Research articleNa+ transporter HKT1;2 reduces flower Na+ content and considerably mitigates the decline in tomato fruit yields under saline conditions
Introduction
Soil salinity, which is a major threat to global food security, affects 2.1% of dry agricultural land and up to 20% of irrigated land, accounting for one third of the world's food production (FAO, 2019). The losses in agricultural production worldwide due to salinity are estimated at 27 billion dollars per year (Munns and Gilliham, 2015). Worldwide, tomato is the most important horticultural crop, with a total production and cultivated area estimated at 164 Mt and 4.76 Mha, respectively (FAOSTAT, 2017). Tomato crops are grown mainly in areas with an arid or semi-arid Mediterranean climate. As tomato requires a large amount of water for its growth (Romero-Aranda et al., 2001; Reina et al., 2005), in areas with a shortage of good-quality water for irrigation, farmers often use low-quality water from wells and groundwater aquifers containing high concentrations of soluble salts, mainly NaCl. In these areas, electrical conductivity (EC) frequently exceeds 3 dS/m (equivalent to 2 g/L NaCl) in soils and irrigation water, which causes a sharp decrease in tomato production (Maas y Grieve, 1990; Romero-Aranda et al., 2002; Reina et al., 2005). All this highlights the necessity of seeking solutions to improve the salt tolerance of the tomato crop (Cuartero et al., 2006). An important strategy to reduce the impact of salinity on tomato plants involves exploiting the halotolerant genetic potential of wild relative species as donors of tolerance, whose beneficial gene alleles, identified by QTL analysis, could be introgressed into elite cultivated tomato (Cuartero et al., 2006). The identification of genes functionally underlying QTLs makes for more efficient marker-assisted selection in genetic improvement programs and facilitates their manipulation through genetic engineering (Arzani and Ashraf, 2016).
For most crop plants, particularly tomato, excessive Na+ accumulation in the aerial part can have highly negative effects on growth and development, as high concentrations of Na+ competitively inhibit K+ uptake systems and negatively affect many K+-dependent physiological plant functions (Kronzucker et al., 2013). Although this Na+ accumulation is considered to be an adaptive advantage in halotolerant accessions of some tomato wild-related species given that Na+ is a biologically inexpensive osmolyte that contributes to maintaining an optimal water balance, disturbances in Na+/K+ concentrations inside the cell can adversely affect plant development in tomato cultivars (Kronzucker et al., 2013; Munns et al., 2016). Thus, maintaining a balanced cytosolic Na+/K+ ratio has become a key salinity tolerance mechanism in tomato (Cuartero et al., 2006). Na+/K+ homeostasis at the whole plant level is a complex mechanism involving multiple genes that participate in a network of transport processes which control absorption, extrusion through the plasma membrane, salt compartmentalization in cellular vacuoles and ion recirculation through the plant organs, thus enabling osmotic adjustment and the maintenance of a high K+/Na+ ratio in the plant cytosol (Pardo and Rubio, 2011). As in Arabidopsis, rice and other crop plants (Pardo and Rubio, 2011; Kronzucker et al., 2013), tomato genes encoding Na+ transporter SOS1 and its regulatory proteins (Olías et al., 2009; Huertas et al., 2012), HKT1-like proteins (Asins et al., 2013; Jaime-Pérez et al., 2017) and certain NHX-type antiporters (Gálvez et al., 2012; Huertas et al., 2013) are mainly involved in regulating Na+ concentrations in various tissues of tomato plants. HKT1-like transporters, which are responsible for removing Na+ from the xylem at root level, preventing its accumulation in the aerial part and indirectly improving K+ accumulation, play an important role in salt tolerance (Ren et al., 2005; Davenport et al., 2007; Munns et al., 2012; Byrt et al., 2014; Suzuki et al., 2016). In addition, in the aerial part, HKT1-like proteins are involved not only in removing Na+ from the xylem, which indirectly boosts the vacuolar accumulation of Na+ in stems and leaves in collaboration with other transporters, but also possibly in its redistribution to other sink tissues via the phloem (Munns et al., 2016). HKT1-like transporters must therefore be related to energy-efficient osmotic adjustments by Na+ ions and to the maintenance of turgor potential in above-ground tissues (Munns et al., 2019). Several HKT1-like genes have been identified as directly associated with QTLs responsible for Na+ and K+ concentrations in aerial parts of monocots such as rice, corn, barley and wheat (Ren et al., 2005; Munns et al., 2012; Zhang et al., 2017) and dicots including Arabidopsis and grapevine (Baxter et al., 2010; Henderson et al., 2018). Similarly, using candidate gene analysis of tomato, we identified two closely linked genes encoding HKT1-like transporters, HKT1;1 and HKT1;2, as candidate genes for a major QTL associated with shoot Na+/K+ homeostasis using two populations of recombinant inbred lines (RILs) derived from S. lycopersicum cv. Cerasiform and the salt-tolerant wild tomato species S. pimpinellifolium and S. cheesmaniae (Asins et al., 2013). Under growth chamber conditions, we evaluated different transgenic lines derived from two near-isogenic lines (NILs) of S. lycopersicum cv. cerasiform differing in terms of their HKT1;1/HKT1;2 alleles from S. lycopersicum and S. cheesmaniae, in which each of these genes is silenced by stable transformation. We found that the Na+ transporter-encoding gene HKT1;2 functionally underlies the major QTL controlling shoot Na+/K+ homeostasis and plays an important role in tomato salt tolerance in terms of vegetative growth, while HKT1; 1 alleles had little effect (Jaime-Pérez et al., 2017).
Given that tomato yield is considered to be the ultimate criterion for salt tolerance, we decided to evaluate plant behaviour under natural greenhouse conditions following standard commercial cultural procedures in order to follow up the previous experiments carried out under controlled growth chamber conditions (Jaime-Pérez et al., 2017). We also investigated the role of two HKT1;2 alleles, lycopersicum and cheesmaniae, in salt tolerance in terms of plant physiological parameters, biomass production, as well as fruit yield and quality, by using both NILs homozygous for either the S. lycopersicum allele (NIL17) or the S. cheesmaniae allele (NIL14) at both HKT1 loci, as well as transgenic lines derived from these NILs in which both S. lycopersicum and S. cheesmaniae HKT1;2 alleles had been silenced by stable transformation. These new findings could potentially be very useful in future strategies for improving tomato productivity under Mediterranean greenhouse conditions.
Section snippets
Plant material
The two tomato near-isogenic lines (NILs), NIL157-14 (NIL14) and NIL157-17 (NIL17), used in this study were obtained as described elsewhere (Asins et al., 2013). Briefly, they were developed by selfing a segregating F6 line (RIL B157), which itself was obtained after five selfing generations of an F1 progeny from a cross between a salt-sensitive genotype of S. lycopersicum, var. Cerasiform, as female parent and a salt-tolerant genotype of S. cheesmaniae (L. Riley) Fosberg as male parent (
Physiological and phenotypic evaluation: Sl/ScHKT1;2–RNAi silenced lines showed a salt-hypersensitive phenotype at the vegetative stage
The gene expression patterns for Sl/ScHKT1;2 allelic variants were analysed in root, leaf and flower tissues (Fig. 1). SlHKT1;2 transcript levels in NIL17 roots were considerably higher than those of ScHKT1;2 in NIL14 roots, while, in shoots (mainly leaves), their expression followed an opposite trend. As expected, HKT1;2 gene expression of each Sc/SlHKT1;2-silenced line was much lower than that of the respective non-silenced NIL plants, regardless of tissue and salt treatment (Fig. 1).
SlHKT1;2 and ScHKT1;2 Na+ transporters prevent Na+ over-accumulation in leaf tissues
The tomato gene HKT1;2 encodes a Na+-selective class I HKT transporter expressed in the vascular system, particularly in the xylem and possibly in the phloem of tomato leaves and roots (Asins et al., 2013; Almeida et al., 2014; Jaime-Pérez et al., 2017). Under commercial greenhouse (natural light and no temperature control) and salinity conditions, HKT1;2 gene expression in non-silenced NILs was very similar to expression patterns observed in previous studies (Fig. 1; Asins et al., 2013;
CRediT authorship contribution statement
María Remedios Romero-Aranda: Formal analysis, Writing - original draft, Supervision. Paloma González-Fernández: Formal analysis. Jacob Rafael Pérez-Tienda: Formal analysis. María Remedios López-Diaz: Formal analysis. Jesús Espinosa: Formal analysis. Espen Granum: Formal analysis. Jose Ángel Traverso: Data curation, Formal analysis. Benito Pineda: Formal analysis. Begoña Garcia-Sogo: Formal analysis. Vicente Moreno: Writing - original draft, Supervision. María José Asins: Writing - original
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We wish to thank Emilio Jaime Fernández (La Mayora, IHMS-CSIC) and Elena Sánchez Romero (EEZ-CSIC) for their technical assistance, the Scientific Instrumentation Service at EEZ-CSIC for their ICP-OES mineral analysis and Michael O'Shea for proofreading the text. The study was funded by EU-cofinanced grants from Agencia Estatal de Investigación, Spanish Ministry of Economy, Industry and Competition (AGL2013-41733-R and AGL2017-82452-C2-1R to A.B., AGL2017-82452-C2-2R to M.J.A.) and the
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