Effects Of Silicon On Cadmium Uptake And Transport By Rice Roots

Rice is not only an important grain crop in China,but also the only bulk grain crop which with competitive advantage in the international grain market.The safe production of rice is of great significance to the sustainable development of food security.However,in recent years,due to the unreasonable application of pesticides and chemical fertilizers and the emission of industrial wastes,heavy metals in the environment are increasing rapidly,and the paddy field system is increasingly polluted by cadmium（Cd）.Cd pollution leads to the reduction of regional rice production and quality,which not only seriously affects the development of agricultural economy,but also threatens human health.Si,an environment friendly element,is beneficial for plant’s growth and plays a pivotal role in ameliorating heavy metal toxicity in plants.However,from previous reports,few paid attention to how exogenous Si regulated cell wall and vacuole to promote Cd resistance in different durations of Cd exposure,and how different forms of silicic acid affected the Cd absorption pathways by rice.In this arcticle,rice was taking as the research object,and inductively coupled plasma-mass spectrometry（ICP-MS）,transmission electron microscopy（TEM）and element mapping,fourier transform infrared spectroscopy（FTIR）,ultra performance liquid chromatography-mass spectrometry（UPLC-MS）,data independent acquisition（DIA）-based proteomics and metabolomics were employed in this study to study the effects of Si on Cd retention in rice roots,the subcellular distribution of Cd in roots,alterations in roots cell wall characteristics and adsorption capacity of Cd,vacuoles retention of Cd,changes in root metabolic pathways and proteins,were systematically explored.More over,the transport of monosilicic and polysilicic acids in the apoplast and symplast of rice as well as the effects of Si supplementation on Cd transport through apoplastic and symplastic pathways were investigated to reveal the influence of Si on the Cd tolerance mechanisms in rice roots.It is expected to explore the mechanisms of Cd tolerance and the key factors affecting Cd tolerance in rice,providing a new theoretical basis for the mitigation and resistance of Cd stress in rice plants,so as to promote the safe production of rice.The main results are summarized as follows:1.The mechanisms of Si on Cd retention in roots under short-term（12h）and long-term（5d）Cd exposure were studied by hydroponic experiments.The results showed that rice roots were the main tissues of Cd retention.Si addition caused no significant changes in root Cd concentrations but decreased shoots Cd contents when compared with sole Cd treatment under short-term and long-term exposure.Three subcellular components were isolated from rice roots by ultra-centrifugation:cell wall,soluble fractions and organelles.The majority of the total Cd（83.4%-95.9%）was presented on the cell wall and soluble fraction,with only a small part stored in the organelles.Under short-term Cd exposure,most of Cd has entered the soluble component,while Si addition significantly increased the concentration of Cd in cell wall,the Cd concentration in the cell wall evidently increased in the presence of Si,with an increase of 24.0%and 23.2%at low and high Cd levels,respectively.This was accompanied by a decline in Cd proportions in the soluble fraction of 21.5%and 20.6%at low and high Cd levels,respectively,indicating Si promoted the redistribution of Cd in cell components.TEM and element mapping results further confirmed that Si increased the deposition of Cd on the cell wall,especially in corners of cells rich in pectin.Under long-term Cd exposure,the retention ability of Cd of cell wall was significantly lower than that under short-term Cd exposure,and most of Cd in root system existed in soluble components（52.5%-79.5%）whether Si was added or not.These indicated that there might be different mechanisms exist in Si alleviating Cd toxicity in rice under short-term and long-term Cd exposure.2.The contents and Cd concentration of root cell wall polysaccharides including cellulose,hemicellulose and pectin were determined.It was found that Cd in cell wall was mainly bound to pectin.Under short-term Cd stress,the addition of Si significantly increased Cd content in CSP and hemicellulose by 48.5%and 53.0%,respectively under short-term exposure.On the contrary,Si addition decreased Cd concentration in CSP and WSP by 33.5%and 26.7%,respectively under long-term exposure.the FTIR spectroscopy analysis of cell wall components showed that the-OH and C-H groups were the main binding sites of Cd²⁺on cell wall in adorption process under short-term Cd expsoure.The addition of Si increased the binding ability of functional groups on cell wall to Cd.Under long-term exposure,with the saturation of ion exchange sites on the cell wall,a substantial amount of Cd inevitably entered into the cytosol.In the cytoplasm,compared with short-term Cd stress,both levels of PC2 and PC3 were triggered under long-term exposure and Si promoted PC2 and PC3 contents.To sum up,our conclusion showed that Si is predominated in restricting the transportion of Cd through the root cell wall under short-term Cd exposure,and promoted vacuoles compartmentalization to reduce the toxicity of Cd under long-term exposure rather than reducing the absorption of Cd by rice roots,so as to reduce the transportion of Cd to the shoots.3.According to the DIA analysis of the expression levels of proteins in rice roots,167DEPs specifically showed expression changes after short-term exposure（STCd RPs）and a total of 473 DEPs specifically showed expression changes under long-term exposure（LTCd RPs）.GO enrichment analysis showed that STCd RPs were significantly enriched in cell wall and in response to wounding.While LTCd RPs were significantly enriched in the cytosol and vacuoles,and mainly involved in stress-response interactions,amino acid metabolisms and secondary metabolisms.These results indicated that STCd RPs located on the cell wall served as emergency response to Cd stress,and with extended Cd exposure,the response of LTCd RPs in the cytosol tended to be triggered.The Peroxidases and germin-like proteins（GLPs）were the main proteins on the cell wall.With the addition of Si,the expressions showed down-regulated with Si supply under short-term exposure,Thus,the production of H₂O₂ would be less generate and the oxidative stress response was alleviated.Under long-term Cd stress,the addition of Si slightly promoted the Cd entering into the vacuoles with low-Cd level,and down regulated the MRP-like ABC transporters,indicating a reduction of Cd efflux of vacuoles with Si supply.Therefore,more Cd was retained in the vacuole by binding with PCs to partition free Cd into the cytosol,and thus restricting Cd translocation.4.The effects of Si on root metabolites and metabolic pathways under short-term and long-term Cd exposure were vary.Under short-term Cd exposure（12 h）,a total of 17differential metabolites（DEMs）were identified,including 7 amino acids,8 fatty acids,1carbohydrate and 1 others,and a total of 98 of DEMs were identified under long-term Cd exposure,including 31 amino acids,28 lipid metabolites,10 nucleotides,19 organic acids and 10 carbohydrate and alcohol metabolites.Roots can reduce cadmium toxicity,oxidative stress and cell membrane damage by regulating amino acid synthesis metabolism,carbohydrate metabolism and lipid metabolism.In this study,most amino acid metabolites were significantly up-regulated under short-term Cd exposure,while Si significantly down regulated expression levels of alanine,phenylalanine,proline,serine and other amino acids.Under long-term Cd stress,Si treatment up-regulated methyl jasmonate and methyl dihydrojasmonate,and increased the expression of malic acid,which was the key intermediate of TCA cycle.It indicated that Si reduced the toxicity of Cd by regulating different metabolites and pathways under different Cd exposure periods.5.The transport of monosilicic and polysilicic acids in the apoplast and symplast of rice as well as the effects of absorption and transportation of monosilicic acid and polysilicic acid on Cd contents in rice roots,stems and leaves were studied by a hydroponic culture in laboratory.In this experiment,soluble Si existed in the form of both monosilicic acid and polysilicic acid in apoplast and symplast.The concentrations of monosilicic acid and polysilicic acid in rice organs were range from 72 to 658 mg kg^-1 and 91 to 850 mg kg^-1,respectively.In general,the concentration of polysilicic acid was higher than that of monosilicic acid.The Cd translocation was very fastly and mainly through apoplastic pathway.Under the condition of Cd solution culture for 24 h,the Cd concentration increased 38-50 times in apoplast and 6-8 times in symplast.Under Cd exposure,the adsorption of Cd in the apoplast of rice roots,stems and leaves was inhibited in the first 2days,and increased rapidly on the 4th day.Therefore,the inhibition of Cd by apoplast barrier was time-limited.While with Si supply,the transport of Cd in root,stem and leaf explants decreased by 50-90%,and ability of apoplastic barriers to impede Cd root-to-leaf transport played an important role in the whole stress period.The absorption kinetic of Cd in the symplast of stem was similar to the apoplast of root,stem and leaves,which increased sharply on the 4th day and then decreased,but different from absorption kinetics of root and leaf symplast.Si treatment significantly reduced the concentration of Cd in stem and leaf symplast.