Co-expression of vacuolar Na + /H + antiporter and H + -pyrophosphatase with an IRES-mediated dicistronic vector improves salinity tolerance and enhances potassium biofortification of tomato Sandra Gouiaa, Habib Khoudi ⇑ Laboratory of Plant Protection and Improvement, Center of Biotechnology of Sfax (CBS), Route Sidi Mansour Km 6, B.P’1177’, 3018 Sfax, Tunisia article info Article history: Received 19 November 2014 Received in revised form 18 May 2015 Accepted 26 May 2015 Available online xxxx Keywords: Tomato Biofortification NHX-type protein Pyrophosphatase Potassium Salt tolerance IRES Dicistronic vector Ionomic analysis abstract Potassium (K) deficiency is a worldwide problem. Thus, the K biofortification of crops is needed to enhance human nutrition. Tomato represents an ideal candidate for such biofortification programs thanks to its widespread distribution and its easy growth on a commercial scale. However, although tomato is moderately tolerant to abiotic stresses, the crop losses due to salinity can be severe. In this study, we generated transgenic tomato plants over-expressing a Na + –K + /H + exchanger gene (TNHXS1), singly or with H + -pyrophosphatase (H + -PPiase) gene using a bicistronic construct. Transgenic tomato lines co-expressing both genes (LNV) significantly showed higher salinity tolerance than the wild-type (WT) plans or those expressing the TNHXS1 gene alone (LN). Indeed, under salt stress conditions, double transgenic plants produced higher biomass and retained more chlorophyll and catalase (CAT) activity. In addition, they showed earlier flowering and produced more fruits. To address K deficiencies in humans, an increase of 50% in K content of vegetable products was proposed. In this study, ion content analysis revealed that, under salt stress, fruits from double transgenic plants accumulated 5 times more potas- sium and 9 times less sodium than WT counterparts. Interestingly, the ionomic analysis of tomato fruits also revealed that LNV had a distinct profile compared to WT and to LN plants. Indeed, LNV fruits accu- mulated less Fe 2+ , Ca 2+ , Mg 2+ and Zn 2+ , but more Mn 2+ . This study demonstrates the effectiveness of bicis- tronic constructs as an important tool for the enhancement of biofortification and salt stress tolerance in crops. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Potassium (K) is one of the eight essential minerals that the human body needs and in relatively large quantities (100 ? 1000 mg/d) (Broadley and White, 2010). The recom- mended daily value for K is 4700 mg (Hoy and Goldman, 2012). Albeit common medical reports on the importance of K in reducing the risk of heart disease, and in preventing renal disease, osteo- porosis and diabetes (He and MacGregor, 2008; Ando et al., 2010), the World Health Organization (WHO) has reported a worldwide deficiency of this key mineral in human diet (WHO, 2003). Therefore, K enhancement in crops is needed to improve human nutrition. Biofortification is an emerging, cost-effective and sustainable approach to alleviate mineral malnutrition by increasing mineral concentrations in edible crops. Crop mineral biofortification can be achieved by agronomic fertilization, but this does not represent a long-term sustainable approach. In fact, it is very costly and not environmentally-friendly. In contrast, crop biofortification by genetic engineering represents a sustainable and low cost means to address mineral deficiencies in human. Genetic engineering has been proven useful for macronutrient biofortification. In fact, over-expression of genes encoding AtCAX1 lacking its inhibitory domain (sCAX1), a modified AtCAX2 (sCAX2) or AtCAX4 in carrot (Park et al., 2004; Morris et al., 2008), lettuce (Park et al., 2009), potato (Park et al., 2005b) and tomato (Park et al., 2005a; Chung et al., 2010) resulted in increased calcium concentration in shoots which could increase dietary calcium delivery. Tomato (Lycopersicon esculentum L.) is the second most impor- tant vegetable crop in the world. It is consumed in many forms such as raw vegetable or as processed products such as paste, whole peeled, diced, juice, sauces and soups. In addition, tomato is a valuable source of health-promoting compounds such as the antioxidant lycopene (Pohar et al., 2003; Rao and Agarwal, 2000). Due to its widespread distribution and its easy growth on a http://dx.doi.org/10.1016/j.phytochem.2015.05.016 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. E-mail address: hkhoudi@hotmail.com (H. Khoudi). Phytochemistry xxx (2015) xxx–xxx Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: Gouiaa, S., Khoudi, H. Co-expression of vacuolar Na + /H + antiporter and H + -pyrophosphatase with an IRES-mediated dicis- tronic vector improves salinity tolerance and enhances potassium biofortification of tomato. Phytochemistry (2015), http://dx.doi.org/10.1016/ j.phytochem.2015.05.016