The Role Of SaPCR2 And SaHMA2 Involved In Zn Uptake And Accumulation In A Hyperaccumulator Sedum Alfredii

Heavy metal is reported to be a severe pollutant for agricultural soils in China.Phytoremediation is considered as an eco-friendliness and cost-effective technology to clean up the soil with heavy metals.Hyperaccumulators can accumulate 100-to1000-fold higher heavy metals than normal plants without toxic consequences and,therefore,are of high scientific interest as a representative plant material to extract heavy metals with great ability.Sedum alfredii Hance,a Chinese native zinc（Zn）/cadmium（Cd）hyperaccumulator belonging to the Crassulaceae family,has great potential for phytoremediation of heavy metal polluted soils.Revealing the hyperaccumulating mechanism in S.alfredii would promote the phytoremediation efficiency by using agronomic measures or genetic engineering methods.However,the current researches mostly concentrated on the physiological mechanism in S.alfredii,while the molecular mechanism of uptake,transport,and accumulation of heavy metals still largely remained unknown.A great number of studies showed that PCR（Plant Cadmium Resistance）family and HMA（Heavy Metal transporting ATPases）family facilitate the transportation of Zn,Cd and other divalent cations in plants.Previous studies of our research group found that SaPCR2 and SaHMA2 play an important role in the uptake and accumulation of Cd in S.alfredii,but their function to Zn wasnot cleared yet.So in this thesis,we investigated the function of SaPCR2 and SaHMA2 in the uptake and accumulation of Zn using molecular biological methodsandμ-XRF（micro X-ray fluorescence）technology.The main results achieved are listed as follow:（1）The results ofreal-time quantitative PCR（RT-q PCR）analysis showed that SaPCR2 was highly expressed in the root of hyperaccumulating ecotype（HE）S.alfredii and the expression levels were induced by Zn deficiency,but slightly reduced under the treatment with the addition of Zn SO₄.SaPCR2 was transformed into the Zn-sensitive Saccharomyces cerevisiaemutantΔzrc1 and Zn-uptake S.cerevisiae deficient mutant ZHY3 using the Li Ac/PEG/ss DNA method.The growth of yeasts showed that the heterologous expression of SaPCR2 inΔzrc1 increased Zn absorption and reduced its tolerance to Zn.In contrast,expressing SaPCR2 in mutant ZHY3could reverse the Zn uptake deficiency.Combining with the early result of"SaPCR2localized to the plasma membrane",we indicate that SaPCR2 is a transporter responsible for the influx of Zn across the plasma membrane in the root of HE S.alfredii.Agrobacterium transformation method was used to overexpress SaPCR2 in non-hyperaccumulating ecotype（NHE）S.alfredii.The concentrations of trace nutrient elements were investigated in transgenic lines（L1 and L2）and their wild type（WT）.Overexpression of SaPCR2 increased Zn accumulation and uptake in the transgeniclines buthadno significant effect on iron（Fe）,manganese（Mn）and copper（Cu）.In vivo analysis of Zn distribution byμ-XRF showed that the signal of Znwas low and mainly distributed in vascular bundles in the stem of WT,but much higher in SaPCR2 overexpressing plants and distributed in vascular bundle,cortex and epidermis.Therefore,the overexpression of SaPCR2 improved Zn uptake in the root of transgenic lines and resulted to the higher Zn accumulation in both root and shoot.In addition,the higher Zn accumulation could be observed in the elongation zone of SaPCR2 overexpressing plants by the in situμ-XRF mapping.Compared with WT plants,Zn exposure had a more significant inhibitory effect on the root growth of SaPCR2 overexpressing plants,especially in the elongation zone.The expression level of SaPCR2 at the root bottom was significantly higher than the root tip.In conclusion,SaPCR2 mediates Zn uptake by the root cells,especially in the elongation zone of HE S.alfredii,which may facilitate the rapid uptake and accumulation of Zn as well as the growth of root cells.（2）The expression pattern of SaHMA2 was investigated in HE S.alfredii.The results showed that SaHMA2 was mainly expressed in the root and stem,and the expression level at the root bottom was also significantly higher than the root tip.But there was no significant expression difference between the young and mature stems/leaves.We also investigated whether Zn could induce SaHMA2 expression.But no significant difference in SaHMA2 expression was observed after Zn treatment.After transforming SaHMA2 into NHE S.alfredii,three independent overexpressing lines were identified（OX2,OX4 and OX5）.Zn concentrations in the xylem sap of transgenic plants were significantly higher than WT,and the Zn accumulations of plants showed the same tendency.Furthermore,overexpression of SaHMA2 also increased Fe and Mn concentrations in xylem sap,but there was no significant difference on the accumulations of Fe and Mn.Zn concentrations in the plants increased after additional Zn exposure,especially in the young leaf and root tip of transgenic lines,and significantly higher than WT.This result showed that SaHMA2overexpressing plants preferred to accumulate Zn in younger organs.All those findings suggest that SaHMA2 is closely related to the xylem loading of Zn in HE S.alfredii and may be involved in phloem remobilization of Zn.Therefore,SaPCR2 and SaHMA2 transporters play an important role in Zn uptake and xylem loading by the roots of HE S.alfredii,so as to hyperaccumulate Zn in the shoots,which may contribute to the development of phytoremediation and food safety in the future.