Differential Salt Tolerance Strategies in Three Halophytes from the Same Ecological Habitat: Augmentation of Antioxidant Enzymes and Compounds.

Understanding the salt tolerance mechanism in obligate halophytes provides valuable information for conservation and re-habitation of saline areas. Here, we investigated the responses of three obligate halophytes namely Arthrocnemum macrostachyum, Sarcocornia fruticosa and Salicornia europaea to salt stress (0, 100, 200, 400 and 600 mM NaCl) during their vegetative growth with regard to biomass, ions contents (Na+, K+ and Ca+2), chlorophyll contents, carotenoids, phenolic compounds, flavonoids, and superoxide dismutase, peroxidase and esterase activities. S. europaea showed the lowest biomass, root K+ content, Chl a/b ratio, and carotenoids under salinity. This reduction of biomass is concomitant with the increase in proline contents and peroxidase activity. On the other hand, the promotion of growth under low salinity and maintenance under high salinity (200 and 400 Mm NaCl) in A. Macrostachyum and S. fruticosa are accompanied by an increase in Chl a/b ratio, carotenoids, phenolics contents, and esterase activity. Proline content was decreased under high salinity (400 and 600 mM NaCl) in both species compared to S. europaea, while peroxidase showed the lowest activity in both plants under all salt levels except under 600 mM NaCl in Arthrocnemum macrostachyum compared to S. europaea. These results suggest two differential strategies; (1) the salt tolerance is due to activation of antioxidant enzymes and biosynthesis of proline in S. europaea, (2) the salt tolerance in A. macrostachyum, S. fruticosa are due to rearrangement of chlorophyll ratio and biosynthesis of antioxidant compounds (carotenoids, phenolics and flavonoids) which their cost seem to need less energy than activation of antioxidant enzymes. The differential behavior in halophytes of the same habitat confirms that the tolerance mechanism in halophytes is species-specific which provides new insight about the restoration strategy of saline areas.

Cloning, expression and nanodiscs assemble of recombinant HIV-1 gp41.

Structural studies of membrane proteins have been hurdled by their difficulty for expression in heterogeneous expression systems due to their intrinsically strong hydrophobicity and requirements for association with other cellular membranes. This study aims to design a construct for expression of membrane proteins. Because of its outstanding interest in HIV-1 vaccine design, transmembrane gp41 amino acid residue 662-723 was chosen as a representative membrane protein. Therefore, we constructed expression vectors for expression of gp41(662-723) alone (pET28a-gp41(662-723)) or coupled with a fusion partner: GB1 (pET30a-GB1-gp41(662-723)) and Trx (pET32a-Trx-gp41(662-723)). For enhancing protein expression, the expression plasmids were transformed into E. coli BL-21 (DE3), E. coli T7 Express lysY/Iq and E. coli Lemo21 (DE3). Interestingly, HIV-1 gp41(662-723) was expressed as a C-terminus fusion to the fusion partner Trx (Trx-gp41(662-723)) with an apparent molecular mass of 21.8 kDa. Trx-gp41(662-723) was overexpressed into E. coli T7 Express lysY/Iq by early induction as OD600 ~0.5 followed by incubation at 20 °C/overnight. Our data demonstrated that almost all recombinant Trx-gp41(662-723) was incorporated into lipid nanodiscs by slowing down the nanodiscs assembly process. Negative-stained electron micrographs revealed homogenous 10 nm Trx-gp41(662-723)-nanodiscs. While the neutralizing epitopes in the purified Trx-gp41(662-723) were accessible and recognizable by anti-MPER bNAbs, these epitopes became less accessibly exposed, particularly in the C-terminal region of MPER, after incorporation of Trx-gp41(662-723) into nanodiscs.