, Grx3-knockdown HeLa cells displayed a significant delay in mitotic exit and had a higher percentage of binucleated cells. Therefore, our findings suggest that the mammalian Grx3 has conserved functions in protecting cells against oxidative stress and deletion of Grx3 in mice causes early embryonic lethality which could be due to defective cell cycle progression during late mitosis.


Subject(s)
Carrier Proteins/physiology , Cell Cycle , Embryonic Development , Glutaredoxins/physiology , Animals , Carrier Proteins/genetics , Cell Line , Embryo, Mammalian/abnormalities , Embryo, Mammalian/metabolism , Female , Gene Expression Regulation, Enzymologic/drug effects , Gene Silencing , Genes, Lethal , Glutaredoxins/genetics , Humans , Isoenzymes/genetics , Isoenzymes/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myoblasts/metabolism , Myoblasts/pathology , Oxidative Stress/drug effects , Protein Disulfide Reductase (Glutathione) , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
3.
J Biol Chem ; 286(23): 20398-406, 2011 Jun 10.
Article in English | MEDLINE | ID: mdl-21515673

ABSTRACT

Global environmental temperature changes threaten innumerable plant species. Although various signaling networks regulate plant responses to temperature fluctuations, the mechanisms unifying these diverse processes are largely unknown. Here, we demonstrate that an Arabidopsis monothiol glutaredoxin, AtGRXS17 (At4g04950), plays a critical role in redox homeostasis and hormone perception to mediate temperature-dependent postembryonic growth. AtGRXS17 expression was induced by elevated temperatures. Lines altered in AtGRXS17 expression were hypersensitive to elevated temperatures and phenocopied mutants altered in the perception of the phytohormone auxin. We show that auxin sensitivity and polar auxin transport were perturbed in these mutants, whereas auxin biosynthesis was not altered. In addition, atgrxs17 plants displayed phenotypes consistent with defects in proliferation and/or cell cycle control while accumulating higher levels of reactive oxygen species and cellular membrane damage under high temperature. Together, our findings provide a nexus between reactive oxygen species homeostasis, auxin signaling, and temperature responses.


Subject(s)
Arabidopsis Proteins/biosynthesis , Arabidopsis/enzymology , Arabidopsis/growth & development , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Plant/physiology , Glutaredoxins/biosynthesis , Hot Temperature , Indoleacetic Acids/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Biological Transport/physiology , Cell Cycle/physiology , Glutaredoxins/genetics , Homeostasis/physiology , Mutation , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Signal Transduction/physiology
4.
Plant Cell ; 23(1): 240-57, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21258004

ABSTRACT

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.


Subject(s)
Antiporters/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Calcium/metabolism , Cation Transport Proteins/metabolism , Vacuoles/metabolism , Antiporters/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cation Transport Proteins/genetics , Cell Wall/metabolism , Gene Expression Profiling , Gene Expression Regulation, Plant , Mutagenesis, Insertional , Mutation , Oligonucleotide Array Sequence Analysis , Phenotype , Plant Leaves/cytology , Plant Leaves/metabolism , Plant Stomata/metabolism , RNA, Plant/genetics , Single-Cell Analysis
5.
J Clin Invest ; 121(1): 212-25, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21123948

ABSTRACT

Cancer cells have an efficient antioxidant system to counteract their increased generation of ROS. However, whether this ability to survive high levels of ROS has an important role in the growth and metastasis of tumors is not well understood. Here, we demonstrate that the redox protein thioredoxin-like 2 (TXNL2) regulates the growth and metastasis of human breast cancer cells through a redox signaling mechanism. TXNL2 was found to be overexpressed in human cancers, including breast cancers. Knockdown of TXNL2 in human breast cancer cell lines increased ROS levels and reduced NF-κB activity, resulting in inhibition of in vitro proliferation, survival, and invasion. In addition, TXNL2 knockdown inhibited tumorigenesis and metastasis of these cells upon transplantation into immunodeficient mice. Furthermore, analysis of primary breast cancer samples demonstrated that enhanced TXNL2 expression correlated with metastasis to the lung and brain and with decreased overall patient survival. Our studies provided insight into redox-based mechanisms underlying tumor growth and metastasis and suggest that TXNL2 could be a target for treatment of breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carrier Proteins/metabolism , NF-kappa B/metabolism , Animals , Base Sequence , Brain Neoplasms/metabolism , Brain Neoplasms/secondary , Breast Neoplasms/genetics , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Cell Line, Tumor , Cell Proliferation , Epithelial-Mesenchymal Transition , Female , Gene Expression , Gene Knockdown Techniques , Homeostasis , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mice , Mice, SCID , Neoplasm Transplantation , Oxidation-Reduction , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Signal Transduction , Transplantation, Heterologous
6.
New Phytol ; 183(1): 95-105, 2009.
Article in English | MEDLINE | ID: mdl-19368667

ABSTRACT

* The Arabidopsis vacuolar CAtion eXchangers (CAXs) play a key role in mediating cation influx into the vacuole. In Arabidopsis, there are six CAX genes. However, some members are yet to be characterized fully. * In this study, we show that CAX4 is expressed in the root apex and lateral root primordia, and that expression is increased when Ni(2+) or Mn(2+) levels are elevated or Ca(2+) is depleted. * Transgenic plants expressing increased levels of CAX4 display symptoms consistent with increased sequestration of Ca(2+) and Cd(2+) into the vacuole. When CAX4 is highly expressed in an Arabidopsis cax1 mutant line with weak vacuolar Ca(2+)/H(+) antiport activity, a 29% increase in Ca(2+)/H(+) antiport is measured. A cax4 loss-of-function mutant and CAX4 RNA interference lines display altered root growth in response to Cd(2+), Mn(2+) and auxin. The DR5::GUS auxin reporter detected reduces auxin responses in the cax4 lines. * These results indicate that CAX4 is a cation/H(+) antiporter that plays an important function in root growth under heavy metal stress conditions.


Subject(s)
Adaptation, Physiological/genetics , Antiporters/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cation Transport Proteins/metabolism , Metals, Heavy/metabolism , Plant Roots/metabolism , Plants, Genetically Modified/metabolism , Antiporters/genetics , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Cation Transport Proteins/genetics , Gene Expression , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Metals, Heavy/toxicity , Mutation , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/growth & development , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Stress, Physiological/genetics , Vacuoles/metabolism
7.
J Biol Chem ; 284(7): 4605-15, 2009 Feb 13.
Article in English | MEDLINE | ID: mdl-19098009

ABSTRACT

In plants, high capacity tonoplast cation/H(+) antiport is mediated in part by a family of CAX (cation exchanger) transporters. Functional association between CAX1 and CAX3 has previously been inferred; however, the nature of this interaction has not been established. Here we analyze the formation of "hetero-CAX" complexes and their transport properties. Co-expressing both CAX1 and CAX3 mediated lithium and salt tolerance in yeast, and these phenotypes could not be recapitulated by expression of deregulated versions of either transporter. Coincident expression of Arabidopsis CAX1 and CAX3 occurs during particular stress responses, flowering, and seedling growth. Analysis of cax1, cax3, and cax1/3 seedlings demonstrated similar stress sensitivities. When plants expressed high levels of both CAXs, alterations in transport properties were evident that could not be recapitulated by high level expression of either transporter individually. In planta coimmunoprecipitation suggested that a protein-protein interaction occurred between CAX1 and CAX3. In vivo interaction between the CAX proteins was shown using a split ubiquitin yeast two-hybrid system and gel shift assays. These findings demonstrate cation exchange plasticity through hetero-CAX interactions.


Subject(s)
Antiporters/biosynthesis , Arabidopsis Proteins/biosynthesis , Arabidopsis/enzymology , Cation Transport Proteins/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Plant/physiology , Antiporters/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cation Transport Proteins/genetics , Flowers/enzymology , Ion Transport/physiology , Seedlings/enzymology , Stress, Physiological/physiology
8.
Plant Physiol ; 148(2): 796-807, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18676662

ABSTRACT

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using (86)Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a K(m) of 136 and 196 microm, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cation Transport Proteins/metabolism , Potassium/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Biological Transport , Cation Transport Proteins/genetics , Cell Membrane/metabolism , Gene Expression Regulation, Plant , Molecular Sequence Data , Phenotype , Plant Roots/genetics , Plant Roots/metabolism , Plasmids , RNA, Plant/genetics , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
9.
FEBS Lett ; 582(6): 848-54, 2008 Mar 19.
Article in English | MEDLINE | ID: mdl-18275854

ABSTRACT

Arabidopsis monothiol glutaredoxin (Grx), AtGRX4, was targeted to chloroplasts/plastids and had high similarity to yeast Grx5. In yeast expression assays, AtGRX4 localized to the mitochondria and suppressed the sensitivity of grx5 cells to oxidants. In addition, AtGRX4 reduced iron accumulation and rescued the lysine auxotrophy of grx5 cells. In planta, AtGRX4 RNA transcripts accumulated in growing tissues. Furthermore, AtGRX4expression was altered under various stresses. Genetic analysis revealed that seedlings of atgrx4 mutants were sensitive to oxidants. Taken together, these results suggest that AtGRX4 may have important functions in plant growth and development under extreme environments.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Chloroplasts/genetics , Glutaredoxins/genetics , Oxidative Stress/genetics , Amino Acid Sequence , Arabidopsis/enzymology , Arabidopsis Proteins/analysis , Arabidopsis Proteins/metabolism , Chloroplasts/enzymology , Genetic Complementation Test , Glutaredoxins/analysis , Glutaredoxins/metabolism , Hydrogen Peroxide/pharmacology , Lysine/genetics , Lysine/metabolism , Mitochondria/enzymology , Molecular Sequence Data , Mutation , Phenotype , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Suppression, Genetic
10.
J Biol Chem ; 281(36): 26280-8, 2006 Sep 08.
Article in English | MEDLINE | ID: mdl-16829529

ABSTRACT

Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H2O2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage.


Subject(s)
Arabidopsis Proteins/metabolism , Carrier Proteins/metabolism , Chloroplasts/metabolism , Oxidoreductases/metabolism , Protein Isoforms/metabolism , Amino Acid Sequence , Animals , Antiporters , Arabidopsis/anatomy & histology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Carrier Proteins/genetics , Chloroplasts/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Glutaredoxins , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidation-Reduction , Oxidative Stress , Oxidoreductases/genetics , Phenotype , Plants, Genetically Modified , Protein Isoforms/genetics , Reactive Oxygen Species/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sulfhydryl Compounds/metabolism
11.
Plant Physiol ; 139(3): 1194-206, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16244156

ABSTRACT

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H(+)/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H(+)/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth.


Subject(s)
Antiporters/metabolism , Arabidopsis , Calcium-Binding Proteins/metabolism , Calcium/metabolism , Cation Transport Proteins/metabolism , Protons , Solanum lycopersicum/metabolism , Antiporters/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Carbohydrates/analysis , Cations/metabolism , Ethylenes/biosynthesis , Fruit/physiology , Gene Expression , Ion Transport , Solanum lycopersicum/genetics , Solanum lycopersicum/growth & development , Phenotype , Plants, Genetically Modified , Substrate Specificity , Time Factors
12.
Plant Physiol ; 138(4): 2048-60, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16055687

ABSTRACT

Cation levels within the cytosol are coordinated by a network of transporters. Here, we examine the functional roles of calcium exchanger 1 (CAX1), a vacuolar H+/Ca2+ transporter, and the closely related transporter CAX3. We demonstrate that like CAX1, CAX3 is also localized to the tonoplast. We show that CAX1 is predominately expressed in leaves, while CAX3 is highly expressed in roots. Previously, using a yeast assay, we demonstrated that an N-terminal truncation of CAX1 functions as an H+/Ca2+ transporter. Here, we use the same yeast assay to show that full-length CAX1 and full-length CAX3 can partially, but not fully, suppress the Ca2+ hypersensitive yeast phenotype and coexpression of full-length CAX1 and CAX3 conferred phenotypes not produced when either transporter was expressed individually. In planta, CAX3 null alleles were modestly sensitive to exogenous Ca2+ and also displayed a 22% reduction in vacuolar H+-ATPase activity. cax1/cax3 double mutants displayed a severe reduction in growth, including leaf tip and flower necrosis and pronounced sensitivity to exogenous Ca2+ and other ions. These growth defects were partially suppressed by addition of exogenous Mg2+. The double mutant displayed a 42% decrease in vacuolar H+/Ca2+ transport, and a 47% decrease in H+-ATPase activity. While the ionome of cax1 and cax3 lines were modestly perturbed, the cax1/cax3 lines displayed increased PO4(3-), Mn2+, and Zn2+ and decreased Ca2+ and Mg2+ in shoot tissue. These findings suggest synergistic function of CAX1 and CAX3 in plant growth and nutrient acquisition.


Subject(s)
Antiporters/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/genetics , Antiporters/genetics , Arabidopsis Proteins/genetics , Calcium Signaling , Calcium-Binding Proteins/metabolism , Cation Transport Proteins/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Ion Transport/genetics , Ion Transport/physiology , Magnesium/metabolism , Mutation , Proton-Translocating ATPases/metabolism , Tissue Distribution
13.
Mol Microbiol ; 54(4): 1104-16, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15522090

ABSTRACT

The Ca(2+)-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger Vcx1p. In yeast and plants, cation/H(+) exchangers are important in shaping cytosolic Ca(2+) levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca(2+), Mn(2+) and Cd(2+). Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca(2+) transporter and may also function in Cd(2+) transport, but cannot transport Mn(2+). Furthermore, direct Ca(2+) transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-S204A/L208P), termed VCX1-M1, was previously found to confer strong Mn(2+) tolerance. Here we demonstrate that this Mn(2+) tolerance is independent of the Ca(2+)/Mn(2+)-ATPase Pmr1p and results from Mn(2+)-specific vacuolar transport activity of Vcx1-M1p. This Mn(2+) transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn(2+) tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.


Subject(s)
Antiporters/metabolism , Calcineurin/metabolism , Protein Isoforms/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Antiporters/genetics , Biological Transport/physiology , Cadmium/metabolism , Calcium/metabolism , Calcium-Transporting ATPases/metabolism , Manganese/metabolism , Molecular Chaperones/metabolism , Point Mutation , Protein Isoforms/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/metabolism
14.
Plant Physiol ; 136(1): 2532-47, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15347787

ABSTRACT

A combined bioinformatic and experimental approach is being used to uncover the functions of a novel family of cation/H(+) exchanger (CHX) genes in plants using Arabidopsis as a model. The predicted protein (85-95 kD) of 28 AtCHX genes after revision consists of an amino-terminal domain with 10 to 12 transmembrane spans (approximately 440 residues) and a hydrophilic domain of approximately 360 residues at the carboxyl end, which is proposed to have regulatory roles. The hydrophobic, but not the hydrophilic, domain of plant CHX is remarkably similar to monovalent cation/proton antiporter-2 (CPA2) proteins, especially yeast (Saccharomyces cerevisiae) KHA1 and Synechocystis NhaS4. Reports of characterized fungal and prokaryotic CPA2 indicate that they have various transport modes, including K(+)/H(+) (KHA1), Na(+)/H(+)-K(+) (GerN) antiport, and ligand-gated ion channel (KefC). The expression pattern of AtCHX genes was determined by reverse transcription PCR, promoter-driven beta-glucuronidase expression in transgenic plants, and Affymetrix ATH1 genome arrays. Results show that 18 genes are specifically or preferentially expressed in the male gametophyte, and six genes are highly expressed in sporophytic tissues. Microarray data revealed that several AtCHX genes were developmentally regulated during microgametogenesis. An exciting idea is that CHX proteins allow osmotic adjustment and K(+) homeostasis as mature pollen desiccates and then rehydrates at germination. The multiplicity of CHX-like genes is conserved in higher plants but is not found in animals. Only 17 genes, OsCHX01 to OsCHX17, were identified in rice (Oryza sativa) subsp. japonica, suggesting diversification of CHX in Arabidopsis. These results reveal a novel CHX gene family in flowering plants with potential functions in pollen development, germination, and tube growth.


Subject(s)
Antiporters/genetics , Antiporters/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Amino Acid Sequence , Animals , Arabidopsis/growth & development , Base Sequence , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , DNA, Plant/genetics , Gene Expression , Gene Expression Profiling , Genes, Plant , Homeostasis , Molecular Sequence Data , Multigene Family , Oligonucleotide Array Sequence Analysis , Oryza/genetics , Phylogeny , Plants, Genetically Modified , Pollen/growth & development , Pollen/metabolism , Potassium/metabolism , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Species Specificity , Water-Electrolyte Balance
15.
Mol Plant Microbe Interact ; 17(6): 583-92, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15195941

ABSTRACT

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.


Subject(s)
Nicotiana/virology , RNA Interference , Tobacco Mosaic Virus/pathogenicity , Viral Proteins/genetics , Biological Transport , Chromosome Mapping , Green Fluorescent Proteins , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mutation , Phenotype , Plant Diseases/virology , Plants, Genetically Modified , Nicotiana/genetics , Tobacco Mosaic Virus/genetics , Tobacco Mosaic Virus/physiology , Viral Proteins/metabolism , Virus Replication
16.
Proc Natl Acad Sci U S A ; 101(16): 6297-302, 2004 Apr 20.
Article in English | MEDLINE | ID: mdl-15079073

ABSTRACT

Nicotiana benthamiana often displays more intense symptoms after infection by RNA viruses than do other Nicotiana species. Here, we examined the role of RNA-dependent RNA polymerases (RdRPs) in N. benthamiana antiviral defense. cDNAs representing only two genes encoding RdRPs were identified in N. benthamiana. One RdRP was similar in sequence to SDE1/SGS2 required for maintenance of transgene silencing, whereas the second, named NbRdRP1m, was >90% identical in sequence to the salicylic acid (SA)-inducible RdRP from Nicotiana tabacum required for defense against viruses. NbRdRP1m expression was induced by SA treatment or challenge with Tobacco mosaic virus, but the gene and transcript sequences differed from those of other SA-inducible RdRPs in that they contained a 72-nt insert with tandem in-frame stop codons in the 5' portion of the ORF. N. benthamiana plants transformed with an SA-inducible RdRP gene from Medicago truncatula were more resistant to infection by Tobacco mosaic virus, Turnip vein-clearing virus, and Sunn hemp mosaic virus (members of Tobamovirus genus), but not to Cucumber mosaic virus and Potato virus X (members of different genera than the tobamoviruses). Our results indicate that N. benthamiana lacks an active SA- and virus-inducible RdRP and thus is hypersusceptible to viruses normally limited in their accumulation by this RdRP. These findings are significant for those studying virus-induced gene silencing, the hypersensitive response and systemic acquired resistance.


Subject(s)
Nicotiana/virology , RNA-Dependent RNA Polymerase/metabolism , Base Sequence , DNA Primers , Enzyme-Linked Immunosorbent Assay , Gene Silencing , Molecular Sequence Data , Plants, Genetically Modified/virology , RNA-Dependent RNA Polymerase/genetics , Reverse Transcriptase Polymerase Chain Reaction
17.
FEBS Lett ; 559(1-3): 99-106, 2004 Feb 13.
Article in English | MEDLINE | ID: mdl-14960315

ABSTRACT

Precise regulation of calcium transporters is essential for modulating the Ca2+ signaling network that is involved in the growth and adaptation of all organisms. The Arabidopsis H+/Ca2+ antiporter, CAX1, is a high capacity and low affinity Ca2+ transporter and several CAX1-like transporters are found in Arabidopsis. When heterologously expressed in yeast, CAX1 is unable to suppress the Ca2+ hypersensitivity of yeast vacuolar Ca2+ transporter mutants due to an N-terminal autoinhibition mechanism that prevents Ca2+ transport. Using a yeast screen, we have identified CAX nteracting Protein 4 (CXIP4) that activated full-length CAX1, but not full-length CAX2, CAX3 or CAX4. CXIP4 encodes a novel plant protein with no bacterial, fungal, animal, or mammalian homologs. Expression of a GFP-CXIP4 fusion in yeast and plant cells suggests that CXIP4 is targeted predominantly to the nucleus. Using a yeast growth assay, CXIP4 activated a chimeric CAX construct that contained specific portions of the N-terminus of CAX1. Together with other recent studies, these results suggest that CAX1 is regulated by several signaling molecules that converge on the N-terminus of CAX1 to regulate H+/Ca2+ antiport.


Subject(s)
Arabidopsis Proteins/physiology , Carrier Proteins/physiology , Amino Acid Sequence , Arabidopsis Proteins/genetics , Carrier Proteins/genetics , Cell Nucleus/chemistry , Green Fluorescent Proteins , Luminescent Proteins/genetics , Nuclear Proteins , Plant Leaves/cytology , Plant Leaves/ultrastructure , Recombinant Fusion Proteins/genetics , Sequence Alignment , Nicotiana/cytology , Nicotiana/ultrastructure , Yeasts/genetics
18.
Plant Mol Biol ; 56(6): 959-71, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15821993

ABSTRACT

The vacuolar sequestration of metals is an important metal tolerance mechanism in plants. The Arabidopsis thaliana vacuolar transporters CAX1 and CAX2 were originally identified in a Saccharomyces cerevisiae suppression screen as Ca2+/H+ antiporters. CAX2 has a low affinity for Ca2+ but can transport other metals including Mn2+ and Cd2+. Here we demonstrate that unlike cax1 mutants, CAX2 insertional mutants caused no discernable morphological phenotypes or alterations in Ca2+/H+ antiport activity. However, cax2 lines exhibited a reduction in vacuolar Mn2+/H+ antiport and, like cax1 mutants, reduced V-type H+ -ATPase (V-ATPase) activity. Analysis of a CAX2 promoter beta-glucoronidase (GUS) reporter gene fusion confirmed that CAX2 was expressed throughout the plant and strongly expressed in flower tissue, vascular tissue and in the apical meristem of young plants. Heterologous expression in yeast identified an N-terminal regulatory region in CAX2, suggesting that Arabidopsis contains multiple cation/H+ antiporters with shared regulatory features. Furthermore, despite significant variations in morphological and biochemical phenotypes, cax1 and cax2 lines both significantly alter V-ATPase activity, hinting at coordinate regulation among transporters driven by H+ gradients and the V-ATPase.


Subject(s)
Antiporters/genetics , Arabidopsis Proteins/genetics , Arabidopsis/genetics , Calcium-Binding Proteins/genetics , Cation Transport Proteins/genetics , Amino Acid Sequence , Antiporters/physiology , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/physiology , Biological Transport , Calcium-Binding Proteins/physiology , Cation Transport Proteins/physiology , Gene Deletion , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genotype , Glucuronidase/genetics , Glucuronidase/metabolism , Manganese/metabolism , Molecular Sequence Data , Mutation , Plants, Genetically Modified , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Vacuolar Proton-Translocating ATPases/metabolism , Vacuoles/metabolism
19.
J Biol Chem ; 279(4): 2922-6, 2004 Jan 23.
Article in English | MEDLINE | ID: mdl-14583601

ABSTRACT

The regulation of ions within cells is an indispensable component of growth and adaptation. The plant SOS2 protein kinase and its associated Ca(2+) sensor, SOS3, have been demonstrated to modulate the plasma membrane H(+)/Na(+) antiporter SOS1; however, how these regulators modulate Ca(2+) levels within cells is poorly understood. Here we demonstrate that SOS2 regulates the vacuolar H(+)/Ca(2+) antiporter CAX1. Using a yeast growth assay, co-expression of SOS2 specifically activated CAX1, whereas SOS3 did not. CAX1-like chimeric transporters were activated by SOS2 if the chimeric proteins contained the N terminus of CAX1. Vacuolar membranes from CAX1-expressing cells were made to be H(+)/Ca(2+)-competent by the addition of SOS2 protein in a dose-dependent manner. Using a yeast two-hybrid assay, SOS2 interacted with the N terminus of CAX1. In each of these yeast assays, the activation of CAX1 by SOS2 was SOS3-independent. In planta, the high level of expression of a deregulated version of CAX1 caused salt sensitivity. These findings suggest multiple functions for SOS2 and provide a mechanistic link between Ca(2+) and Na(+) homeostasis in plants.


Subject(s)
Antiporters/metabolism , Arabidopsis/enzymology , Calcium-Binding Proteins/metabolism , Calcium/metabolism , Cation Transport Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Arabidopsis Proteins , Enzyme Activation , Ion Transport , Plant Proteins/metabolism , Sodium/metabolism
20.
Plant Cell ; 15(2): 347-64, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12566577

ABSTRACT

The Arabidopsis Ca(2+)/H(+) transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca(2+) levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca(2+)/H(+) antiport activity, a 40% reduction in tonoplast V-type H(+)-translocating ATPase activity, a 36% increase in tonoplast Ca(2+)-ATPase activity, and increased expression of the putative vacuolar Ca(2+)/H(+) antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn(2+) and Mg(2+) stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.


Subject(s)
Antiporters/metabolism , Arabidopsis/growth & development , Calcium-Binding Proteins/metabolism , Plant Growth Regulators/pharmacology , Alleles , Antiporters/genetics , Arabidopsis/genetics , Arabidopsis/metabolism , Calcium/metabolism , Calcium/pharmacology , Calcium-Binding Proteins/genetics , Cation Transport Proteins/metabolism , Gene Expression Regulation, Plant/drug effects , Homeostasis/drug effects , Ion Transport/drug effects , Mutation , Phenotype , Vacuolar Proton-Translocating ATPases/metabolism , Vacuoles/metabolism
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