Functional Analysis Of OsPP2C02 Under Mercury, Cadmium And Lead Stress

Mercury,cadmium and lead are harmful heavy metals,which have detrimental effects on the growth and development of rice.However,the detoxification and tolerance mechanisms of rice to mercury,cadmium and lead stress are still not fully understood.The study on the functions and mechanisms of genes related to mercury,cadmium and lead stress in rice provideds a theoretical basis for cultivating rice varieties related to mercury,cadmium and lead tolerance.Reversible protein phosphorylation is a common regulation in organisms.PP2C,as a protein phosphatase,plays an important role in plant response to stress.However,there are few studies on the tolerance of OsPP2C to mercury,cadmium and lead stress in rice.In this paper,the overexpression of OsPP2C02 in rice,the knockdown of OsPP2C02 in rice obtained in our laboratory and the wild type Nipponbare were used as experimental materials to study the function of OsPP2C02 in tolerance to mercury,cadmium and lead.The main results of this paper are as follows:1.Compared with the wild type,the overexpression of OsPP2C02 in rice decreased the tolerance of the plants to mercury stress,with significantly lower survival rate,while the knockdown of OsPP2C02 in rice enhanced stress tolerance,with significantly higher survival rate.These results indicate that OsPP2C02 acts as a negative regulator of rice’s tolerance to mercury stress.Compared with the wild type,the chlorophyll content of OsPP2C02 overexpressed lines decreased significantly and the relative ion permeability increased significantly under mercury treatment.2.The transcriptomic analysis of rice leaves under mercury stress showed that compared with the wild type,differentially expressed genes in response to mercury stress in OsPP2C02 overexpressed lines and OsPP2C02 interference lines were mainly related to copper ion transporter.The differentially expressed genes in response to mercury stress in OsPP2C02 interference line were mainly related to metal transporter,auxin signaling,Ca²⁺signaling and ABA signaling.The number of differentially expressed the protein serine/threonine kinas genes in the knockdown of OsPP2C02 in rice were increased compared to the wild-type by co-functional gene networks.14 differentially expressed genes in response to mercury stress were randomly chosen in OsPP2C02 overexpression line and OsPP2C02 interference line,and their expression levels were verified by real-time quantitative PCR.The quantitative results were basically consistent with transcriptome sequencing results,with a correlation coefficient of 0.92.The results show that OsPP2C02 interference lines can enhance the tolerance to Hg²⁺by decreasing Hg²⁺absorption,chelation and compartmentalization to alleviate the damaging effects of Hg toxicity.3.After cadmium treatment,compared with the wild type,the OsPP2C02overexpressed lines were more sensitive to cadmium,with significantly reduced survival rate,while the OsPP2C02 interference lines were tolerant to cadmium,with significantly increased survival rate.These results indicate that the expression level of OsPP2C02 is negatively correlated with cadmium tolerance of rice.After cadmium treatment,compared with the wild type,the chlorophyll contents of OsPP2C02overexpressed lines decreased significantly,and the relative ion permeability increased significantly.Histochemical staining showed that the contents of hydrogen peroxide and superoxide anion in leaves of OsPP2C02 were lower than those of wild type under cadmium treatment.The accumulation of cadmium in root and leave in the knockdown of OsPP2C02 rice were less than the wild type.The expression levels of genes related to cadmium accumulation and transport from root to shoot were down-regulated in root of OsPP2C02 interference lines by real-time quantitative PCR.These results indicate that OsPP2C02 interference lines could enhance the tolerance to cadmium by decreasing Cd²⁺absorption and inhibiting Cd²⁺transport.4.There was no significant difference in phenotype between Nipponbare,OsPP2C02 overexpressed lines and OsPP2C02 interference lines under 4 m M Pb²⁺treatment.These results indicate that OsPP2C02 gene can’t enhance the tolerance of rice under 4 m M Pb²⁺stress.