Overexpression of OsHsp 18.0 in rice enhanced tolerance to heavy metal stress

OsHsp18.0 plays a key role in cross-protection of rice seedlings from damages to photochemical systems and cellular membranes, caused by Cd and Cu stresses.

Under heat stress, plants synthesize multiple families of sHSPs with monomer sizes ranging from 12-42 kDa, that are more diverse and abundant than other organisms (Wu et al. 2022).In Arabidopsis, RNAi suppression of certain sHSPs revealed essential and distinct functions of sHSPs in plant's recovery from severe heat stress (10 h at 45 °C), and heat tolerance.Moreover, plant sHSPs also function in other cellular processes under normal conditions (Wu et al. 2022;Liu et al. 2023).Decades of research revealed conditions, and stages of plant growth development for various sHSPs to confer stress protection (Waters and Vierling 2020).Besides, certain plant sHSPs can be induced by other stressors, such as heat, heavy metal, and oxidative stresses (Wu et al. 2022).Heat and heavy metal stresses increased rice sHSPs mRNA (Tseng et al. 1993, cf. supplementary references).Heat pretreatments protect rice from toxicity of subsequent cadmium exposure (Hsu and Kao 2007), suggesting a role for sHSPs in heavy metal protection (Hall 2002 cf.supplementary references).
Both rice OsHsp16.9A and OsHsp18.0 are highly similar, and belong to same Class I sHSP group, however, they function differently in thermoprotection (Liu et al. 2023).Overexpressed OsHsp18.0 conferred thermoprotection for rice seedlings survival only; overexpressed OsHsp16.9Athermo-protected rice in all stages investigated.Therefore, we further explored sensitivities of OsHsp18.0-overexpressinglines to heavy metal stress of copper (Cu) and cadmium (Cd) treatments.Presence of excess Cu in river beds surveyed, and excess Cd causing incidents of rice field contamination have been recorded locally.
We further compared electrolyte leakage among WT, OsHsp16.9A-OE, and OsHsp18.0-OEseedlings from Cu and Cd treatments at 28℃ (Fig. 1f and g).Results of Cu, Cd treatments showed lower conductivity of OsHsp18.0-OEsincubation medium than WT (24.6% ~ 37.6% and The second leaves were excised from rice seedlings, after incubation at 28 ℃ in shaking buffer with or without 150 or 250 μM CuS O4 and 1 or 2 μM CdC l2 , respectively.Detached leaves were then used to determine the contents of chlorophyll (b), (d) and malondialdehyde (MDA) (c), (e) as indicated.f, g Percentage of electrolyte leakage was calculated from ten etiolated rice seedlings with cotyledons removed, that were incubated at 28℃, in de-ionized water with or without 150 or 250 μM CuSO4 and 1 or 2 μM CdCl2, respectively.h-k Detached leaves were then used to determine the activities of catalase (CAT) and the activities of ascorbate peroxidase (APX) respectively (30 leaves in each experiment).h, i Catalase and j, k Ascorbate peroxidase activities were upregulated in OsHsp18.0 transgenic rice seedlings under Cu and Cd stresses.All the experiments were carried out with rice seedling grown under control conditions for 12 days.Data are mean ± SD from five independent experiments (30 leaves in each experiment).Bars with the same letter are not significantly different at p < 0.05 1 3 25.7% ~ 34.6% reduction, respectively), whereas overexpressed OsHsp16.9A did not suppress the cellular leakage after Cu and Cd treatments (Fig. 1f and g), indicating that OsHsp18.0-OEstransgenic lines maintained higher cell membrane integrity than WT and OsHsp16.9A-OElines.Further analyses on antioxidant enzymes: catalase (CAT), and ascorbate peroxidase (APX) were performed, and the results showed that, under non-stress conditions, no difference in the activity of these antioxidant enzymes, among all lines examined (Fig. 1 h-k), while CAT activity was significantly higher in OsHsp18.0-OEthan WT and OsHsp 16.9A-OE (p < 0.05, Fig. 1h); and APX activity exhibited significant differences among OsHsp18.0-OEand OsHsp 16.9A-OE leaves after exposure to 150 µM and 250 µM of CuS O4 (Fig. 1j).With Cd treatments, CAT activity was significantly higher in OsHsp 18.0-OE than WT and OsHsp 16.9A-OE leaves (p < 0.05, Fig. 1i).Similarly, APX activity was significantly elevated in OsHsp 18.0-OE leaves after 1 and 2 µM Cd treatments (p < 0.05, Fig. 1k).These results revealed OsHsp18.0 plays a key role in maintaining cell membrane integrity against Cu and Cd stress by scavenging generated reactive oxygen species (ROS); and are consistent with finding that OsHsp18.0 was induced by ROS-generating chemicals (Guan et al. 2004).
Since Cd is not an essential metal ion, no specific uptake systems for this metal reported; rice cultivars may have evolved to limit and sequester Cd uptake (Ueno, et al. 2010).However, Cd still negatively impacts rice growth, and yields (Kanu et al. 2017).Similar to protecting effects of Lycopersicon peruvianum sHSP17 against Cd stress (Neumann et al. 1994), overexpressed OsHsp18.0 also enhanced rice's tolerance to Cu and Cd toxicity.Besides the chaperon activity, overexpressed OsHsp18.0 may exert protection by associating with other protein partners; much like the overexpressed OsHsp16.9A-OsHsp101interaction provided thermotolerance for rice (Liu et al. 2023).Related experimentations are needed for definitive conclusions.Our studies showed that OsHsp18.0'sresistant function in heavy metal stress, is a suitable target for oxidative stress related engineering.

Fig. 1
Fig.1Transgenic rice seedling overexpressing OsHsp16.9A and OsHsp18.0 showed tolerance to copper (Cu) and cadmium (Cd) stress a Map of pPZP/35H Tibased vector carrying the DNA fragments of OsHsp16.9A and OsHsp18.0 for overexpression.b-k Seedlings of transgenic rice overexpressing OsHsp18.0 showed tolerance to copper (Cu) and cadmium (Cd) treatments.The second leaves were excised from rice seedlings, after incubation at 28 ℃ in shaking buffer with or without 150 or 250 μM CuS O4 and 1 or 2 μM CdC l2 , respectively.Detached leaves were then used to determine the contents of chlorophyll (b), (d) and malondialdehyde (MDA) (c), (e) as indicated.f, g Percentage of electrolyte leakage was calculated from ten etiolated rice seedlings with cotyledons removed, that were incubated at 28℃, in de-ionized water with or without 150 or 250 μM CuSO4 and 1 or 2 μM CdCl2, respectively.h-k Detached leaves were then used to determine the activities of catalase (CAT) and the activities of ascorbate peroxidase (APX) respectively (30 leaves in each experiment).h, i Catalase and j, k Ascorbate peroxidase activities were upregulated in OsHsp18.0 transgenic rice seedlings under Cu and Cd stresses.All the experiments were carried out with rice seedling grown under control conditions for 12 days.Data are mean ± SD from five independent experiments (30 leaves in each experiment).Bars with the same letter are not significantly different at p < 0.05