Over-expression of a plasma membrane H+-ATPase SpAHA1 conferred salt tolerance to transgenic Arabidopsis.

The SpAHA1 gene, encoding a plasma membrane (PM) H+-ATPase (AHA) in Sesuvium portulacastrum, was transformed into Arabidopsis plants, and its expression increased salinity tolerance of transgenic Arabidopsis plants: seed germination ratio, root growth, and biomass of transgenic plants were greater compared to wild-type plants under NaCl treatment condition. Upon salinity stress, both Na+ and H+ effluxes in the roots of SpAHA1 expressing plants were faster than those of untransformed plants. Transformed plants with SpAHA1 had lower Na+ and higher K+ contents relative to wild-type plants when treated with NaCl, resulting in greater K+/Na+ ratio in transgenic plants than in wild-type plants under salt stress. Extent of oxidative stress increased in both transgenic and wild-type plants exposed to salinity stress, but overexpression of SpAHA1 could alleviate the accumulation of hydrogen peroxide (H2O2) induced by NaCl treatment in transgenic plants relative to wild-type plants; the content of malondialdehyde (MDA) was lower in transgenic plants than that in wild-type plants under salinity stress. These results suggest that the higher H+-pumping activity generated by SpAHA1 improved the growth of transgenic plants via regulating ion and reactive oxygen species (ROS) homeostasis in plant cells under salinity stress.

An assessment of the capacity for phosphoenolpyruvate carboxykinase to contribute to C4 photosynthesis.

Three C4 acid decarboxylases, phosphoenolpyruvate carboxykinase (PEPCK), NADP-malic enzyme (NADP-ME), and NAD-malic enzyme (NAD-ME) were recruited from C3 plants to support C4 photosynthesis. In Poaceae, there are established lineages having PEPCK type species, and some NADP-ME lineages in which PEPCK contributes to C4. Besides family Poaceae, recently PEPCK has been reported to function in C4 photosynthesis in eudicot species including Cleome gynandra (Cleomaceae), Trianthema portulacastrum and Zaleya pentandra (Aizoaceae). We evaluated PEPCK by enzyme assay and western blots in representatives of Poaceae, Aizoaceae, Cleomaceae, and Chenopodiaceae compared to that in the PEPCK type C4 grass Spartina anglica. Eragrostis nutans was identified as the first NAD-ME type C4 grass having substantial amounts of PEPCK. In the eudicots, including C. gynandra, Cleome angustifolia, T. portulacastrum, Z. pentandra, and nine C4 members of family Chenopodiaceae (which has the most C4 species and diversity in forms among eudicot families), amounts of PEPCK were generally very low (barely detectable up to 4% of that in S. anglica). Based on these results, C4 species can be classified biochemically according to the dominant decarboxylase recruited for C4 function; and, Poaceae remains the only family in which PEPCK is known to have a significant role in C4 photosynthesis.

Quantitative proteomics of Sesuvium portulacastrum leaves revealed that ion transportation by V-ATPase and sugar accumulation in chloroplast played crucial roles in halophyte salt tolerance.

Physiological and proteomic responses of Sesuvium portulacastrum leaves under salinity were investigated. Different from glycophytes, this halophyte had optimal growth at 200-300mM NaCl and accumulated more starch grains in chloroplasts under high salinity. Increased contents of soluble sugars, proline, and Na(+) were observed upon salinity. X-ray microanalysis revealed that Na(+) was mainly compartmentalized into cell vacuole. Quantitative proteomics produced 96 salt responsive proteins, and the majority was chloroplast-located proteins. Gene ontology analysis revealed that proteins involved in ion binding, proton transport, photosynthesis and ATP synthesis were overrepresented. The expressions of a Na(+)/H(+) antiporter and several ATP synthase subunits were activated upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased upon NaCl whereas vacuolar H(+)-pyrophosphatase and plasma membrane P-ATPase activities were not increased, which indicated that sodium compartmentalization was mainly performed by enhancing V-ATPase activity rather than P-ATPase and H(+)-pyrophosphatase. Accumulation of soluble sugars as well as sodium compartmentalization maintained the osmotic balance between vacuole and cytoplasm, which finally established ionic homeostasis in saline cells in true halophytes. BIOLOGICAL SIGNIFICANCE: Physiological and proteomic analyses of S. portulacastrum leaves under different salinities were investigated. This true halophyte accumulated more soluble sugars, starch, proline and Na(+) under high salinity. Differential proteomics produced 96 salt responsive proteins and the majority was involved in ion binding, proton transport, photosynthesis, and ATP synthesis. A Na(+)/H(+) antiporter and several ATP synthase subunits were induced upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased whereas vacuolar H(+)-pyrophosphatase and plasma membrane ATPase activities were stable upon NaCl. These findings demonstrated that the increased Na(+) was compartmentalized into vacuole by enhancing V-ATPase activity rather than H(+)-ATPase.

Growth, osmolyte concentration and antioxidant enzymes in the leaves of Sesuvium portulacastrum L. under salinity stress.

In this study, growth and osmolyte concentration in the leaves of halophyte, Sesuvium portulacastrum, were studied with respect to salinity. Therefore, the changes in shoot growth, leaf tissue water content, osmolyte concentration (proline content, glycine betaine) and antioxidant enzymes [polyphenol oxidase (PPO), superoxide dismutase (SOD) and catalase (CAT)] were investigated. The 30-day old S. portulacastrum plants were subjected to 100, 200, 300, 400, 500 and 600 mM NaCl for 28 days. The plant growth was steadily increased up to 500 mM NaCl stress at 28 days. TWC was higher in 300 mM NaCl treated leaves than that of 600 mM NaCl. Salinity stress induced the accumulation of osmolyte concentration when compared to control during the study period. The antioxidant enzymes PPO, CAT and SOD were increased under salinity.

Cloning and molecular characterization of fructose-1,6-bisphosphate aldolase gene regulated by high-salinity and drought in Sesuvium portulacastrum.

Sesuvium portulacastrum, a mangrove plant from seashore, is a halophyte species well adapted to salinity and drought. Some efforts have been made to describe its physiological and structural characteristics on salt and drought-tolerance, but the underlying molecular mechanism and key components have not yet been identified. Here, a fructose-1,6-bisphosphate aldolase gene, designated SpFBA, was isolated and characterized from S. portulacastrum roots in response to seawater. The SpFBA cDNA has a total length of 1452 bp with an open reading frame of 1071 bp, and is predicted to encode a precursor protein of 357 amino acid residues sharing high degree of homology with class I FBAs from other plants. Semi-quantitative RT-PCR analysis indicated that the SpFBA was more strongly expressed in roots than in leaves and stems, and the abiotic stimuli such as Seawater, NaCl, ABA, and PEG, could trigger a significant induction of SpFBA in S. portulacastrum roots within 2-12 h. Overproduction of Recombinant SpFBA resulted in an increased tolerance to salinity in transgenic Escherichia coli. All these results suggest that the SpFBA plays very important roles in responding to salt stress and related abiotic stimuli, and in improving the survival ability of S. portulacastrum under high salinity and drought.

Site-directed mutagenesis and protein 3D-homology modelling suggest a catalytic mechanism for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis.

In livingstone daisy (Dorotheanthus bellidiformis), betanidin 5-O-glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site-directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP-glucose binding site, and His22, located close to the N-terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP-glucose-dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN-1 reaction, as one plausible alternative to the generally proposed SN-2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.