Amiotic stresses from the outside world can produce large amounts of ROS in the plants, destroy the macromolecular material in the organism, and thus affect the plant growth and development. We cloned an antioxidant-related gene Tpx. Under salt stress, the specific mechanism of Tpx under salt stress is still unclear. This study explored the role of tomato Tpx in salt stress response. The results of multiple sequence alignment showed that tomato Tpx had high sequence similarity with potato and tobacco (Fig. 1).
Tpx is an important enzyme that improves the antioxidant capacity of plants by removing excess H2O2 in plants. Many studies have been shown the effect of overexpression of Tpx genes on cellular metabolism of multiple organisms (Broin and M., 2003). Under environmental pressure, Tpx plays an important role. Increasing evidence suggests that overexpression of Tpx enhances plant tolerance to methyldopsin-induced oxidative stress and salt-induced osmotic stress (Dietz et al., 2002). In this study, the level and activity of Tpx transcripts increased significantly after NaCl treatment (Fig. 2).
We transformed tobacco through Agrobacterium-mediated transformation, and analyzed the obtained plants by genomic PCR, western blot and qPCR, and obtained three transgenic plants(Fig. 3). After collecting the offspring, the germination rate of the overexpressed tobacco seeds was higher than that of the WT tobacco seeds after the tobacco seed germination test was carried out in the medium containing NaCl(Fig. 4A-D). The results showed that overexpression of Tpx from transgenic tobacco seeds played an important role in improving the germination rate of tobacco seeds under NaCl stress. At the same time, we also did a tobacco germination test under H2O2 and MV stress(Fig. 8), and the germination rate of the transgenic plants was also higher than that of the WT plants, indicating that the antioxidant capacity of the transgenic plants was improved. Significant reduced ROS and superoxide anion content in transgenic plants by DAB and NBT staining indicate that overexpressed transgenic plants have a stronger ability to remove ROS than WT plants and can effectively alleviate oxidative damage in tobacco plants upon stress(Fig. 4E and F).
In an organism, free radicals act on lipid peroxidation reaction. The oxidation end product is maldialaldehyde, which will cause crosslinking polymerization of vital macromolecules such as proteins and nucleic acids, and is cytotoxic (Hongbo et al., 2005). When the ROS content increases in plants, it is usually accompanied by an increased MDA content. MDA content is an important parameter to reflect the body's potential ability to resist oxidation, which can reflect the body's lipid peroxidation rate and intensity, and can also indirectly reflect the degree of tissue peroxidation damage (Alessio et al., 1988; Huang et al., 2009). We determined MDA content and found less MDA in transgenic overexpression after stress than WT plants(Fig. 5B), meeting our expectations, further validating the ability of Tpx to clear H2O2.
Heterologous expressed Tpx can also increased salt and low temperature tolerance in Arabidopsis (Jing et al., 2006). Tpx, a key component of H2O2 clearance in the Trx system (Adriani et al., 2021), its transgenic lines suggest that Tpx increases the clear efficiency of ROS by regulating antioxidant enzyme activity, maintains ROS homeostasis, and subsequently plays a key role in stress regulation (Kowaltowski et al., 2000).In this study, after NaCl stress on the obtained Tpx transgenic tobacco, it showed an improved activity of SOD, POD, CAT, and APX as compared to the WT plants(Fig. 6A-D).
As osmoprotective substances, proline and soluble sugars are important components of increasing permeability solutes and play an important role in the resistance physiology of plants (Xiao et al., 2005). Sugars are small molecules that regulate osmotic stress as an important member of increased permeability solutes when plants subjected to stress (Berkowitz and Masmoudi, 2007). Proline, however, is involved in the synthesis by sugar and phosphorylation under stress (Roger, 2001), and the glutamate pathway is the main pathway of proline synthesis under osmotic stress (Delauney and Verma, 1993). This paper studied the soluble sugar and proline content of transgenic tobacco under salt stress(Fig. 6E-F), which higher than wild plants, and the results showed that the overexpression of Tpx gene improved the synthesis of soluble sugar and proline, and then stabilized the osmotic pressure of plants, and parodied the growth of transgenic plants under salt stress. At the same time, we also measured the gene expression levels of P5CS, LEA5 and Osmotin(Fig. 6G-I), three proteins related to osmoregulation, and found that the gene expression levels in transgenic plants were higher than those in WT. This indicated that the Tpx transgenic plants had improved salt tolerance. At the same time, we also constructed a Tpx prokaryotic expression vector(Fig. 9), and analyzed the oxidative stress tolerance of the recombinant bacteria to further verify the function of the Tpx protein.
In recent years, studies of gene-regulated plants improving resistance under stress have gradually matured, and reports of redox homeostatic balance have gradually increased.Tpx is involved in the regulation of the antioxidant system by clearing H2O2 and is an important enzyme for maintaining redox homeostasis in plants (Koh et al., 2007). Here, by measuring the ROS in the roots after stress, we found that the ROS in the root tips of the overexpressing transgenic plants were significantly smaller than in the WT, and it is speculated that ROS metabolism may be related to the antioxidant system. It has been shown that heterologous expression of Tpx can improve the photosynthesis efficiency of cyanobacteria (Kim et al., 2018), but no studies has been investigated in tomato and tobacco.