Plants synthesis multiple families of sHSPs with monomer sizes ranging from 12 to 42 kDa, are the most diverse and more abundant than in other organisms (Wu et al., 2022). In addition to heat tolerance, the sHSPs suggesting that they may play important roles other cellular processes under normal conditions (Wu et al., 2022). Over the past decades, it has been observed that the expression of certain plant sHSPs is induced by a range of stressors, including heat, salt, drought, osmotic, hormonal, heavy metal, oxidative stresses, as well as developmental signals specific to plants (Wu, et al. 2022). In addition, a multitude of investigation have involved the overexpression of various sHSPs in both homologous and heterologous plant systems, including E. coli and Yeast models (Santhanagopalan, et al. 2015; Waters and Vierling 2020). The outcomes of many of these experiments have revealed that stress protection conferred by these proteins is restricted to specific conditions and narrow range of plant growth stages. On contrast, suppression of CI or CII sHSPs in A. thaliana by RNAi showed that these sHSPs were required for recovery from a severe heat stress (10 h at 45°C) and have independent functions (McLoughlin, et al. 2016; Wu, et al. 2022). However, additional investigations are needed to uncover further applications for these proteins.
In this work, the DNA fragments of OsHsp16.9A and OsHsp18.0 for overexpression were cloned into the binary expression vector pCYH8 (Liu, et al. 2023) in the downstream region of the ubiquitin 1 promoter (Ubi1). The recombinant constructs were then introduced into the pPZP/35H Ti-based vector, which was constructed by inserting 35S-hph-tml fragment into the HindIII site of pPZP200 (Supplementary material). The recombinant constructs for overexpression of OsHsp16.9A and OsHsp18.0 were individually transferred into rice genome by Agrobacterium-mediated transformation (Liu, et al. 2023) (Fig 1a). Two independent T3 homozygous transgenic lines OsHsp16.9A-OXs (OsHsp16.9A-OX1, and OsHsp16.9A-OX3) and OsHsp18.0-OXs (OsHsp18.0-OE1, and OsHsp18.0-OE3) were selected for heavy metal stress analysis. Because OsHsp16.9A and OsHsp18.0 showed distinct resistance to high temperature in rice vegetative and reproductive stages (Liu, et al. 2023). As compared with WT, constitute expression of OsHsp18.0-OXs leaves maintained higher chlorophyll content (p < 0.05, Fig. 1b) and lower MDA level (p < 0.05, Fig. 1c) under 150 µM and 250 µM Cu stress, whereas OsHsp16.9A-OXs leaves showed resistance to only 150µM Cu toxicity (p < 0.05, Fig. 1b). This result indicates that the alleviation of imbalance in reactive oxygen species caused by Cu stress reduce the accumulation of reactive oxygen species. In addition, constitutive expression of OsHsp18.0 showed higher tolerance for Cd-induced detrimental effects than OsHsp16.9A accumulation in rice leaves (p < 0.05, Fig. 1d and e). Because Cd is a nonessential metal ion, plants are not expected to have specific uptake systems for this metal (Ueno, et al. 2010). It has been hypothesized that some cultivars evolved to selectively limit Cd uptake and sequestration into vacuoles (Ueno, et al. 2010). Therefore, the results suggest in agreement with previous report that the elevated expression of OsHsp18.0 was able to confer enhance antioxidant enzyme activities tolerance to Cu and Cd toxicity (Kanu, et al. 2017).
We compared electrolyte leakage among WT, OsHsp16.9A-OX, and OsHsp18.0-OX seedlings under Cu and Cd treatment (Fig.1f and g). The results showed that the, the conductivity of the incubation medium of OsHsp18.0-OXs was significantly less than the WT (24.6% ~ 37.6% and 25.7% ~ 34.6% reduction, respectively), whereas overexpression of OsHsp16.9A-OXs was not able to act efficiently on suppression of the cellular leakage after 12 h or 24h Cu and Cd treatment (Fig. 1f and g). These results confirmed that OsHsp18.0-OXs transgenic lines proved to be effective in maintaining higher cell membrane integrity than the WT and OsHsp16.9A -OE lines. Further analysis on antioxidant assay (CAT, APX) was performed, and the results showed that the activity of these antioxidant enzymes was not changed in WT, OsHsp16.9A-OX, and OsHsp 18.0-OX leaves under non-stress control treatment (Fig. 1 h-k). With Cu treatment, CAT activity was significantly higher in OsHsp18.0-OE than WT and OsHsp 16.9A-OE leaves (p < 0.05, Fig. 1h) and APX activity was exhibited significant differences among OsHsp18.0-OE and OsHsp 16.9A-OE leaves after expose to 150µM and 250 µM treatment CuSO4, respectively (Figure 1j). With Cd treatment, CAT activity was significantly higher in OsHsp 18.0-OE than WT and OsHsp 16.9A-OE leaves (p < 0.05, Fig. 1i) and APX activity was significantly elevated in OsHsp 18.0-OE leaves after 1 and 2 µM Cd treatment (p < 0.05, Fig. 1k). These results revealed that OsHsp16.9A confers thermoprotection in rice seeds and seedlings, whereas OsHsp18.0 plays a key role in cross-tolerance to protect rice seedling cells against heat and Cd as well as Cu stress by maintaining redox homeostasis, to scavenge ROS. Prior findings demonstrated that, OsHsp16.9A conferred chaperone activity to protect denatured proteins, and OsHsp18.0 was induced by ROS-generating chemicals (Guan, et al. 2004). Few reports on the functional role of sHSPs has been associated with conferring tolerance to abiotic and biotic stress tolerance (Ju, et al. 2017; Wang, et al. 2015). For instance, OsHsp18.0-CI enhanced resistance against bacterial pathogenic Xoc, salt, cadmium and viral RdRp (Ju, et al. 2017). Moreover, this study shows that OsHsp18.0-CI exhibits the ability to augment resistance against heavy metal stress. Together with above evidence in conjunction with the aforementioned findings, it can be concluded that the development-activated OsHsp16.9A may function as a molecular marker for thermotolerance of rice seeds, and OsHsp18.0 could be used as a candidate target gene for engineering oxidative stress tolerance in rice seedlings.