Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger “AlSAP” gene isolated from the halophyte grass Aeluropus littoralis

Abstract

We describe here the isolation of a novel gene, designated AlSAP, from A. littoralis in a first step to exploit the potential of this halophyte grass as a genetic resource to improve salt and drought tolerance in plants and, particularly, in cereals. The Aeluropus genome contains a single AlSAP gene which has an intron at its 5'UTR. Sequence homology analysis showed that the AlSAP protein is characterized by the presence of two conserved zinc-finger domains A20 and AN1. AlSAP is induced not only by various abiotic stresses such as salt, osmotic, heat and cold but, also by abscisic acid (ABA) and salicylic acid (SA). Tobacco plants expressing the AlSAP gene under the control of the duplicated CaMV35S promoter exhibited an enhanced tolerance to abiotic stresses such as salinity (350 mM NaCl), drought (soil Relative Water Content (RWC) = 25%), heat (55 degrees C for 2.5 h) and freezing (-20 degrees C for 3 h). Moreover, under high salt and drought conditions, the transgenic plants were able to complete their life cycle and to produce viable seeds while the wild-type plants died at the vegetative stage. Measurements of the leaf RWC and of the root and leaf endogenous Na(+) and K(+) levels in AlSAP transgenic lines compared to wild-type tobacco, showed an evident lower water loss rate and a higher Na(+) accumulation in senescent-basal leaves, respectively. Finally, we found that the steady state levels of transcripts of eight stress-related genes were higher in AlSAP transgenic lines than in wild-type tobacco. Taken together, these results show that AlSAP is a potentially useful candidate gene for engineering drought and salt tolerance in cultivated plants.

Promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs developmental-regulated, stress-inducible, and organ-specific gene expression in transgenic tobacco

“…Sequence analysis revealed the presence of potential abiotic and biotic stress responsive cis-elements, transcription factor-binding sites such as MYB, MYC, Dof, and WRKY and several tissue-specific expression and light-responsive elements ( Table 2). The presence of these ciselements related to various stresses is consistent with our previous finding that AlSAP is induced not only by various abiotic stresses (for example salt, osmotic, heat, and cold) but also by abscisic acid (ABA) and salicylic acid (SA) (Ben Saad et al 2010). In the same way, in-silico analysis of the region upstream of the OsSAP genes (OsSAP1, OsSAP6, OsSAP8, and OsSAP9) encoding AN1/A20 zinc finger proteins in rice revealed the presence of various cis-acting regulatory elements involved in abiotic stress-responsive gene expression (Vij and Tyagi 2006;Huang et al 2008;Kanneganti and Gupta 2008).…”

“…When b-glucuronidase activity was monitored by histochemical staining in these transgenic events, comparable patterns of gusA gene expression were observed in the T2 generation. A representative transgenic line (L4) Pr AlSAP is a stress-inducible, age-dependent, and organ and tissue-specific promoter It has previously been reported that expression of the AlSAP gene is induced by abiotic and hormonal stresses in A. littoralis (Ben Saad et al 2010). Here, temporal and spatial b-glucuronidase (GUS) expression were examined under control, NaCl, mannitol, ABA, or SA treatment at different developmental stages in transgenic tobacco harboring the Pr AlSAP -gusA construct (Fig.…”

“…In the same way, in-silico analysis of the region upstream of the OsSAP genes (OsSAP1, OsSAP6, OsSAP8, and OsSAP9) encoding AN1/A20 zinc finger proteins in rice revealed the presence of various cis-acting regulatory elements involved in abiotic stress-responsive gene expression (Vij and Tyagi 2006;Huang et al 2008;Kanneganti and Gupta 2008). Transcript profiling indicates a strong response to abiotic stress at least of a subgroup of the SAP family, suggesting a role of members of this family in stress acclimation (Mukhopadhyay et al 2004;Tyagi 2006, 2008;Jin et al 2007;Huang et al 2008;Kanneganti and Gupta 2008;Ben Saad et al 2010).…”

“…This gene was shown to be induced by salt, drought, cold, heat, ABA, and SA. Furthermore, its overexpression in tobacco, under the control of the CaMV-35S promoter, improved tolerance to continuous salt and drought stresses under greenhouse conditions (Ben Saad et al 2010). To the best of our knowledge, despite the demonstrated role of some genes belonging to the SAP family in abiotic stress responses, no experimental functional analysis of their promoters has yet been reported.…”

The E3 Ligase TaSAP5 Alters Drought Stress Responses by Promoting the Degradation of DRIP Proteins

“…Accordingly, some SAPs have been shown to be potential candidates for improving abiotic stress tolerance through molecular breeding without yield penalty under normal growth conditions. For example, overexpression of AlSAP from Aeluropus littoralis in tobacco and rice resulted in yield maintenance under stress conditions, which was accompanied by no observed yield penalty under normal growth conditions (Ben-Saad et al, 2010, 2012b. In contrast, OSISAP8 overexpression in tobacco and rice, which conferred higher yield under drought stress than in wild-type plants, resulted in a 50% yield penalty under normal growth conditions (Kanneganti and Gupta, 2008).…”