BackgroundStructural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.Methodology/Principal FindingsCodon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from Tm ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.ConclusionThe significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins.

Soil salinity is an increasingly serious problem worldwide that reduces agricultural output potential. Selected beneficial soil bacteria can promote plant growth and augment tolerance to biotic and abiotic stresses. Bacillus subtilis strain GB03 has been shown to confer growth promotion and abiotic stress tolerance in the model plant Arabidopsis thaliana. Here we examined the effect of this beneficial soil bacterium on salt tolerance in the legume forage crop, white clover. Plants of white clover (Trifolium repens L. cultivar Huia) were grown from seeds with or without soil inoculation of the beneficial soil bacterium Bacillus subtilis GB03 supplemented with 0, 50, 100, or 150 mM NaCl water into soil. Growth parameters, chlorophyll content, malondialdehyde (MDA) content and osmotic potential were monitored during the growth cycle. Endogenous Na+ and K+ contents were determined at the time of harvest. White clover plants grown in GB03-inoculated soil were significantly larger than non-inoculated controls with respect to shoot height, root length, plant biomass, leaf area and chlorophyll content; leaf MDA content under saline condition and leaf osmotic potential under severe salinity condition (150 mM NaCl) were significantly decreased. Furthermore, GB03 significantly decreased shoot and root Na+ accumulation and thereby improved K+/Na+ ratio when GB03-inoculated plants were grown under elevated salt conditions. The results indicate that soil inoculation with GB03 promotes white clover growth under both non-saline and saline conditions by directly or indirectly regulating plant chlorophyll content, leaf osmotic potential, cell membrane integrity and ion accumulation.

Salinity is one of the major abiotic stresses that impacts plant growth and reduces the productivity of field crops. Compared to field plants, test tube plantlets offer a direct and fast approach to investigate the mechanism of salt tolerance. Here we examined the ultrastructural and physiological responses of potato (Solanum tuberosum L. c.v. “Longshu No. 3”) plantlets to gradient saline stress (0, 25, 50, 100, and 200 mM NaCl) with two consequent observations (2 and 6 weeks, respectively). The results showed that, with the increase of external NaCl concentration and the duration of treatments, (1) the number of chloroplasts and cell intercellular spaces markedly decreased, (2) cell walls were thickened and even ruptured, (3) mesophyll cells and chloroplasts were gradually damaged to a complete disorganization containing more starch, (4) leaf Na and Cl contents increased while leaf K content decreased, (5) leaf proline content and the activities of catalase (CAT) and superoxide dismutase (SOD) increased significantly, and (6) leaf malondialdehyde (MDA) content increased significantly and stomatal area and chlorophyll content decline were also detected. Severe salt stress (200 mM NaCl) inhibited plantlet growth. These results indicated that potato plantlets adapt to salt stress to some extent through accumulating osmoprotectants, such as proline, increasing the activities of antioxidant enzymes, such as CAT and SOD. The outcomes of this study provide ultrastructural and physiological insights into characterizing potential damages induced by salt stress for selecting salt-tolerant potato cultivars.

Crop plants often have challenges of biotic and abiotic stresses, and they adapt sophisticated ways to acclimate and cope with these through the expression of specific genes. Changes in chromatin, histone, and DNA mostly serve the purpose of combating challenges and ensuring the survival of plants in stressful environments. Epigenetic changes, due to environmental stress, enable plants to remember a past stress event in order to deal with such challenges in the future. This heritable memory, called “plant stress memory”, enables plants to respond against stresses in a better and efficient way, not only for the current plant in prevailing situations but also for future generations. Development of stress resistance in plants for increasing the yield potential and stability has always been a traditional objective of breeders for crop improvement through integrated breeding approaches. The application of epigenetics for improvements in complex traits in tetraploid and some other field crops has been unclear. An improved understanding of epigenetics and stress memory applications will contribute to the development of strategies to incorporate them into breeding for complex agronomic traits. The insight in the application of novel plant breeding techniques (NPBTs) has opened a new plethora of options among plant scientists to develop germplasms for stress tolerance. This review summarizes and discusses plant stress memory at the intergenerational and transgenerational levels, mechanisms involved in stress memory, exploitation of induced and natural epigenetic changes, and genome editing technologies with their future possible applications, in the breeding of crops for abiotic stress tolerance to increase the yield for zero hunger goals achievement on a sustainable basis in the changing climatic era.