Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland

Joshi M (2013) Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland. PLoS ONE 8(8): e71136. doi:10.1371/journal.pone.0071136 Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland Bhavanath Jha 0 Avinash Mishra 0 Anupama Jha 0 Mukul Joshi 0 Jean Peccoud, Virginia Tech, United States of America 0 Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute , Bhavnagar, Gujarat , India Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na+/H+ antiporter gene that compartmentalises excess Na+ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas. - Funding: The financial assistance received from CSIR (www.csir.res.in), New Delhi (CSC0102: TapCoal) is duly acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Jatropha is a second generation biofuel resource valued for its high oil content, low seed cost, land reclamation and easy adaptation to different kinds of marginal and semi marginal lands. The broad potential of this plant and multiple uses of different plant parts have made this species quite profitable for cultivation [1,2]. As fossil fuels pose a great threat to energy security and also adversely affect the environment, initiatives are being taken for the partial replacement of fossil fuels by biofuels [3]. Recently, attention has been drawn to the high oil content (up to 50%) of its seeds that can be easily processed to partially or fully replace petroleum based diesel fuel [3,4]. Jatropha is a non-food crop that separates this plant from fuel vs food controversy. Its oil properties include flash point 235uC and calorific value 39.63 MJ kg21 that make this oil suitable as biofuel [1]. Jatropha oil contains 45.79% oleic acid (18:1), 32.27% linoleic acid (18:2), 13.37% palmitic acid (16:0) and 5.43% stearic acid (18:0) that is comparable with peanut, palm and corn oil [5]. Jatropha ranks next to oil palm in oil production per hectare, encouraging its cultivation worldwide [6]. There are concerns that Jatropha investors may drive its cultivation from marginal or degraded lands towards agricultural lands in order to reduce financial risk [7] but this possibility was recently ruled out [8]. Jatropha is well distributed in India [9]; this encourages its use as an alternative source for energy security in the country. India has a plan to expand biodiesel production and substitute 20% of diesel consumption by 2020 [10]. The land required to achieve this substitution target ranges from 4.2466.98 million hectares (Mha) depending on the potential yield of the plant varieties and further its improvement programs. This target is feasible because of the extent of available wastelands in India [11]. CSIR-CSMCRI has been recognized worldwide and is actively working on Jatropha elite accessions selection, cultivation, genetic improvement and biodiesel production [6,12]. Over 800 Mha of land throughout the world are affected by salt [13]. In India, the total arable land area is about 184 Mha, out of which 8.6 Mha is salt affected [14]. Salinity imposes various constraints on plant growth and yield from osmotic stress created due to high concentrations of Na+ and Cl- ions [15]. There are some major effects of salt stress, i.e. water potential reduction, ionic imbalance or disturbances in ion homeostasis and ion toxicity, which inhibits enzymatic functions in key biological processes [15,16].Salt tolerance depends on a range of physiological, biochemical and molecular adaptations activated by gene(s). The adaptive response to salinity is multigenic in nature, however a single gene can also increase the salt tolerance of a plant species [17,18,19]. Antiporters Na+/H+ are expressed by functional gene(s) and play an important role in plant salt tolerance. The basic strategy of salt tolerance is maintenance of Na+ homeostasis in the cytosol [15]; however it varies with the plant species. Ion homeostasis is carried out either by sequestration of excess sodium into the vacuoles via vacuolar Na+/H+ antiporters (NHX1) energized by the proton gradient generated by vacuolar membrane H+-ATPase and H+-pyrophosphatases [15,17], or by active exclusion through Na+/H+ antiporters (SOS1) located on the plasma membrane [18,19]. Therefore, the ability to maintain lower Na+ and Cl- in the cytosol may be a key determinant of salt tolerance. The previous study that vacuolar NHX proteins are capable of Na+ and H+ exchange across the tonoplast [15,17], has been modified based on the biochemistry of the NHX proteins. Recently it was reported that NHX proteins do not discriminate between Na+ and K+ or have a preference for K+ transport [20,21]. These reports suggest that NHX1 and NHX2 are vacuolar Na+K+/H+ exchangers essential for active K+ uptake at the tonoplast, osmotic adjustment, turgor regulation, stomata function and cell expansion [22,23]. Jatropha growth and yield are adversely affected by increasing salt concentration beyond 3050 mM [24,25,26]. Therefore, Jatropha is considered to be a moderately salt-tolerant plant and its salinity tolerance is comparable with major crops such as soybean or wheat [27]. Development of transgenic Jatropha plants by the overexpression of selected salt-responsive gene(s) is a better choice for genetic improvement of the plant for enhanced salt tolerance and fastening the breeding of improved plants. There are several methods available for genetic transformation of plants but the widely used methods (...truncated)