The Roles Of OsNRAMPs In Cadmium Uptake And Translocation In Rice

Cadmium（Cd）is a toxic heavy metal pollutant widely present in the environment.With rapid growth in industry and urbanization in China,the problem of soil Cd pollution has become increasingly prominent.Cd is easily taken up by plants and can enter the human body through the food chain,seriously threatening human health.Compared with other cereal crops,rice roots possess a higher Cd uptake capacity.Rice is a major dietary source of the toxic metal Cd for Chinese and Asian populations.In some areas of China,soil pollution and acidification further increase the bioavailability of Cd in the soil,which leads to excessive Cd accumulation in rice grains.It is therefore important for food safety to study the mechanisms of Cd uptake and translocation in rice,and potential strategies for reducing Cd accumulation in rice grain.The thesis consists of three parts.（1）The first is a functional analysis of OsNRAMP1（Natural Resistance-Associated Macrophage Proteins 1）.OsNRAMP1 is highly homologous with OsNRAMP5.It is known that OsNRAMP5 is the main transport protein of manganese（Mn）and Cd in rice roots.However,the in planta function of OsNRAMP1remains unknown.The reverse genetics and detailed molecular genetics and physiological analyses were used to anslysze the function of the OsNRAMP1.（2）The second is the effects of OsNRAMP5-overexpression on accumulation of Cd in rice.Owing to the important role of OsNRAMP5 in Cd uptake in rice roots,we hypothesized that overexpression of OsNRAMP5 might increase Cd uptake,and offering plant materials for Cd phytoextraction of Cd-contaminated soils.Therefore,here we investigated the effects of OsNRAMP5-overexpression on accumulation of Cd and Mn in rice through hydroponics,Cd-contaminated soil pot experiments and molecular biology experiments.（3）The third part is the interaction between zinc（Zn）and Cd.There is interaction between Zn and Cd in geochemical environment,and the uptake and transport processes in organisms.However,their interactions in rice plants are not fully understood.Finally,the effects of exogenous Zn on Cd uptake and translocation in rice were investigated by increasing Zn in hydroponic culture and gene expression analysis,foliar spraying Zn in pot experiments.The main results are summarized as follows:（1）OsNRAMP1 is a close homologue of OsNRAMP5 with 72.8%similarity in the amino acid sequences.Here,we showed that OsNRAMP1 was able to transport Cd and Mn when expressed in yeast,but did not transport Fe（Ⅱ）or As（Ⅲ）.OsNRAMP1 was mainly expressed in roots and leaves and encoded a plasma membrane-localized protein.OsNRAMP1 expression level was induced by Cd treatment and Fe deficiency.The transgenic rice plants expressing p OsNRAMP1::OsNRAMP1-e GFP were used for immunostaining.Immunohistological staining of OsNRAMP1-e GFP showed that OsNRAMP1 was localized in all root cells except the central vasculature,as well as in the leaf mesophyll cells.Moreover,different from the polar localization of OsNRAMP5,the OsNRAMP1-GFP fusion proteins appears to be localized to the plasma membrane in a non-polar fashion.We obtained four independent lines of osnramp1 knockout mutant and two independent lines of osnramp5 knockout mutant in the cv.ZH11 background using the CRISPR/Cas9 system.Hydroponic experiments and measurements of the transient Cd²⁺fluxes showed that knockout of OsNRAMP1 resulted in significant decreases in root uptake of Cd and accumulation in rice shoots.Knockout of OsNRAMP1 also resulted in decreased Mn concentrations in rice roots,shoots and grains,and increased sensitivity to Mn deficiency.Knockout of OsNRAMP1 had smaller effects on Cd and Mn uptake than knockout of OsNRAMP5,while knockout of both genes resulted in large decreases in the uptake of the two metals.In addition,knockout OsNRAMP1 did not affect rice yield grown in a paddy field.There was also no significant effect on the concentrations of Fe,Zn,Cu and other trace elements in rice grains.Taken together,OsNRAMP1 contributes significantly to the uptake of Mn and Cd in rice and the functions of OsNRAMP1 and OsNRAMP5 are similar but not redundant.（2）We generated transgenic rice in two difference indica and japonica cultivars overexpressing OsNRAMP5 under the control of the OsActin1 or maize Ubiquitin promoter,and tested the effect on Cd uptake and translocation.BG367（W4）is an indica cultivar with high Cd accumulation in the shoots and grain due to a total loss of function of OsHMA3,whereas Zhonghua11（ZH11）is a japonica cultivar with a functional OsHMA3.Immunohistological staining against an OsNRAMP5 antibody showed that overexpressed OsNRAMP5 protein was localized to the plasma membrane of all cell types in the root tips and lateral root primordia without polarity,which was different from the polar distribution of the native OsNRAMP5 at the distal exodermis of wild-type mature roots.Hydroponic experiments with different levels of Cd showed that overexpression of OsNRAMP5increased Cd and Mn uptake into the roots,but decreased Cd concertation in the shoots and xylem sap markedly,and had a relatively small effect on Mn accumulation in the shoots.Synchrotron X-ray fluorescence mapping and Cd green fluorescens showed that the overexpression lines accumulated more Cd in the root tips and lateral root primordia compared with wild type.When grown in three Cd-contaminated paddy soils,OsNRAMP5 overexpression decreased grain Cd and Mn concentration by 49-94%and 20-40%,respectively,compared with wild type.OsNRAMP5 overexpression had a relatively small effect on the concentrations of other trace metals.Overexpressing OsNRAMP5 proteins disrupt the polar distribution pattern of the native OsNRAMP5 transporter at rice roots.Thus,overexpression of OsNRAMP5 decreased Cd translocation from roots to shoots and the accumulation of Cd in the grain by disrupting radial transport of Cd into the stele for xylem loading.OsMTP9,which is localized to the plasma membrane in the proximal side of the exodermis and endodermis cells,functions to transport Mn out of the cells and toward the stele,and could alleviate the effect of overexpression of OsNRAMP5 on Mn.Our results show that the polar localization of transporters is important for the mineral element distribution in the roots;disruption of this localization pattern can cause unpredictable results.Overexpression of OsNRAMP5 also offers a potential solution for reducing Cd accumulation in rice grain.（3）The chemical properties of Zn and Cd are similar,and they are often associated in nature.No matter whether the soils is polluted or not,the total Zn concentration in soil is always more than that of Cd,so the present study only investigated the effect of increasing Zn concentration on Cd uptake and translocation in rice.The present study showed that increasing Zn concentration in hydroponic solution and foliar Zn application significantly promoted the translocation of Cd from roots to shoots,and increased the accumulation of Cd in shoots.Furthermore,Zn caused a synergistic effect on Cd translocation from roots to shoots in an element specific fashion.Using RNA sequencing of rice roots,we found that elevated Zn concentration up-regulated the expression of genes involved in the glutathione synthesis and metabolism pathway and the Zn/Cd transporter gene OsHMA2,while down-regulated the expression of the genes related to Zn uptake.Knockout of OsIRT1,OsIRT2,OsZIP9 or OsCAL1 did not affect the synergistic effect of Zn on the translocation of Cd from roots to shoots.The contribution of OsNRAMP5 to Cd uptake in low Zn condition was much lower than that in high Zn condition.Both knockout OsNRAMP5 and high Zn treatment led to the phenotype of increased Cd translocation to shoots,although increasing Zn concentration still increased Cd translocation to shoots of osnramp5 mutant.The loss of OsHMA3 function and overexpression of OsHMA3 protein also did not affect the phenotype of increased Cd translocation to shoot at higher Zn concentration conditions.However,knockout OsHMA2 result in increased accumulation of Zn and Cd in roots and reduced the translocation of the two metals to the shoots.Meanwhile,knockout OsHMA2 abolished the synergistic effect of Zn on Cd translocation to shoots,and that was mainly achieved through OsHMA2 mediated xylem loading and translocation.The contribution of OsHMA2 to Cd translocation was larger than that of Zn.Previous studies have investigated the effect of foliar spraying Zn on Cd accumulation in grains,but the mechanism is not clear.However,it was found that foliar spraying Zn at the flowering stage effectively increased the Zn concentrations in straw and grain,but reduced the accumulation of Cd in the grains of plants grown in our soil pot experiments.OsHMA2 played a key role in the interaction between the two elements.Therefore,exogenous Zn cause synergistic effect on Cd at the seedling stage in hydroponic solution,while Zn had antagonistic effect on Cd translocation from leaves to grains.Taken together,Zn has the opposite dual-effect on Cd transportation in rice.In conclution,our study elucidated that OsNRAMP1 contributes to the uptake of Cd and Mn in rice roots.It was found that overexpression of OsNRAMP5 could distrup the radial translocation of Cd in rice roots,and then greatly dereeased Cd concentration in rice grains.Our study also elucidated that the dual-effects of Zn on the promotion of Cd was translacted to shoots and the inhibition of Cd translocated to grains.The synergistic effect of Zn on Cd translocation to shoots was mainly achieved through OsHMA2 mediated xylem loading and translocation.