Constitutive Overexpressing At.TC Improves Drought-mediated Oxidative Tolerance in Transgenic Brassica Napus L.

Background: Environmental stresses are the most important factors limiting crops production in worldwide. Tocopherol, belonging to family of vitamin E compounds, is an amphipathic antioxidants involved in oxidative responses. In the current study, we generated transgenic canola plants overexpressing Arabidopsis VTE1 gene (At.TC) through Agrobacterium tumefaciens system. Methods and results: The putative transgenic plants were successfully regenerated and acclimated in greenhouse conditions. The transcriptional activity of the At.TC gene was evaluated by RT-PCR. In addition, the relative gene expression analysis by qRT-PCR confirmed an increased expression pattern of the transformed gene in canola transgenic lines, with the highest level in R. Line1. Given the results, the transgenic plants, particularly H. Line1 and R. Line2 showed a lower lipid peroxidation compared to WTs under FC 30%. Moreover, two ROS scavenging enzymes including CAT and PPO were up-regulated in transgenic lines; however, no significant pattern was observed for Ascorbate Peroxidase. Also, the amount of leaf tocopherol was significantly more in all T1 lines under drought stress (FC 30%). Conclusion: Taken together, here we successfully developed transgenic lines overexpressing At.TC gene constituently throughout the plant. The results confirmed that the generated transgenic plants are resistant to drought stress, thereby paving the way toward introducing canola plants to deal with the climate change and water shortage.

The critical role of MetR/MetB/MetC/MetX in cysteine and methionine metabolism, fungal development and virulence of Alternaria alternata

Methionine is a unique sulfur-containing amino acid, which plays an important role in biological protein synthesis and various cellular processes. Here, we characterized the biological functions of AaMetB, AaMetC, and AaMetX in the tangerine pathotype of Alternaria alternata. Morphological analysis showed that the mutants lacking AaMetB, AaMetC, or AaMetX resulted in less aerial hypha and fewer conidia in artificial media. Pathogenicity analysis showed that AaMetB, AaMetC, and AaMetX are required for full virulence. The defects in vegetative growth, conidiation and virulence of ΔMetB, ΔMetC, and ΔMetX can be restored by exogenous methionine and homocysteine, indicating that AaMetB, AaMetC, and AaMetX are required for methionine biosynthesis. However, exogenous cysteine only restored the growth and virulence defects of ΔMetR but not ΔMetB/C/X, suggesting that AaMetR is essential for cysteine biosynthesis. Oxidant sensitivity assay showed that only ΔMetR is sensitive to H2O2 and many ROS-generating compounds, indicating that AaMetR is essential for oxidative tolerance. Interestingly, fungicides indoor bioassays showed that only the ΔMetR mutants are susceptive to chlorothalonil, a fungicide that could bind to the cysteine of glyceraldehyde-3-phosphate dehydrogenase. Comparative transcriptome analysis showed that the inactivation of MetB, MetC, MetX, or MetR significantly affected the expression of methionine metabolism-related genes. Moreover, the inactivation of AaMetR significantly affected the expression of many genes related to glutathione metabolism, which is essential for ROS tolerance. Taken together, our study provides genetic evidence to define the critical roles of AaMetB, AaMetC, AaMetX, and AaMetR in cysteine and methionine metabolism, fungal development and virulence of Alternaria alternata. IMPORTANCE The transcription factor METR regulating methionine metabolism is essential for reactive oxygen species (ROS) tolerance and virulence in many phytopathogenic fungi. However, the underlying regulatory mechanism of METR involved in this process is still unclear. In the present study, we generated AaMetB, AaMetC and AaMetX deletion mutants and compared these mutants with AaMetR disrupted mutants. Interestingly, we found that AaMetB, AaMetC and AaMetX are required for vegetative growth, conidiation, and pathogenicity in Alternaria alternata, but not for ROS tolerance and cysteine metabolism. Furthermore, we found that METR is involved in the biosynthesis of cysteine, which is an essential substrate for the biosynthesis of methionine and glutathione. This study emphasizes the critical roles of MetR, MetB, MetC, MetX in the regulation of cysteine and methionine metabolism, as well as the cross-link with glutathione-mediated ROS tolerance in phytopathogenic fungi, which provides a foundation for future investigations.

Endosymbiont bacteria Holospora undulata confers oxidative tolerance in host Paramecium caudatum

Paramecium is a genus of ciliated protozoan that, while unicellular, has a complex intracellular structure, comparable to Metazoan cells, which has made them excellent models for the study of genetics and cellular functions. Holospora undulata is a bacterial endosymbiont specific to the species Paramecium caudatum; they are unable to grow outside of P. caudatum. The presence of this endosymbiont has proven to have an effect on the subsequent gene expression and cellular maintenance of its host cells. Recent studies have demonstrated that infection by H. obtusa increases the expression of host heat-shock genes and leads to both resistance at normally-lethal high temperatures and heat resistance in ciliary movement (Fujishima, Kawai, & Yamamoto, 2005; Hori & Fujishima, 2003). Heat-shock resistance occurs because bacterial DNA triggers the upregulation of its P. caudatum host’s heat-shock genes (i.e., hsp60 and hsp70), although the mechanisms are not known (Hori & Fujishima, 2003). These studies demonstrate that infection of P. caudatum by H. undulata (a closely-related species to H. obtusa) induces heat-shock resistance, but fail to address whether H. undulata protects against other common environmental stressors such as oxidative damage. To determine if infection by H. undulata has the ability to induce additional tolerances, we examined differences in oxidative tolerance, based on percent survival, between P. caudatum with and without H. undulata infection. Samples of both lines were treated with increasing concentrations of hydrogen peroxide, the number of surviving cells were counted, and the percent survivability of each sample was calculated. There was an approximate 20% increase in survival when P. caudautum was infected with H. undulata—thus H. undulata infections confer oxidative tolerance. Further studies will be conducted to determine if an increase in survivability occurs in response to other damaging mechanisms. Future work will also investigate if the genes responsible for oxidative damage repair are upregulated, in addition to the already characterized heat-shock genes.