Purification and properties of Manganese Superoxide Dismutase (MnSOD) from Tamarix aphylla L.

Superoxide dismutase (SOD) of the Tamarix aphylla leaves were detected at optimum conditions that collected in April, May and June. Results indicated the specific activity in the crude extract reaching to 36.76 unit/ mg protein. Crude SOD was purified by several techniques, precipitation with ammonium sulfate (50-75) %, Ion exchange chromatography using DEAE-cellulose and two steps of size exclusion chromatography on sephacryl S-200 column. The obtained specific activity (310 unit/mg protein) and purification fold 7.91. The purified enzyme revealed one band by SDS-polyacrylamide gel electrophoresis with molecular mass 85.703 kDa. while 89.125 kDa by Sephacryl S-200. The optimal pH and temperature for enzyme activity were 7.5, and 50ºC respectively. EDTA, SDS and NaN3 reduced activity, contrariwise of H2O2 and KCN, pointed to the studied SOD is MnSOD. Michalis constant Km and maximum velocity Vmax values were 0.016 mM and 55.86 mM/min, respectively by using Pyrogallol as substrate. According to the results, we conclude Tamarix aphylla produce MnSOD which can have purified by serial purification techniques for better activity and characterized for further studies.

A ThDREB gene from Tamarix hispida improved the salt and drought tolerance of transgenic tobacco and T. hispida.

Dehydration-responsive element-binding (DREB) transcription factors are important abiotic stress tolerance related genes, and some reports on the roles of DREB have primarily addressed herbal plants. To explore the abiotic stress tolerance role of DREB (ThDREB) from Tamarix hispida, a ThDREB gene with a complete ORF of 783 bp that encodes a 28.74 kDa protein with 260 amino acids, was isolated and functionally annotated. ThDREB expression was highly induced by NaCl, PEG, NaHCO3 and CdCl2 treatments, and the highest expression level (369.2-fold of control) was found for the roots that were under NaCl stress for 6 h. The tobacco plants that were transformed by ThDREB were conferred with higher germination rates, fresh weights and root lengths than the wild type (WT) tobacco plants under NaCl and mannitol treatments. The total chlorophyll content (tcc), superoxide dismutase (SOD) and peroxidase (POD) activities were also higher in the transgenic lines in comparison with the WT, and the malondialdehyde (MDA) and H2O2 content, electrolyte leakage (EL) rate and ROS as tracked by staining were generated to a lesser degree in ThDREB transgenic plants than in the WT under NaCl and mannitol stress. Furthermore, the transient overexpression analysis of ThDREB in T. hispida also improved plant salt and drought tolerance in comparison with the empty vector-transformed lines. Our results indicated that ThDREB expression could effectively improve tolerance to salt and drought stress by enhancing the antioxidase activity that keeps the ROS at a low accumulation level and makes them easy to scavenge.

The Reaumuria trigyna leucoanthocyanidin dioxygenase (RtLDOX) gene complements anthocyanidin synthesis and increases the salt tolerance potential of a transgenic Arabidopsis LDOX mutant.

Reaumuria trigyna is a typical, native desert halophyte that grows under extreme conditions in Inner Mongolia. In a previous transcriptomic profiling analysis, flavonoid pathway-related genes in R. trigyna showed significant differences in transcript abundance under salt stress. Leucoanthocyanidin dioxygenase (LDOX, EC 1.14.11.19) is one of three dioxygenases in the flavonoid pathway that catalyzes the formation of anthocyanidins from leucoanthocyanidins. In this study, we cloned the full-length cDNA of R. trigyna LDOX (RtLDOX), and found RtLDOX recombinant protein was able to replace flavanone-3-hydroxylase (F3H, EC 1.14.11.9), another dioxygenase in the flavonoid pathway, to convert naringenin to dihydrokaempferol in vitro. R. trigyna LDOX can complement the Arabidopsis LDOX mutant transparent testa11 (tt11-11), which has reduced proanthocyanin (PA) and anthocyanin levels in seeds, to accumulate these two compounds. Thus, RtLDOX acts as a multifunctional dioxygenase to effect the synthesis of PA and anthocyanins and can perform F3H dioxygenase activities in the flavonoid biosynthesis pathway. The RtLDOX promoter harbored many cis-acting elements that might be recognized and bound by transcription factors related to stress response. RtLDOX expression was strongly increased under salt stress, and RtLDOX transgenic Arabidopsis mutant under NaCl stress accumulated the content of flavonoids leading to an increased antioxidant activities and plant biomass. These results suggest that RtLDOX as a multifunctional dioxygenase in flavonoid biosynthesis involves in enhancing plant response to NaCl stress.

Regulation of flavanone 3-hydroxylase gene involved in the flavonoid biosynthesis pathway in response to UV-B radiation and drought stress in the desert plant, Reaumuria soongorica.

Flavonoid are known to have various functions in growth, development, reproduction, and also involved in diverse stress responses in plants. However, little is known about the roles of the key enzymes in the flavonoid biosynthetic pathway in response to environmental stress, such as UV-B radiation and drought. To understand this problem, we investigated the participation of flavanone 3-hydroxylase gene (F3H), a key enzyme in flavonoid biosynthetic pathway under UV-B radiation and drought stress in the desert plant Reaumuria soongorica. A novel cDNA sequence, named as RsF3H, was isolated from R. soongorica. The deduced amino acids showed high identities to other F3Hs. A phylogenetic analysis indicated that RsF3H appeared to be most homologous to F3H from Malus domestica (MdF3H). RsF3H protein structure contained all five conserved motifs for 2-oxoglutarate-dependent dioxygenases (2-ODDs) and an Arg-X-Ser motif, all of which were also found in other F3Hs. Quantitative real-time RT-PCR analysis showed that there was a rapid increase in gene expression of RsF3H under stress. Both UV-B radiation and drought stress induced an increase in RsF3H enzyme activity and the accumulation of the products in the flavonoid biosynthetic pathway (total flavonoid and anthocyanin). The antioxidant ability (inhibition of lipid oxidation) of total flavonoid was enhanced during this study. The results suggested that one explanation of the stress tolerance of R. soongorica may be a combination of an increase in RsF3H gene expression, RsF3H enzyme activity and the anti-oxidative ability of the metabolic end products in the flavonoid biosynthetic pathway in response to UV-B radiation and drought.

Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from Tamarix androssowii.

Superoxide dismutases (SODs) play important role in stress tolerance of plants. In this study, an MnSOD gene (TaMnSOD) from Tamarix androssowii, under the control of the CaMV35S promoter, was introduced into poplar (Populus davidiana x P. bolleana). The physiological parameters, including SOD activity, malondialdehyde (MDA) content, relative electrical conductivity (REC) and relative weight gain, of transgenic lines and wild type (WT) plants, were measured and compared. The results showed that SOD activity was enhanced in transgenic plants, and the MDA content and REC were significantly decreased compared to WT plants when exposed to NaCl stress. In addition, the relative weight gains of the transgenic plants were 8- to 23-fold of those observed for WT plants after NaCl stress for 30 days. The data showed that the SOD activities that increased in transgenic lines are 1.3-4-folds of that increased in the WT plant when exposed to NaCl stress. Our analysis showed that increases in SOD activities as low as 0.15-fold can also significantly enhance salt tolerance in transgenic plants, suggesting an important role of increased SOD activity in plant salt tolerance