Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance.

Hydrogen peroxide (H2O2) is known to accumulate in plants during abiotic stress conditions and also acts as a signalling molecule. In this study, Arabidopsis thaliana transgenics overexpressing cytosolic CuZn-superoxide dismutase (PaSOD) from poly-extremophile high-altitude Himalayan plant Potentilla atrosanguinea, cytosolic ascorbate peroxidase (RaAPX) from Rheum australe and dual transgenics overexpressing both the genes were developed and analyzed under salt stress. In comparison to wild-type (WT) or single transgenics, the performance of dual transgenics under salt stress was better with higher biomass accumulation and cellulose content. We identified genes involved in cell wall biosynthesis, including nine cellulose synthases (CesA), seven cellulose synthase-like proteins together with other wall-related genes. RNA-seq analysis and qPCR revealed differential regulation of genes (CesA 4, 7 and 8) and transcription factors (MYB46 and 83) involved in secondary cell wall cellulose biosynthesis, amongst which most of the cellulose biosynthesis gene showed upregulation in single (PaSOD line) and dual transgenics at 100 mM salt stress. A positive correlation between cellulose content and H2O2 accumulation was observed in these transgenic lines. Further, cellulose content was 1.6-2 folds significantly higher in PaSOD and dual transgenic lines, 1.4 fold higher in RaAPX lines as compared to WT plants under stress conditions. Additionally, transgenics overexpressing PaSOD and RaAPX also displayed higher amounts of phenolics as compared to WT. The novelty of present study is that H2O2 apart from its role in signalling, it also provides mechanical strength to plants and aid in plant biomass production during salt stress by transcriptional activation of cellulose biosynthesis pathway. This modulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create plants with enhanced cellulose content for biofuel use.

Expression of SOD and APX genes positively regulates secondary cell wall biosynthesis and promotes plant growth and yield in Arabidopsis under salt stress.

Abiotic stresses cause accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) in plants. Sophisticated mechanisms are required to maintain optimum level of H2O2 that acts as signalling molecule regulating adaptive response to salt stress. CuZn-superoxide dismutase (CuZn-SOD) and ascorbate peroxidase (APX) constitute first line of defence against oxidative stress. In the present study, PaSOD and RaAPX genes from Potentilla atrosanguinea and Rheum australe, respectively were overexpressed individually as well as in combination in Arabidopsis thaliana. Interestingly, PaSOD and dual transgenic lines exhibit enhanced lignin deposition in their vascular bundles with altered S:G ratio under salt stress. RNA-seq analysis revealed that expression of PaSOD gene in single and dual transgenics positively regulates expression of lignin biosynthesis genes and transcription factors (NACs, MYBs, C3Hs and WRKY), leading to enhanced and ectopic deposition of lignin in vascular tissues with larger xylem fibres and alters S:G ratio, as well. In addition, transgenic plants exhibit growth promotion, higher biomass production and increased yield under salt stress as compared to wild type plants. Our results suggest that in dual transgenics, ROS generated during salt stress gets converted into H2O2 by SOD and its optimum level was maintained by APX. This basal level of H2O2 acts as messenger for transcriptional activation of lignin biosynthesis in vascular tissue, which provides mechanical strength to plants. These findings reveal an important role of PaSOD and RaAPX in enhancing salt tolerance of transgenic Arabidopsis via increased accumulation of compatible solutes and by regulating lignin biosynthesis.

Improved callus induction, shoot regeneration, and salt stress tolerance in Arabidopsis overexpressing superoxide dismutase from Potentilla atrosanguinea.

Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radicals (O2( ·-)) to molecular oxygen (O2) and hydrogen peroxide (H2O2). Previously, we have identified and characterized a thermo-tolerant copper-zinc superoxide dismutase from Potentilla atrosanguinea (PaSOD), which retains its activity in the presence of NaCl. In the present study, we show that cotyledonary explants of PaSOD overexpressing transgenic Arabidopsis thaliana exhibit early callus induction and high shoot regenerative capacity than wild-type (WT) explants. Growth kinetic studies showed that transgenic lines have 2.6-3.3-folds higher growth rate of calli compared to WT. Regeneration frequency of calli developed from transgenic cotyledons was found to be 1.5-2.5-folds higher than that of WT explants on Murashige and Skoog medium supplemented with different concentrations of naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP) within 2 weeks. A positive regulatory effect of PaSOD and H2O2 was observed on different stages of callusing and regeneration. However, this effect was more pronounced at the early stages of the regeneration processes in transgenic lines as compared to WT. These results clearly indicate that plant regeneration is regulated by endogenous H2O2 and by factors, which enhance its accumulation. Transgenics also exhibited salt stress tolerance with higher SOD activity, chlorophyll content, total soluble sugars, and proline content, while lower ion leakage and less reduction in relative water content, as compared to WT. Thus, it appears that the activation of PaSOD at regeneration stage accompanied by increased H2O2 production can be one of the mechanisms controlling in vitro morphogenesis.

Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications.

Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.