Mechanism of hydrogen peroxide regulating cadmium tolerance and distribution in rice

Objectives By exploring the effects of different concentrations of hydrogen peroxide(H₂O₂) on the growth, root development, reactive oxygen species and Cd concentration of rice seedlings under Cd stress, we aimed to elucidate the mechanism of H₂O₂ signaling on cadmium tolerance and uptake in rice.

Methods Hydroponic experiment was conducted using japonica rice (Oryza sativa L., ZH11) as test material. The rice nutrient solution was added with 10 μmol/L CdCl₂ to make Cd stress, along with H₂O₂ of 0, 20, and 80 μmol/L, respectively. Normal nutrient solution was used as control (CK). Endogenous H₂O₂ and superoxide anion content in rice seedlings were investigated by taking fresh samples of roots after treatment for one day. The SPAD value of the penultimate leaf, plant height, root morphology, dry matter weight and Cd ion content in rice seedlings were investigated after treatment for thirty days. At the same time, Fe-plaque on the root surface and subcellular fractions of the root were extracted. To verify the role of H₂O₂ in cadmium stress, a H₂O₂ scavenger addition experiment was set up, and rice seedlings were treated with 10 µmol/L CdCl₂+20 µmol/L KI and 20 µmol/L KI. The SPAD value of the penultimate leaf and plant height in rice seedlings were investigated after treatment for twenty days. The total RNA was extracted from rice roots treated with CK, 10 µmol/L CdCl₂, or 10 µmol/L CdCl₂+20 µmol/L H₂O₂ for one day.

Results Rice growth was inhibited under Cd stress, and exogenous H₂O₂ significantly alleviated Cd stress in rice, but the alleviating effect diminished with increasing concentration. KI exacerbated the inhibitory effect caused by Cd stress. Plant height, SPAD Value, shoot and root dry matter weights significantly increased by 35.1%, 50.5%, 227.0% and 339.0%, respectively under 20 μmol/L H₂O₂ treatment, compared with no H₂O₂. Moreover, root morphometric parameters (total root length, root surface area, root volume, and number of root tips) were significantly increased. Cd stress led to a significant increase in the superoxide anion content in the roots of rice seedlings, and exogenous addition of H₂O₂ significantly reduced the superoxide anion concentration. Exogenous H₂O₂ reduced Cd uptake in rice by down-regulating the expression value of OsNRAMP5, resulting in a significant reduction in Cd ion content in both roots and shoots. In addition, H₂O₂ treatment promoted the formation of Fe-plaque on the root surface of rice, which adsorbed more Cd ions on the root surface, thus inhibiting the transfer of Cd from the root surface to the root. Within root, H₂O₂ treatment up-regulated pectin synthesis and pectin methylesterase expression, which promoted compartmentalization of Cd in the cell wall and reduced Cd content in the cytoplasm, thereby attenuating Cd cytotoxicity.

Conclusions Exogenous addition of appropriate levels of H₂O₂ can reduce the oxidative damage caused by O₂⁻, as well as down-regulate the expression level of OsNRAMP5 to reduce the uptake of Cd in rice. Exogenous H₂O₂ also promotes the formation of Fe-plaque on the root surface of rice and inhibits the translocation of Cd²⁺ from the root surface to the root. Furthermore, exogenous H₂O₂ promotes the compartmentalization of Cd in the cell wall and interfere with the movement of Cd in the cytoplasm, thus alleviating the cytotoxicity of Cd and improving the resistance of rice to Cd stress. However, the effect of exogenous high levels of H₂O₂ in alleviating Cd stress in rice was greatly diminished, which might be related to its inhibition of the formation of iron film on the root surface.