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Plant function and evolutionary biology
RESEARCH ARTICLE

Expressing Arabidopsis thaliana V-ATPase subunit C in barley (Hordeum vulgare) improves plant performance under saline condition by enabling better osmotic adjustment

Getnet D. Adem A , Stuart J. Roy B C , Yuqing Huang D , Zhong-Hua Chen D , Feifei Wang A , Meixue Zhou A , John P. Bowman A , Paul Holford D and Sergey Shabala A E
+ Author Affiliations
- Author Affiliations

A School of Land and Food, University of Tasmania, Private Bag 54, Hobart, Tas. 7001, Australia.

B Australian Centre for Plant Functional Genomics, Private Mail Bag 1, Glen Osmond, SA 5064, Australia.

C School of Agriculture, Food and Wine, University of Adelaide, Private Mail Bag 1, Glen Osmond, SA 5064, Australia.

D School of Science and Health, Hawkesbury Institute for the Environment, Western Sydney University, Penrith, NSW 2751, Australia.

E Corresponding author. Email: sergey.shabala@utas.edu.au

Functional Plant Biology 44(12) 1147-1159 https://doi.org/10.1071/FP17133
Submitted: 4 May 2017  Accepted: 28 July 2017   Published: 27 September 2017

Abstract

Salinity is a global problem affecting agriculture that results in an estimated US$27 billion loss in revenue per year. Overexpression of vacuolar ATPase subunits has been shown to be beneficial in improving plant performance under saline conditions. Most studies, however, have not shown whether overexpression of genes encoding ATPase subunits results in improvements in grain yield, and have not investigated the physiological mechanisms behind the improvement in plant growth. In this study, we constitutively expressed Arabidopsis Vacuolar ATPase subunit C (AtVHA-C) in barley. Transgenic plants were assessed for agronomical and physiological characteristics, such as fresh and dry biomass, leaf pigment content, stomatal conductance, grain yield, and leaf Na+ and K+ concentration, when grown in either 0 or 300 mM NaCl. When compared with non-transformed barley, AtVHA-C expressing barley lines had a smaller reduction in both biomass and grain yield under salinity stress. The transgenic lines accumulated Na+ and K+ in leaves for osmotic adjustment. This in turn saves energy consumed in the synthesis of organic osmolytes that otherwise would be needed for osmotic adjustment.

Additional keywords: organic osmolytes, osmotic adjustment, potassium, salinity stress tolerance, sodium, vacuolar sequestration.


References

Adem GD, Roy SJ, Zhou M, Bowman JP, Shabala S (2014) Evaluating contribution of ionic, osmotic and oxidative stress components towards salinity tolerance in barley. BMC Plant Biology 14, 113
Evaluating contribution of ionic, osmotic and oxidative stress components towards salinity tolerance in barley.Crossref | GoogleScholarGoogle Scholar |

Adem G, Roy S, Plett D, Zhou M, Bowman J, Shabala S (2015) Expressing AtNHX1 in barley (Hordium vulgare L.) does not improve plant performance under saline conditions. Plant Growth Regulation 77, 289–297.
Expressing AtNHX1 in barley (Hordium vulgare L.) does not improve plant performance under saline conditions.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXmt12itL0%3D&md5=53c7cc50cb599b931056fc50fd0b85baCAS |

Allen GJ, Chu SP, Schumacher K, Shimazaki CT, Vafeados D, Kemper A, Hawke SD, Tallman G, Tsien RY, Harper JF, Chory J, Schroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant. Science 289, 2338–2342.
Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXmvF2ru7o%3D&md5=6a59040df5a0ecd3455da99eb31064d8CAS |

Anschütz U, Beckera D, Shabala S (2014) Going beyond nutrition: regulation of potassium homoeostasis as a common denominator of plant adaptive responses to environment. Journal of Plant Physiology 171, 670–687.
Going beyond nutrition: regulation of potassium homoeostasis as a common denominator of plant adaptive responses to environment.Crossref | GoogleScholarGoogle Scholar |

Apse MP, Blumwald E (2007) Na+ transport in plants. FEBS Letters 581, 2247–2254.
Na+ transport in plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXls1aiurY%3D&md5=bb91df374c86d7235a69927048acb968CAS |

Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258.
Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXls1Sju7s%3D&md5=5d3bfb350e4abb4f0fcc3a9277402b32CAS |

Armbrüster A, Svergun DI, Coskun U, Juliano S, Bailer SM, Grüber G (2004) Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution. FEBS Letters 570, 119–125.
Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution.Crossref | GoogleScholarGoogle Scholar |

Baisakh N, RamanaRao MV, Rajasekaran K, Subudhi P, Janda J, Galbraith D, Vanier C, Pereira A (2012) Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel. Plant Biotechnology Journal 10, 453–464.
Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xot1ygs7s%3D&md5=4cfb5a71753170fd16ffc9095eb5563bCAS |

Barkla BJ, Zingarelli L, Blumwald E, Smith JAC (1995) Tonoplast Na+/H+ antiport activity and its energization by the vacuolar H+-ATPase in the halophytic plant Mesembryanthemum crystallinum L. Plant Physiology 109, 549–556.
Tonoplast Na+/H+ antiport activity and its energization by the vacuolar H+-ATPase in the halophytic plant Mesembryanthemum crystallinum L.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXoslOqtrc%3D&md5=15bf65222374334bd711ea54925fdb4aCAS |

Barragan V, Leidi EO, Andres Z, Rubio L, De Luca A, Fernandez JA, Cubero B, Pardo JM (2012) Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis. The Plant Cell 24, 1127–1142.
Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xmsl2jtL4%3D&md5=85113ace15c3f714e56907ed2dc9ef89CAS |

Barrett-Lennard E, Setter T (2010) Developing saline agriculture: from traits and genes to systems. Functional Plant Biology 37, iii–iv.
Developing saline agriculture: from traits and genes to systems.Crossref | GoogleScholarGoogle Scholar |

Bassil E, Tajima H, Liang YC, Ohto MA, Ushijima K, Nakano R, Esumi T, Coku A, Belmonte M, Blumwald E (2011) The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction The Plant Cell 23, 3482–3497.
The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproductionCrossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsVGqtrvP&md5=b06a8dd0c7d589e23369e331df749dbdCAS |

Bayat F, Shiran B, Belyaev DV (2011) Overexpression of HvNHX2, a vacuolar Na+/H+ antiporter gene from barley, improves salt tolerance in Arabidopsis thaliana. Australian Journal of Crop Science 5, 428–432.

Blumwald E, Poole RJ (1985) Na+/H+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris. Plant Physiology 78, 163–167.
Na+/H+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2MXktVags74%3D&md5=8e75e24ad9cda9c0ef2f5254d8190eacCAS |

Bonales-Alatorre E, Pottosin I, Shabala L, Chen Z-H, Zeng F, Jacobsen S-E, Shabala S (2013a) Differential activity of plasma and vacuolar membrane transporters contributes to genotypic differences in salinity tolerance in halophyte species, Chenopodium quinoa. International Journal of Molecular Sciences 14, 9267–9285.
Differential activity of plasma and vacuolar membrane transporters contributes to genotypic differences in salinity tolerance in halophyte species, Chenopodium quinoa.Crossref | GoogleScholarGoogle Scholar |

Bonales-Alatorre E, Shabala S, Chen Z-H, Pottosin I (2013b) Reduced tonoplast fast-activating and slow-activating channel activity is essential for conferring salinity tolerance in a facultative halophyte, quinoa. Plant Physiology 162, 940–952.
Reduced tonoplast fast-activating and slow-activating channel activity is essential for conferring salinity tolerance in a facultative halophyte, quinoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXps1Oqs7g%3D&md5=a8869980568d3ab1547f591d4b93c3c5CAS |

Bose J, Rodrigo-Moreno A, Shabala S (2014) ROS homeostasis in halophytes in the context salinity stress tolerance. Journal of Experimental Botany 65, 1241–1257.
ROS homeostasis in halophytes in the context salinity stress tolerance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXks12htbY%3D&md5=0e9bfe786dfba2f3a3bf74a46da82d1aCAS |

Chen Z, Cuin TA, Zhou M, Twomey A, Naidu BP, Shiabala S (2007) Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance. Journal of Experimental Botany 58, 4245–4255.
Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXitlymurg%3D&md5=eb90624fe98b2124e320a0b4d6be5732CAS |

Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiology 133, 462–469.
A gateway cloning vector set for high-throughput functional analysis of genes in planta.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXosVaqtrw%3D&md5=175088d0c2056c01e842de3c0a1065d0CAS |

Demidchik V, Cuin TA, Svistunenko D, Smith SJ, Miller AJ, Shabala S, Sokolik A, Yurin V (2010) Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death. Journal of Cell Science 123, 1468–1479.
Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXnt1KmtL0%3D&md5=1ea5171aa64a36e614598240e5e6d344CAS |

Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19, 1349
A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXitVWju74%3D&md5=5341044301d35e7e48cf4bf5e0c8d213CAS |

Flowers TJ, Colmer TD (2008) Salinity tolerance in halophytes. New Phytologist 179, 945–963.
Salinity tolerance in halophytes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtFWqur%2FE&md5=a36e6fda1d4e8df4e7a168c06e633bccCAS |

Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proceedings of the National Academy of Sciences of the United States of America 96, 1480–1485.
The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXhsFSru7o%3D&md5=18e2dea9a24d684103d05f67bb400147CAS |

Guo SL, Yin HB, Zhang X, Zhao FY, Li PH, Chen SH, Zhao YX, Zhang H (2006) Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene, SsVP, from the halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis. Plant Molecular Biology 60, 41–50.
Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene, SsVP, from the halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtFynsbc%3D&md5=0032a13ac1029c349d0e4460daa90c97CAS |

He XL, Huang X, Shen YZ, Huang ZJ (2014) Wheat V-H+-ATPase subunit genes significantly affect salt tolerance in Arabidopsis thaliana. PLoS One 9, e86982
Wheat V-H+-ATPase subunit genes significantly affect salt tolerance in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Hughes FM, Cidlowski JA (1998) Glucocorticoid-induced thymocyte apoptosis: Protease-dependent activation of cell shrinkage and DNA degradation. The Journal of Steroid Biochemistry and Molecular Biology 65, 207–217.
Glucocorticoid-induced thymocyte apoptosis: Protease-dependent activation of cell shrinkage and DNA degradation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXkvVKqsrs%3D&md5=ee0915a34b0aa15c5c3ffe302cc04cedCAS |

Hughes FM, Cidlowski JA (1999) Potassium is a critical regulator of apoptotic enzymes in vitro and in vivo. Advances in Enzyme Regulation 39, 157–171.

Inan G, Zhang Q, Li PH, Wang ZL, Cao ZY, Zhang H, Zhang CQ, Quist TM, Goodwin SM, Zhu JH, Shi HH, Damsz B, Charbaji T, Gong QQ, Ma SS, Fredricksen M, Galbraith DW, Jenks MA, Rhodes D, Hasegawa PM, Bohnert HJ, Joly RJ, Bressan RA, Zhu JK (2004) Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiology 135, 1718–1737.
Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmtVOqsbg%3D&md5=b4c21c0614c0a20a1b9b71b51e9dc47cCAS |

Jacobs A, Lunde C, Bacic A, Tester M, Roessner U (2007) The impact of constitutive heterologous expression of a moss Na+ transporter on the metabolomes of rice and barley. Metabolomics 3, 307–317.
The impact of constitutive heterologous expression of a moss Na+ transporter on the metabolomes of rice and barley.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFOntbjL&md5=41c67873af60757f80d4252c9bde30a0CAS |

James RA, Munns R, Von Caemmerer S, Trejo C, Miller C, Condon T (2006) Photosynthetic capacity is related to the cellular and subcellular partitioning of Na+, K+ and Cl– in salt-affected barley and durum wheat. Plant, Cell & Environment 29, 2185–2197.
Photosynthetic capacity is related to the cellular and subcellular partitioning of Na+, K+ and Cl in salt-affected barley and durum wheat.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtVaisA%3D%3D&md5=bbe95fed983e525b452e3074e400da38CAS |

Krebs M, Beyhl D, Gorlich E, Al-Rasheid KAS, Marten I, Stierhof YD, Hedrich R, Schumacher K (2010) Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation. Proceedings of the National Academy of Sciences of the United States of America 107, 3251–3256.
Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXis1Cgurw%3D&md5=45dfe7dcf8e8c2d0d33d26a9eb475ac7CAS |

Leidi OE, Barragan V, Rubio L, El-Hamdaoui A, Ruiz TM, Cubero B, Fernandez AJ, Bressan AR, Hasagawa MP, Quintero JF, Pardo MJ (2010) The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato. The Plant Journal 61, 495–506.
The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXitFGqtL0%3D&md5=85de7b47efcd7b4303bf1372421da28aCAS |

Maeshima M (2000) Vacuolar H+-pyrophosphatase. Biochimica et Biophysica Acta (BBA) Biomembranes 1465, 37–51.
Vacuolar H+-pyrophosphatase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXit1Wgtr4%3D&md5=c1c787a2ab4d887782dabf9e19d91861CAS |

Maksimović JD, Zhang JY, Zeng FR, Živanović BD, Shabala L, Zhou MX, Shabala S (2013) Linking oxidative and salinity stress tolerance in barley: can root antioxidant enzyme activity be used as a measure of stress tolerance? Plant and Soil 365, 141–155.
Linking oxidative and salinity stress tolerance in barley: can root antioxidant enzyme activity be used as a measure of stress tolerance?Crossref | GoogleScholarGoogle Scholar |

Munns R (2002) Comparative physiology of salt and water stress. Plant, Cell & Environment 25, 239–250.
Comparative physiology of salt and water stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xhslakurw%3D&md5=2f4c6c7e2f9463e9f6a3ba186035af1dCAS |

Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651–681.
Mechanisms of salinity tolerance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXntFaqtrw%3D&md5=b30f07d810b3303a6dcacfac2b459d6cCAS |

Panta S, Flowers T, Lane P, Doyle R, Haros G, Shabala S (2014) Halophyte agriculture: success stories. Environmental and Experimental Botany 107, 71–83.
Halophyte agriculture: success stories.Crossref | GoogleScholarGoogle Scholar |

Pantoja O, Dainty J, Blumwald E (1989) Ion channels in vacuoles from halophytes and glycophytes. FEBS Letters 255, 92–96.
Ion channels in vacuoles from halophytes and glycophytes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1MXmt1Ogsb8%3D&md5=fc0a61d6bf45c60c7817491c398a2de0CAS |

Parks GE, Dietrich MA, Schumaker KS (2002) Increased vacuolar Na+/H+ exchange activity in Salicornia bigelovii Torr. in response to NaCl. Journal of Experimental Botany 53, 1055–1065.
Increased vacuolar Na+/H+ exchange activity in Salicornia bigelovii Torr. in response to NaCl.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XjsFagu7k%3D&md5=f490d70efaa256004b76686ec12ae877CAS |

Puniran-Hartley N, Hartley J, Shabala L, Shabala S (2014) Salinity-induced accumulation of organic osmolytes in barley and wheat leaves correlates with increased oxidative stress tolerance: in planta evidence for cross-tolerance. Plant Physiology and Biochemistry 83, 32–39.
Salinity-induced accumulation of organic osmolytes in barley and wheat leaves correlates with increased oxidative stress tolerance: in planta evidence for cross-tolerance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXht1KlsL7N&md5=139f1b25c74fc2c17a5d19ea17b1a494CAS |

Qadir M, Quillérou E, Nangia V, Murtaza G, Singh M, Thomas RJ, Drechsel P, Noble AD (2014) Economics of salt-induced land degradation and restoration. Natural Resources Forum 38, 282–295.
Economics of salt-induced land degradation and restoration.Crossref | GoogleScholarGoogle Scholar |

Qiu NW, Chen M, Guo JR, Bao HY, Ma XL, Wang BS (2007) Co-ordinate up-regulation of V-H+-ATPase and vacuolar Na+/H+ antiporter as a response to NaCl treatment in a C3 halophyte Suaeda salsa. Plant Science 172, 1218–1225.
Co-ordinate up-regulation of V-H+-ATPase and vacuolar Na+/H+ antiporter as a response to NaCl treatment in a C3 halophyte Suaeda salsa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXkvFGitbo%3D&md5=4aafc44170ddec3f33d73171b904f0e3CAS |

Raven JA (1985) Regulation of pH and generation of osmolarity in vascular plants – a cost-benefit analysis in relation to efficiency of use of energy, nitrogen and water. New Phytologist 101, 25–77.
Regulation of pH and generation of osmolarity in vascular plants – a cost-benefit analysis in relation to efficiency of use of energy, nitrogen and water.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2MXmtFWjt7g%3D&md5=2e1b69b49e6dafb61f02bde3d53d4cc7CAS |

Rodríguez-Rosales MP, Jiang XY, Gálvez FJ, Aranda MN, Cubero B, Venema K (2008) Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization. New Phytologist 179, 366–377.
Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization.Crossref | GoogleScholarGoogle Scholar |

Roy SJ, Huang W, Wang XJ, Evrard A, Schmockel SM, Zafar ZU, Tester M (2013) A novel protein kinase involved in Na+ exclusion revealed from positional cloning. Plant, Cell & Environment 36, 553–568.
A novel protein kinase involved in Na+ exclusion revealed from positional cloning.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhs1ais7c%3D&md5=e7560c1fa7da78ae87b067326faa0805CAS |

Scheuring D, Schöller M, Kleine-Vehn J, Löfke C (2015) Vacuolar staining methods in plant cells. In ‘Plant cell expansion: methods and Protocols. Methods in molecular biology. Vol. 1242’. (Ed. J Estevez) pp. 83–92. (Humana Press: Clifton, NJ, USA).

Shabala S (2013) Learning from halophytes: physiological basis and strategies to improve abiotic stress tolerance in crops. Annals of Botany 112, 1209–1221.
Learning from halophytes: physiological basis and strategies to improve abiotic stress tolerance in crops.Crossref | GoogleScholarGoogle Scholar |

Shabala S, Mackay A (2011) Ion transport in halophytes. Advances in Botanical Research 57, 151–199.
Ion transport in halophytes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXps1Sit74%3D&md5=2d715f8077e1330e11408ba9452e7346CAS |

Shabala S, Cuin TA, Prismall L, Nemchinov LG (2007) Expression of animal CED-9 anti-apoptotic gene in tobacco modifies plasma membrane ion fluxes in response to salinity and oxidative stress. Planta 227, 189–197.
Expression of animal CED-9 anti-apoptotic gene in tobacco modifies plasma membrane ion fluxes in response to salinity and oxidative stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtlyku7zF&md5=4d1e8fec9d499fa7c9c94fa479736102CAS |

Silva P, Facanha AR, Tavares RM, Geros H (2010) Role of tonoplast proton pumps and Na+/H+ antiport system in salt tolerance of Populus euphratica Oliv. Journal of Plant Growth Regulation 29, 23–34.
Role of tonoplast proton pumps and Na+/H+ antiport system in salt tolerance of Populus euphratica Oliv.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXjtFWlt7c%3D&md5=0959e88755d3b46bba81722ad3275089CAS |

Singh RR, Kemp JA, Kollmorgen JF, Qureshi JA, Fincher GB (1997) Fertile plant regeneration from cell suspension and protoplast cultures of barley (Hordeum vulgare cv. Schooner). Plant Cell, Tissue and Organ Culture 49, 121–127.
Fertile plant regeneration from cell suspension and protoplast cultures of barley (Hordeum vulgare cv. Schooner).Crossref | GoogleScholarGoogle Scholar |

Skoog D, West D, Holler F, Crouch S (2000) ‘Analytical chemistry: an introduction.’ (Saunders College Publishing: Philadelphia, PA, USA).

Sze H, Schumacher K, Muller M, Padmanaban S, Taiz L (2002) A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H+-ATPase. Trends in Plant Science 7, 157–161.
A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H+-ATPase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XjtVKnsbg%3D&md5=59d689c8ada417c3c37b557fd32eb984CAS |

Tingay S, McElroy D, Kalla R, Fieg S, Wang MB, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. The Plant Journal 11, 1369–1376.
Agrobacterium tumefaciens-mediated barley transformation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXkslajurw%3D&md5=1379b25997fb3cefae0173a5f1f7fad9CAS |

Tomos AD, Leigh RA, Shaw CA, Jones RGW (1984) A comparison of methods for measuring turgor pressures and osmotic pressures of cells of red beet storage tissue. Journal of Experimental Botany 35, 1675–1683.
A comparison of methods for measuring turgor pressures and osmotic pressures of cells of red beet storage tissue.Crossref | GoogleScholarGoogle Scholar |

Vera-Estrella R, Barkla BJ, Bohnert HJ, Pantoja O (1999) Salt stress in Mesembryanthemum crystallinum L cell suspensions activates adaptive mechanisms similar to those observed in the whole plant. Planta 207, 426–435.
Salt stress in Mesembryanthemum crystallinum L cell suspensions activates adaptive mechanisms similar to those observed in the whole plant.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXhtlOgtro%3D&md5=44e4c6a5f08bf03eb9a46fda9de0ed02CAS |

Vera-Estrella R, Barkla BJ, Garcia-Ramirez L, Pantoja O (2005) Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance. Plant Physiology 139, 1507–1517.
Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXht1Ogu73F&md5=d8b8d45f96f506507c155087b2bedb00CAS |

Wang BS, Luttge U, Ratajczak R (2001) Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa. Journal of Experimental Botany 52, 2355–2365.
Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXptVeisL0%3D&md5=ab2a637d7ccb22816ce1ac4d895f4bddCAS |

Wang L, He XL, Zhao YJ, Shen YZ, Huang ZJ (2011) Wheat vacuolar H+-ATPase subunit B cloning and its involvement in salt tolerance. Planta 234, 1–7.
Wheat vacuolar H+-ATPase subunit B cloning and its involvement in salt tolerance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXotVeltr8%3D&md5=1a9d3e31fd2c9b84ec7d21ccd47f9fb1CAS |

Wu HH, Shabala L, Barry K, Zhou MX, Shabala S (2013) Ability of leaf mesophyll to retain potassium correlates with salinity tolerance in wheat and barley. Physiologia Plantarum 149, 515–527.
Ability of leaf mesophyll to retain potassium correlates with salinity tolerance in wheat and barley.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhslyhtLnL&md5=8aab4fbea16efe03468b821dc99f2ea5CAS |

Wu H, Zhu M, Shabala L, Zhou M, Shabala S (2014) K+ retention in leaf mesophyll, an overlooked component of salinity tolerance mechanism: a case study for barley. Journal of Integrative Plant Biology 57, 1–15.

Wu H, Shabala L, Liu X, Azzarello E, Zhou M, Pandolfi C, Chen ZH, Bose J, Mancuso S, Shabala S (2015) Linking salinity stress tolerance with tissue-specific Na+ sequestration in wheat roots. Frontiers in Plant Science 6, 1–13.
Linking salinity stress tolerance with tissue-specific Na+ sequestration in wheat roots.Crossref | GoogleScholarGoogle Scholar |

Xu CX, Zheng L, Gao CQ, Wang C, Liu GF, Jiang J, Wang YC (2011) Ovexpression of a Vacuolar H+-ATPase c subunit gene mediates physiological changes leading to enhanced salt tolerance in transgenic tobacco. Plant Molecular Biology Reporter 29, 424–430.
Ovexpression of a Vacuolar H+-ATPase c subunit gene mediates physiological changes leading to enhanced salt tolerance in transgenic tobacco.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXltFaqsL8%3D&md5=18f9545df186bac16b0eaf95f0a9ec15CAS |

Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nature Biotechnology 19, 765–768.
Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXlslektLw%3D&md5=50ce757907071620eade0fd76db6f532CAS |

Zhang HX, Hodson JN, Williams JP, Blumwald E (2001) Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proceedings of the National Academy of Sciences of the United States of America 98, 12832–12836.
Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXotFahsLs%3D&md5=1b88ad09ca0c797ef1c04770a0fa5f1fCAS |

Zhang HY, Niu XL, Liu J, Xiao FM, Cao SQ, Liu YS (2013) RNAi-directed downregulation of vacuolar H+-ATPase subunit A results in enhanced stomatal aperture and density in rice. PLoS One 8, 1–18.

Zhao Q, Zhao YJ, Zhao BC, Ge RC, Li M, Shen YZ, Huang ZJ (2009) Cloning and functional analysis of wheat V-H+-ATPase subunit genes. Plant Molecular Biology 69, 33–46.
Cloning and functional analysis of wheat V-H+-ATPase subunit genes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsVagsbzO&md5=aa293bdb360414de509ab947bf174a97CAS |