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

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An Erratum to this article was published on 09 August 2014

Abstract

Main conclusion

The present study demonstrates the first direct evidence of the novel role of OsACA6 in providing Cd 2+ stress tolerance in transgenic tobacco by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway.

Abstract

Cadmium, a non-essential toxic heavy metal, interferes with the plant growth and development. It reaches the leaves through xylem and may become part of the food chain, thus causing detrimental effects to human health. Therefore, there is an urgent need to develop strategies for engineering plants for Cd2+ tolerance and less accumulation. The members of P-type ATPases family transport metal ions including Cd2+, and thus play important role an ion homeostasis. The present study elucidates the role of P-type 2B Ca2+ ATPase (OsACA6) in Cd2+ stress tolerance. The transcript levels of OsACA6 were up-regulated upon Cd2+, Zn2+ and Mn2+ exposure. Transgenic tobacco expressing OsACA6 showed tolerance towards Cd2+ stress as demonstrated by several physiological indices including root length, biomass, chlorophyll, malondialdehyde and hydrogen peroxide content. The roots of the transgenic lines accumulated more Cd2+ as compared to shoot. Further, confocal laser scanning microscopy showed that Cd2+ exposure altered Ca2+ uptake in OsACA6 transgenic plants. OsACA6 expression in tobacco also protected the transgenic plants from oxidative stress by enhancing the activity of enzymatic (SOD, CAT, APX, GR) and non-enzymatic (GSH and AsA) antioxidant machinery. Transgenic lines also tolerated Zn2+ and Mn2+ stress; however, tolerance for these ions was not as significant as observed for Cd2+ exposure. Thus, overexpression of OsACA6 confers Cd2+ stress tolerance in transgenic lines by maintaining cellular ion homeostasis and modulating reactive oxygen species (ROS)-scavenging pathway. The results of the present study will help to develop strategies for engineering Cd2+ stress tolerance in economically important crop plants.

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Abbreviations

AsA:

Ascorbate

APX:

Ascorbate peroxidase

CaM:

Calmodulin

CAT:

Catalase

GR:

Glutathione reductase

GSH:

Tripeptide glutathione

HM:

Heavy metals

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

References

  • Aebi H (1984) Catalase in vitro. Method Enzymol 105:121–126

    Article  CAS  Google Scholar 

  • Anderson ME (1985) Determination of glutathione and glutathione disulfide in biological samples. Method Enzymol 113:548–570

    Article  CAS  Google Scholar 

  • Axelsen KB, Palmgren MG (1998) Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol 46:84–101

    Article  PubMed  CAS  Google Scholar 

  • Barabasz A, Krämer U, Hanikenne M, Rudzka J, Antosiewicz DM (2010) Metal accumulation in tobacco expressing Arabidopsis halleri metal hyperaccumulation gene depends on external supply. J Exp Bot 61:3057–3067

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Barabasz A, Mills RF, Trojanowska E, Williams LE, Antosiewicz DM (2011) Expression of AtECA3 in tobacco modifies its responses to manganese, zinc and calcium. Environ Exp Bot 72:202–209

    Article  CAS  Google Scholar 

  • Barabasz A, Wilkowska A, Ruszczyńska A, Bulska E, Hanikenne M (2012) Metal response of transgenic tomato plants expressing P1B-ATPase. Physiol Plant 145:315–331

    Article  PubMed  CAS  Google Scholar 

  • Barabasz A, Wilkowska A, Tracz K, Ruszczyńska A, Bulska E et al (2013) Expression of HvHMA2 in tobacco modifies Zn–Fe–Cd homeostasis. J Plant Physiol 170:1176–1186

    Article  PubMed  CAS  Google Scholar 

  • Beyersmann D, Hartwig A (2008) Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms. Arch Toxicol 82:493–512

    Article  PubMed  CAS  Google Scholar 

  • Biagioli M, Pifferi S, Ragghianti M, Bucci S, Rizzuto R et al (2008) Endoplasmic reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis. Cell Calcium 43:184–195

    Article  PubMed  CAS  Google Scholar 

  • Bovet L, Feller U, Martinoia E (2005) Possible involvement of plant ABC transporters in cadmium detoxification: a cDNA submicroarray approach. Environ Int 31:263–267

    Article  PubMed  CAS  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • DalCorso G, Farinati S, Maistri S, Furini A (2008) How plants cope with cadmium: staking all on metabolism and gene expression. J Integr Plant Biol 50:1268–1280

    Article  PubMed  CAS  Google Scholar 

  • DalCorso G, Farinati S, Furini A (2010) Regulatory networks of cadmium stress in plants. Plant Signal Behav 5:663–667

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Djebali W, Gallusci P, Polge C, Boulila L, Galtier N et al (2008) Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants. Planta 227:625–639

    Article  PubMed  CAS  Google Scholar 

  • Elmayan T, Tepfer M (1994) Synthesis of a bifunctional metallothionein/beta-glucuronidase fusion protein in transgenic tobacco plants as a means of reducing leaf cadmium levels. Plant J 6:433–440

    Article  PubMed  CAS  Google Scholar 

  • Ferrari D, Pinton P, Szabadkai G, Chami M, Campanella M et al (2002) Endoplasmic reticulum, Bcl-2 and Ca(2+) handling in apoptosis. Cell Calcium 32:413–420

    Article  PubMed  CAS  Google Scholar 

  • Filek M, Keskinen R, Hartikainen H, Szarejko I, Janiak A et al (2008) The protective role of selenium in rape seedlings subjected to cadmium stress. J Plant Physiol 165:833–844

    Article  PubMed  CAS  Google Scholar 

  • Foyer CH, Halliwell B (1976) The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133:21–25

    Article  PubMed  CAS  Google Scholar 

  • Garg B, Jaiswal JP, Misra S, Tripathi BN, Prasad M (2012) A comprehensive study on dehydration-induced antioxidative responses during germination of Indian bread wheat (Triticum aestivum L. em Thell) cultivars collected from different agro climatic zones. Physiol Mol Biol Plants 18:217–228

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Giannopolitis CN, Ries SK (1997) Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol 59:307–314

    Google Scholar 

  • Gill SS, Tuteja N (2011) Cadmium stress tolerance in crop plants: probing the role of sulphur. Plant Signal Behav 6:215–222

    Article  PubMed  CAS  Google Scholar 

  • Gill SS, Khan NA, Tuteja N (2011) Differential cadmium stress tolerance in five Indian mustard (Brassica juncea L.) cultivars: an evaluation of the role of antioxidant machinery. Plant Signal Behav 6:293–300

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Gill SS, Khan NA, Tuteja N (2012) Cadmium at high dose perturbs growth, photosynthesis and nitrogen metabolism while at low dose it up regulates sulfur assimilation and antioxidant machinery in garden cress (Lepidium sativum L.). Plant Sci 182:112–120

    Article  PubMed  CAS  Google Scholar 

  • Gill SS, Tajrishi M, Madan M, Tuteja N (2013) A DESD-box helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. PB1). Plant Mol Biol 82:1–22

    Article  PubMed  CAS  Google Scholar 

  • Gussarson M, Asp H, Adalsteinsson S, Jensen P (1996) Enhancement of Cd effects on growth and nutrient composition of birch (Betula pendula) by buthionine sulphoxinine (BSO). J Exp Bot 47:211–215

    Article  Google Scholar 

  • Huda KMK, Banu MSA, Tuteja R, Tuteja N (2013a) Global calcium transducer P-type Ca2+-ATPases open new avenues for agriculture by regulating stress signalling. J Exp Bot 64:3099–3109

    Article  PubMed  CAS  Google Scholar 

  • Huda KMK, Yadav S, Banu MSA, Trivedi DK, Tuteja N (2013b) Genome-wide analysis of plant-type II Ca2+ATPases gene family from rice and Arabidopsis: potential role in abiotic stresses. Plant Physiol Biochem 65:32–47

    Article  Google Scholar 

  • Huda KMK, Banu MSA, Garg B, Tula S, Tuteja R, Tuteja N (2013c) OsACA6, a P-type IIB Ca2+ATPase promotes salinity and drought stress tolerance in tobacco by ROS scavenging and enhancing the expression of stress-responsive genes. Plant J 76:997–1015

    Article  PubMed  CAS  Google Scholar 

  • Islam MM, Hoque MA, Okuma E, Banu MNA, Shimoishi Y et al (2009) Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells. J Plant Physiol 166:1587–1597

    Article  PubMed  CAS  Google Scholar 

  • Kawachi M, Kobae Y, Mimura T, Maeshima M (2008) Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn2+/H+ antiporter of Arabidopsis thaliana, stimulates the transport activity. J Biol Chem 283:8374–8383

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Law ME, Charles SA, Halliwell B (1983) Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts: the effect of hydrogen peroxide and of paraquat. Biochem J 210:899–903

    PubMed  CAS  PubMed Central  Google Scholar 

  • Li S, Yu J, Zhu M, Zhao F, Luan S (2012) Cadmium impairs ion homeostasis by altering K+ and Ca2+ channel activities in rice root hair cells. Plant Cell Environ 35:1998–2013

    Article  PubMed  CAS  Google Scholar 

  • Liu DH, Zou JH, Wang M, Jiang WS (2008) Hexavalent chromium uptake and its effects on mineral uptake, antioxidant defence system and photosynthesis in Amaranthus viridis L. Bioresour Technol 99:2628–2636

    Article  PubMed  CAS  Google Scholar 

  • Liu YT, Chen ZS, Hong CY (2011) Cadmium-induced physiological response and antioxidant enzyme changes in the novel cadmium accumulator, Tagetes patula. J Hazard Mater 189:724–731

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Lu YY, Deng XP, Kwak SS (2010) Overexpression of CuZn superoxide dismutase (CuZn SOD) and ascorbate peroxidase (APX) in transgenic sweet potato enhances tolerance and recovery from drought stress. Afr J Biotech 9:8378–8391

    CAS  Google Scholar 

  • Lucca N, Leon G (2012) Arabidopsis ACA 7 , encoding a putative auto-regulated Ca2+ATPase, is required for normal pollen development. Plant Cell Rep 31:651–659

    Article  PubMed  CAS  Google Scholar 

  • Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch Biochem Biophys 444:139–158

    Article  PubMed  CAS  Google Scholar 

  • Maksymiec W, Wianowska D, Dawidowicz AL, Radkiewicz S, Mardarowicz M et al (2005) The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus plants under heavy metal stress. J Plant Physiol 162:1338–1346

    Article  PubMed  CAS  Google Scholar 

  • Mhamdi A, Queval G, Chaouch S, Vanderauwera S, Van Breusegem F, Noctor G (2010) Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models. J Exp Bot 61:4107–4320

    Article  Google Scholar 

  • Mielniczki-Pereira AA, Hahn AB, Bonatto D, Riger CJ, Eleutherio EC et al (2011) New insights into the Ca2+-ATPases that contribute to cadmium tolerance in yeast. Toxicol Lett 207:104–111

    Article  PubMed  CAS  Google Scholar 

  • Mills RF, Francini A, Ferreira da Rocha PSC, Baccarini PJ, Aylett M et al (2005) The plant P1B-type ATPase AtHMA4 transports Zn and Cd and plays a role in detoxification of transition metals supplied at elevated levels. FEBS Lett 579:783–791

    Article  PubMed  CAS  Google Scholar 

  • Mills RF, Doherty ML, Marqués RLL, Weimar T, Dupree P et al (2008) ECA3, a Golgi localized P2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis. Plant Physiol 146:116–128

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Mills RF, Peaston KA, Runions J, Williams LE (2012) HvHMA2, a P1B-ATPase from barley, is highly conserved among cereals and functions in Zn and Cd transport. PLoS One 7:e42640. doi:10.1371/journal.pone.0042640

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880

    CAS  Google Scholar 

  • Pomponi M, Censi V, Di Girolamo V, De Paolis A, di Toppi LS et al (2006) Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd(2+) tolerance and accumulation but not translocation to the shoot. Planta 223:180–190

    Article  PubMed  CAS  Google Scholar 

  • Qudeimat E, Faltusz AMC, Wheeler G, Lang D, Brownlee C et al (2008) A PIIB-type Ca2+ATPase is essential for stress adaptation in Physcomitrella patens. Proc Natl Acad Sci USA 105:19555–19560

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Salt DE, Prince RC, Pickering IJ, Raskin I (1995) Mechanisms of cadmium mobility and accumulation in Indian mustard. Plant Physiol 109:1427–1433

    PubMed  CAS  PubMed Central  Google Scholar 

  • Satoh-Nagasawa N, Mori M, Nakazawa N, Kawamoto T, Nagato Y et al (2012) Mutations in rice (Oryza sativa) heavy metal ATPase 2 (OsHMA2) restrict the translocation of zinc and cadmium. Plant Cell Physiol 53:213–224

    Article  PubMed  CAS  Google Scholar 

  • Shabala S, Bækgaard L, Shabala L, Fuglsang A, Babourina O et al (2011) Plasmamembrane Ca2+ transporters mediate virus induced acquired resistance to oxidative stress. Plant Cell Environ 34:406–417

    Article  PubMed  CAS  Google Scholar 

  • Siemianowski O, Mills RF, Williams LE, Antosiewicz DM (2011) Expression of the P1B-type ATPase AtHMA4 in tobacco modifies Zn and Cd root to shoot partitioning and metal tolerance. Plant Biotech J 9:64–74

    Article  CAS  Google Scholar 

  • Siemianowski O, Barabasz A, Weremczuk A, Ruszczyńska A, Bulska E et al (2013) Development of Zn-related necrosis in tobacco is enhanced by expressing AtHMA4 and depends on the apoplastic Zn levels. Plant Cell Environ 36:1093–1104

    Article  PubMed  CAS  Google Scholar 

  • Tran TU, Popova LP (2013) Functions and toxicity of cadmium in plants: recent advances and future prospects. Turk J Bot 37:1–13

    CAS  Google Scholar 

  • Verbruggen N, Hermans C, Schat H (2009) Mechanisms to cope with arsenic or cadmium excess in plants. Curr Opin Plant Biol 12:1–9

    Article  Google Scholar 

  • Wang SS, Chen L, Xia SK (2007) Cadmium is acutely toxic for murine hepatocytes: effects on intracellular free Ca2+ homeostasis. Physiol Res 56:193–201

    PubMed  CAS  Google Scholar 

  • Wang Y, Itaya A, Zhong X, Wu Y, Zhang J et al (2011) Function and evolution of a microRNA that regulates a Ca2+ATPase and triggers the formation of phased small interfering RNAs in tomato reproductive growth. Plant Cell 23:3185–3203

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Yadav DK, Islam SM, Tuteja N (2012) Rice heterotrimeric G-protein gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress. Plant Signal Behav 7:733–740

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Yadav DK, Shukla D, Tuteja N (2013) Rice heterotrimeric G-protein alpha subunit (RGA1): in silico analysis of the gene and promoter and its upregulation under abiotic stress. Plant Physiol Biochem 63:262–271

    Article  PubMed  CAS  Google Scholar 

  • Yang T, Poovaiah BW (2003) Calcium/calmodulin-mediated signal network in plants. Trends Plant Sci 8:505–512

    Article  PubMed  CAS  Google Scholar 

  • Yeh CM, Hsiao LJ, Huang HJ (2004) Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice cell. Plant Cell Physiol 45:1306–1312

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Department of Science and Technology (DST) and Department of Biotechnology (DBT), Government of India for financial support. We also thank to Dr. P. K. Trivedi (NBRI, Lucknow) for his help in cadmium accumulation experiments and critically reading the manuscript. N. T. thanks to ICGEB for awarding “The Arturo Falaschi ICGEB Pre-doctoral Fellowship” to K. M. K. H and M. S. A. B.

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  1. Devesh Shukla

    Present address: Department of Biology, Western Kentucky University, Bowling Green, KY, 42101-1080, USA

Authors and Affiliations

  1. International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Devesh Shukla, Kazi Md. Kamrul Huda, Mst. Sufara Akhter Banu, Renu Tuteja & Narendra Tuteja

  2. Centre for Biotechnology, Stress Physiology and Molecular Biology Lab, MD University, Rohtak, 124 001, India

    Sarvjeet Singh Gill

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  1. Devesh Shukla
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Correspondence to Narendra Tuteja.

Additional information

D. Shukla and K. Md. K. Huda contributed equally to this work.

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Fig. S1

Demonstration of growth tolerance of transgenic tobacco plants expressing OsACA6 relative to WT under without heavy metal stress (0 µM). Sterilized seeds of WT and transgenic lines expressing OsACA6 were grown on Petri plates containing half-MS medium with 1.5 % sucrose. Plates were kept vertically and grown in a culture room. Fresh weight per 10 seedlings and root length of ten seedlings were measured after 21 days as growth parameter. Supplementary material 1 (TIFF 2,824 kb)

Fig. S2

Effect of Mn2+ (500 μM) and Zn2+ (300 μM) exposure on growth of transgenic tobacco plants expressing OsACA6 relative to WT. a Tolerance in growth was shown by transgenic seedlings under Mn2+ (upper panel) and Zn2+ (lower panel) ion exposure. Sterilized seeds of WT and transgenic lines expressing OsACA6 were grown on Petri plates containing half-MS medium with 1.5 % sucrose supplemented with MnCl2 and ZnCl2. b Fresh weight per 10 seedlings of WT and OsACA6 in response to MnCl2 treatment. c Fresh weight per 10 seedlings of WT and OsACA6 in response to ZnCl2 treatment. The data shown are the average ± SD of three independent experiments, with each experiment consisting of 10 individual plants. *P < 0.05 differ significantly from their respective controls according to Student’s paired t test. Supplementary material 2 (TIFF 4,920 kb)

Fig. S3

Quantitative real-time PCR analysis of the expression of antioxidant enzymes in the absence or presence of 100 μM CdCl2. a Expression of NtAPX, NtDHAR, NtFeSOD was decreased relative to WT in absence of CdCl2. b Expression of the APX and DHAR genes was increased. There was no change in expression of Fe and Cu–Zn SOD under 100-μM CdCl2. Statistical analysis was performed using paired Student t test to compare the mean differences in fold change expression of NtAPX, NtDHAR, Fe–SOD and Cu–Zn SOD during different time points of exposure with respect to control (WT). *, ** indicate values that differ significantly at P < 0.05 and P < 0.01, respectively. Supplementary material 3 (TIFF 214 kb)

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Shukla, D., Huda, K.M.K., Banu, M.S.A. et al. OsACA6, a P-type 2B Ca2+ ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load. Planta 240, 809–824 (2014). https://doi.org/10.1007/s00425-014-2133-z

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