The yield reduction due to the direct effect of drought, salt and cold stresses, was estimated up to 70 % [1]. Therefore the enhancing of crop tolerance to theses stresses has long remained a challenge. These stresses affect plant growth and development by way of osmotic stress, nutrient imbalance and toxic effects caused by excess of Na+ and Cl– ions [2]. To alleviate abiotic stresses, plants use many mechanisms such as production of antioxidants and transport and compartmentalization of toxic Na+. Despite the majority of plants cannot thrive in the presence of high concentration of salt in the soil, the extremophile halophytes which represent less than 0.2% of plant species are able to grow and reproduce in these conditions. These halophytes can be used as an important model plants and genetic resources to decipher the molecular, biochemical and physiological mechanisms of salt tolerance in plants. This will contribute to advance the improvement of salt tolerance in important economically crops. Indeed, the identification of the key genes and promoters from these extremophiles could be used to engineer plant abiotic stress tolerance either by overexpressing, silencing or editing (using the CRISPR/Cas9) [3]. For our final objective to improve the tolerance to abiotic stresses in cereals, we are using the halophyte grass Aeluropus littoralis as a source of candidate genes and their regulatory elements (promoter). This extremophile previously described by Zouari et al [4], is a perennial C4 photosynthesis plant, salt-secreting, rhizomatous and characterized by a small haploid genome of 349 Mb (2n=2X=10). To understand the genetic basis of salt tolerance mechanisms in A. littoralis at the genomic level, we have isolated, sequenced and annotated 492 salt stress regulated transcripts with a size ranging from ESTs (Expressed Sequence Tag) to full length cDNAs using SSH technology [4]. These ESTs shared high similarities with sequences from rice, maize and sorghum [4] and the functional analysis of their full-length genes in tobacco can allow the identification of the key candidate genes to be transferred to rice and wheat. As a demonstration of this strategy, we reported previously the isolation of AlSAP gene and its promoter [5, 6]. The constitutively expression of AlSAP in the model plants tobacco, wheat and rice have resulted in enhanced tolerance to multiple abiotic stresses including drought, salinity, cold, heat, and oxidative stress [5, 7, 8].
The maintenance of intracellular ion and osmotic homeostasis is crucial to the physiological processes of living cells. Indeed, cells often maintain high concentration of K+ and low concentration of Na+ in the cytosol, which is important for activities of many cytosolic enzymes [9]. Under high salinity in the soil, it was shown that plants use mechanisms to maintain ion homeostasis such as ion compartmentalization, osmotic adjustment, selective ion uptake and transport, succulence and salt inclusion or secretion [10, 11]. The plant uses all these complex mechanisms to accommodate a balance between cellular requirements and ion homeostasis via specialized transporter proteins that are classified as pumps, carriers and channels [12]. At cellular level, plants use ion channels and ion transporters to maintain an appropriate Na+, K+ and Ca2+ in the cell through active and diffusion mechanisms of ion transport (reviewed in [13]). In fact, some genes such as NHX-type antiporters have been isolated and identified to be responsible for removing Na+ from the cytoplasm either by exclusion of Na+ from plant cells (plasma membrane group: SOS1 (NHX7) from Arabidopsis) or by the sequestration of Na+ in vacuoles (vacuole group: NHX1–NHX4 from Arabidopsis) [14]. However, others genes of plasma membrane Na+ influx systems into plant cells have been identified like Na+ uptake transporters in wheat HKT1 (TaHKT2.1)[15, 16] and in Arabidopsis thaliana AtHKT1 (AtHKT1.1) [17]. The first HKT gene, TaHKT2 (TaHKT2.1), originally cloned from wheat, was shown to mediate K+ and Na+ co-transport in yeast and Xenopus oocytes [15, 16]. During the last decade, some genes known to be responsible for ion transport and which catalyze ion influx were identified. From these there are the nonselective cation channels (NSCCs), ion transporters and membrane-potential modulators (such as a proton pump) [18, 19]. The NSCCs can be divided into: cyclic-nucleotide-gated NSCCs (CNGSs), amino-acid gated NSCCs (AAG-NSCCs) and reactive-oxygen-species-activated [18].
During the recent years a yeast small hydrophobic peptide (55 amino-acids) encoded by a Pmp3p gene was suggested to be involved in preventing Na+ entry to the cells [20]. This protein is localized in the plasma membrane and it was shown to modulate the membrane potential [20, 21]. These authors have demonstrated that due to membrane hyperpolarization the Δpmp3 yeast mutant is sensitive to both Na+ and the cationic hygromycin B [20]. PMP3 belongs to an evolutionarily conserved family of small plasma membrane proteins which are highly conserved in bacteria, yeast, nematode and plants [22]. It was shown that RCI2A gene of Arabidopsis complements the sodium sensitivity caused by a deletion of the homologous yeast gene SNA1 [20]. Many orthologues to PMP3 were found in barley [23], rice [24], wheat [25], maize [9], red sage [26], Arabidopsis [27] and the halophyte Aneurolepidium chinense [28]. All these genes were shown to be transcriptionally induced in response to stress, such as low temperature, salinity, and H2O2 [23, 24, 27, 28]. All members of PMP3 family proteins have two highly hydrophobic domains, separated by a predicted short loop, and appear to be integral membrane proteins [29]. It was shown by using the TMHMM prediction that both the N- and C-termini of the native WPI6 of wheat are located in the apoplastic space, whereas the internal loop separating the transmembrane domains is cytoplasmic [25]. Several analyses of complementation in yeast pmp3 mutants have shown that the expression of many plant homologs such as OsLTi6a, OsLTi6b from rice, AtRCI2a, AtRCI2b from Arabidopsis, AcPMP3-1 from Aneurolepidium chinese, PutPMP3-1, PutPMP3-2 from Puccinellia tenuiflora, ZmPMP3-1 from maize and MsRCI2A, MtRCI2(A-C) from Medicago could functionally complement the salt sensitivity resulting from PMP3 deletion [9, 30, 31]. In Arabidopsis thaliana plants, the disruption of RCI2A leads to over-accumulation of Na+ and increased salt sensitivity [32]. Furthermore, the over-expression of RCI2A gene decreases Na+ uptake and mitigates salinity-induced damages causing an enhanced salt-tolerant phenotype in Arabidopsis thaliana [33]. It was also shown that over-expression of Musa paradisica RCI (MpRCI) in the AtRCI2A knockout mutant increases Na+ tolerance and K+ sensitivity under NaCl or KCl treatments, respectively. This result suggests that MpRCI plays an essential role in Na+/K+ flux in plant cells [34]. On the other hand, over-expression of ZmPMP3.1 enhanced growth of transgenic Arabidopsis under salt condition. This tolerance was likely achieved through diminishing oxidative stress due to the possibility of ZmPMP3.1’s involvement in the regulation of ion homeostasis [9]. More recently, it has been shown that over-expression of MsRCI2A in alfalfa plants showed improved salt tolerance [30]. The overexpression of OsLti6b increased rice tolerance to cold but not to salt or drought [35]. On the other hand, overexpression of OsRCI2-5, which share 60 % sequence identity with OsLti6b, increased drought tolerance [36]. Taken together, these data suggest that PMP3 family proteins play an important role in maintaining intracellular ion homeostasis, membrane potential and membrane organization. Although these high similarities between PMP3 genes different studies reported that these genes are not functionally equivalent.
In order to decipher the efficient regulation systems for ion homeostasis and osmoregulation under salt stress developed by the halophyte grass Aeluropus littoralis, we report here the isolation and characterization of a gene (AlTMP1, KY321744) encoding for a PMP3 protein. In A. littoralis, the AlTMP1 was shown to be induced by abscisic acid (ABA), cold, salt and osmotic stresses. By stress assays, we have found that the overexpression of the AlTPM1 gene in tobacco improved tolerance more efficiently to continuous drought than to salinity under greenhouse conditions. In addition, these transgenic tobacco plants showed significantly improved tolerance to heat and cold stresses. Interestingly, AlTMP1 plays a role in maintaining high membrane stability and low electrolytes leakage. Finally the AlTMP1 was shown to affect some stress related genes under control and stress conditions which probably prime the plants to tolerate stresses.