br Materials and methods br Results br Discussion Cysteine
Materials and methods
Discussion Cysteine proteases are upregulated under oxidative stress and play an important role in the preservation of cellular metabolism (Usui et al., 2007). Cysteine proteases are also important under abiotic stresses for plants to degrade proteins denatured as a result of stress or redirect metabolism by increasing protein turnover (Stroeher et al., 1997). The 8-fold increase in the expression of the cysteine protease gene SmCP in the roots under salt stress might represent a mechanism to degrade these denatured proteins the stress result in. However, a high concentration of proteases can damage MJ33 lithium salt (Turk et al., 2002). Therefore, the expression levels and activities of proteases need to be tightly regulated and controlled by specific inhibitors. SmCP expression was found to be dynamically increased after S. matsudana plants were treated with 100mM NaCl solution. SmCP is the first PCD-associated gene cloned from S. matsudana, and little is known about the function of PCD in salix plants towards environmental stresses especially for salt. We found that SmCP has conserved cysteine protease domains and is transcriptionally induced by salt stresses. To further investigate the function of SmCP and explore the initial role of PCD in salix plants upon salt stress, we generated recombinant E. coli and transgenic Arabidopsis plants that over-expressed SmCP (OE lines). SmCP overexpression was associated with enhanced salt tolerance in both species. ROS-scavenging systems were investigated and stronger SOD enzyme activities and more non-enzymatic antioxidants in transgenic plants than WT were founded. Activity of SOD increased more greatly in transgenic plants than WT. The higher amounts of SOD measured in our transgenic lines might be explained by constructive activation of removing misfolded and oxidized proteins via SmCP overexpression. The enhanced antioxidative activity might have contributed to less oxidative damage and less accumulations of oxidate observed in the transgenics. As one of the crucial primary metabolism, photosynthesis could be affected by salt stress. Abiotic stress-generated ROS damaged the photosynthetic apparatus and inhibit photosynthetic repair resulting in an imbalance in the redox system in chloroplasts. ROS accumulation on photosynthesis could be reduced because of SmCP overexpression by the production of ROS-scavenging enzymes such as SOD and increasing the levels of antioxidants. The enhancement of total chlorophyll, chlorophyll a and chlorophyll b contents that we found in transgenic lines might be a result of balanced antioxidant environment under salt stress. Thus, SmCP overexpression enhanced salt tolerance by increasing PCD-associated degradation, which is thought to be took part in the degradation of oxidized proteins and the regulation of ROS levels under salt stress. The NMT data showed greater Na+ efflux and H+ influx in the roots of transgenic plants compared with the WT after salt stress (Fig. 12), which indicated stronger activity of the Na+/H+ antiporter salt overly sensitive 1(SOS1) in the transgenic Arabidopsis (Jian, 2009, Sun et al., 2009). The possible function of SmCP is unclear and may not be in association with the ion fluxs. AtSOS1 is localized in epidermal cells at the root tip and at the xylem/symplast boundary in roots, stems and leaves where it controls long-distance Na+ transport (Min et al., 2016, Shi et al., 2002). No particular mechanism for this observed phenotype has been hypothesized, although it is believed that cysteine proteases have a number of roles in plant responses to abiotic stress factors. Above all, we have characterized SmCP by over-expressing it in Arabidopsis. Transgenic plants had enhanced salt tolerance, possibly result from a more-reductive redox state. We propose that active PCD-associated protein degradation might contribute to better quality control of proteins and a balanced antioxidant environment under salt stress. Our results provide an infusive combination between PCD and ROS-scavenging system. These data confirm and highlight the functions of SmCP in enhancing plant salt tolerance. However, more work is needed to understand the role of SmCP in Salix salinity tolerance, which might allow SmCP to be used to improve salt tolerance in other plant species through genetic engineering.