Molecular mechanism of cadmium hypertolerance on the cadmium/zinc hyperaccumulator Sedum plumbizincicola

H. Zhaoa, H. Liua,b, L. Wuc, F-J. Zhaob and Wenzhong Xua

aKey Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, China

bState Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, China

c Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, China

xuwzh@ibcas.ac.cn

Cadmium (Cd) is a non-essential heavy metal element that is highly toxic to almost all organisms, including plants and humans. But some rare plant species can hyperaccumulate Cd in aboveground tissues to 100-fold higher than normal plant species without suffering from toxicity, which are called Cd hyperaccumulators. Sedum plumbizincicola is a Crassulaceae species native to the Cd/Zn mining areas in southeast China. Because of its strong resistance and hyperaccumulation to Cd/Zn, it is a main phytoremediation species of Cd pollution soil restoration test. However, as a consequence of the lack of transgenic methods for this species, the mechanism underlying high resistance to Cd remains unclear. We isolated SpHMA3 (Heavy Metal ATPase 3) from S. plumbizincicola, and first developed a genetic transformation system for S. plumbizincicola and the nonhyperaccumulating ecotype of S. alfredii, so RNA interference (RNAi)-mediated silencing and overexpression of SpHMA3 were performed in the Sedum species. We found that SpHMA3 protein was tonoplast-localized and showed Cd-specific transport activity, and SpHMA3 was highly expressed in the shoots of S. plumbizincicola. The SpHMA3-RNAi lines were hypersensitive to Cd, and the growth of shoots and young leaves was severely inhibited by Cd, but did not affect the accumulation of Cd in the aboveground. Furthermore, a homolog gene of HMA1 (Heavy Metal ATPase 1), SpHMA1, was also isolated and functionally identified from the S. plumbizincicola. The SpHMA1 protein was localized to the chloroplast envelope. SpHMA1 RNA interference (RNAi) transgenic plants and CRISPR/Cas9-induced mutant lines showed significantly increased Cd accumulation in the chloroplasts compared with wild-type plants. Chlorophyll fluorescence imaging analysis revealed that the photosystem II of SpHMA1 knockdown and knockout lines suffered from a much higher degree of Cd toxicity than wild type. These results suggest that SpHMA1 functions as a chloroplast Cd exporter and protects photosynthesis by preventing Cd accumulation in the chloroplast in S. plumbizincicola. Taken together, our study showed that SpHMA3 and SpHMA1 are highly expressed in shoots to enable an efficient detoxification of Cd in the leaves. SpHMA1, as a Cd transporter localized at the chloroplast envelope, functions to export Cd from the chloroplasts to the cytoplasm and protect the photochemical reactions in leaf cells of the Cd hyperaccumulator S. plumbizincicola. To keep the cytosolic Cd at low levels, another heavy metal ATPase, SpHMA3, functions to transport Cd from the cytoplasm into the vacuoles. Thus, both SpHMA1 and SpHMA3 are important for Cd hypertolerance in S. plumbizincicola.

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