VcLON2 Is Involved In Abiotic Stress Tolerance In Blueberry

Environmental stresses are major challenges for sustainable food production. Exposure of plants to environmental stress conditions such as temperature extremes, drought or high salinity, perturbs the balance between the production of reactive oxygen species (ROS), often resulting in oxidative damage to the proteins. LON2 protease is an ATP depending serine protease located in peroxisomes. As indicated by the studies on the model plants such as Arabidopsis, LON2 was found greatly involved in protein turnover, degradation and the autophagy of peroxisomes. Despite our understanding of the function of the LON2 protease in peroxisomal protein degradation, less is known regarding its function in the regulation of cellular ROS homeostasis and in controlling the levels of peroxisomal protein oxidation, especially in plants under environmental stress conditions. In this study, based on the isolation of VcLON2 cDNA from blueberry (Vaccinium spp.), the temporal and spatial expression patterns of VcLON2 were assayed by qRT-PCR. Then, the anti-stress related biofunctions of VcLON2 were analyzed through over-expression VcLON2 and RNAi silence of its homologous gene (NbLON2) in nicotiana benthamian. The results showed using overexpression and gene silencing strategies that a LON2 protease from blueberry is involved in the regulation of the activity of peroxisomal antioxidant enzymes necessary for the alleviation of oxidative stress damage. The main results are as follows:The full-length 2,667 bp VcLON2 cDNA encodes a putative 888 amino acid protein with a predicted molecular mass of 98.28 kDa and a pI of 6.68 that shares a significant degree of homology (84% amino acid similarity) to the Arabidopsis thaliana LON2 protein. The deduced amino acid sequence of the VcLON2 protein contains a canonical C-terminal PTS1 (SKL-COOH), peroxisomal targeting signal. Quantitative Real-Time (RT) PCR analysis demonstrated that VcLON2 mRNA was expressed under standard growth conditions in all tissues analyzed. Transcript levels were, however, found to be generally higher in aged or ripened organs relative to younger and/or immature organs. Exposure to abiotic stress conditions, including NaCl, drought and heat, caused a significant induction of VcLON2 expression in blueberry leaves, relative to control condition. The results showed that VcLON2 may be related to plant senescence and stress resistance.To further examine the role of VcLON2 in stress tolerance, we generated transgenic tobacco lines that constitutively overexpress VcLON2. Phenotypically, the T1 tobacco lines (P1, P2 and P5) appeared similar to the WT counterparts when grown under non-stress conditions. Overexpression of VcLON2 in tobacco led to the lower range of decrease of growth parameters and greener compared with wild type (WT) grown under salt (200 mM NaCl), osmotic (2%PEG6000) and heat (45℃,1.5 h) stress. The chlorophyll content of transgenic VcLON2-overexpressed lines was 2 folds of WT. The MDA content and protein carbonyl levels in the leaves significantly increased in leaves of tobacco plants, but VcLON2-overexpressed lines was only 32.5%-75.2% of WT. In addition, VcLON2 over-expressed lines showed significantly lower accumulation of H2O2 under the various stress conditions compared with WT plants. The levels of activity of antioxidant enzymes (CAT, APX, etc.) in VcLON2-overexpressed tobacco seedlings were higher than that in WT. These data indicate that LON2’s role(s) in peroxisomal quality control are critical for a variety of abiotic stress tolerance responses. LON2 function in the elimination of oxidized peroxisomal proteins. It plays a pivotal role in peroxisome H2O2 homeostasis potentially by regulating the activities and/or levels of active peroxisomal antioxidant enzymes such as CAT.A complimentary approach to the overexpression analysis was to generate transgenic plant lines in which the endogenous expression of the NbLON2 gene, which encodes a protein with a high degree of sequence similarity to VcLON2, was reduced by RNAi gene silencing. Although NbLON2-silenced seedlings (S4, S5 and S6) display a minor reduction in growth, no significant differences in growth parameters were detected between NbLON2-silenced and WT plants under standard growth conditions. Furthermore, NbLON2-silenced seedlings led to the higher range of decrease of growth parameters and yellower compared with wild type (WT) grown under salt (100 mM NaCl), osmotic (1%PEG6000) and heat (45℃,1.0 h) stress. The relative water content of NbLON2-si\enced seedlings significantly decreased following treatment with heat by 19.29%-22.90%, but WT tobacco had no significant changes or decreased a little. The development trend of changing of chlorophyll content and relative water content is consistent. In addition, all abiotic stresses tested resulted in a significant higher accumulation of MDA as well as protein carbonyl group levels in the NbLON2 RNAi silenced lines relative to WT tobacco. NbLON2 RNAi silenced lines showed significantly higher accumulation of H2O2 under the various stress conditions compared with WT plants, and the activity in NbLON2-slienced lines was lower than that in WT. NbLON2-slienced tobacco seedlings showed increased susceptibility to abiotic stress and significantly higher accumulation of ROS and oxidation products. These data indicate that LON2 protease plays a pivotal role in peroxisome H2O2 homeostasis.Finally, in order to prepare for the further studies on the physical and chemical characters, VcLON2 were expressed in Escherichia coli. Firstly, a fusion expression vector VcLON2-pET30a (+) was constructed and the expression strains as well as IPTG concentrations in the prokaryotic expression system were also screened. The results showed that a fusion protein was successfully expressed in E. coli strain when treated by Rosetta (DE3) with 0.5 mM IPTG, which was purified by affinity chromatography through HIS-Select nickel gel. However, the purity of purifed VcLON2 protease failed to meet expectations. The expression condition was still need to be optimized and the subsequent analysis is still in progress.