Effect Of Low Silicon Gene1(Lsi1)on Regulating Rice(Oryza Sativa L.)in Resistance To Cadmium Stress

In agriculture,soil,as the carrier for plant growth,continuously made exchange of matter and energy with the outside environment.However,soil suffered from different pollution due to excessive human activities while the soil-plant system provides powerful productivity.With high biological toxicity and long half-life,heavy metal Cd easily threatened to human health through the food chain.Rice,as the staple food of half the population of the world,was subjected to the threat of Cd pollution.It is well known that Si is second most abundant element in the crust followed by oxygen,most major crops are regarded as Si accumulators.Moreover,Si plays an important role in plants against adversity stress.At present,the gene controlling Si influx has been identified.The research was conducted by wild type Lemont rice(WT)and its mutants with Lsi1-overexpression transgenic Lemont rice(Lsi1-OE line)and Lsil-RNAi transgenic Lemont rice(Lsi1-RNAi line).First,we conducted expression profile and Label-free proteome analysis to uncover differences in all lines in molecular levels.Then we analyzed transport pathways of Cd in three rice lines through symplastic pathway and apoplastic transport pathways,and kinetics features of Cd absorption,five different hormones,the capacity of antioxidant enzyme and Cd accumulation in different parts of plants were observed.Based on Cd accumulation difference among three rice lines,the differences in rice yield and Cd content in seeds among three lines of rice were observed,the results of the study are as follows:1.The DGE analysis in three lines roots under Cd stress accompanied with Si addition showed that Lsi1-OE line and Lsi1-RNAi line detected 1010 and 1561 differentially expressed genes respectively,as compared with WT,it indicated that the transgenic rice with Lsi1 genetic modification owed different response process of gene expression regulation.qRT-PCR analysis showed that Cd+ Si treatment had a significant regulation effect on the expression of the stress related genes under Cd stress,confirming that Lsi1 genetically modified transgenetic lines had higher or lower Cd tolerance than that of WT.GO enrichment analysis showed that differentially expressed genes mainly enriching on biological progress between Lsi1-OE line and WT are involved in response to stresses,such as environmental stimulus,defense,oxidation-reduction,salt stress,osmotic stress and peptide hormone,etc,and those enriching on molecular function included ion binding,catalytic activity,peroxidase activity,oxidoreductase activity.The differentially expressed genes mainly enriching on biological progress between Lsi1-RNAi line and WT included both the pathway that response to biotic and abiotic stimulus,defense,oxidation-reduction,oxidative stress,salt stress,osmotic stress,and chemical reaction,etc.The enrichment on Molecular function included iron binding,enzyme(peroxidase and REDOX enzyme),ADP binding,tetrapyrrole binding,heme-binding,peptidase and endopeptidase regulator activity,etc.There were same and different response process in Lsi1-OE line and Lsi1-RNAi line.Coordinate expression of these differentially expressed genes was responsible to Lsi1 mediated Lsi1-OE line increasing resistance to Cd stress.2.Label-free quantitative proteomic analysis shows that Lsi1-OE line has 11 differently up-expressed proteins,55 differently down-expressed proteins,and Lsi1-RNAi line has 21 differently up-expressed proteins,8 differently down-expressed proteins,when compared with WT,and we based on the criterion that differently expression fold greater than 2 was considered as upregulated expression proteins,and those lower than 0.5 was downregulated expression proteins.Functional classification of differently proteins in according to GO classification system shows that Lsi1-OE line included that threonine-type peptidase activity,threonine-type endopeptidase activity,NAD binding,translation factor activity and malate dehydrogenase activity,etc.,but Lsi1-RNAi line mainly involved in structural molecule activity,antioxidant activity,peroxidase activity,oxidoreductase activity,S-adenosylmethionine-dependent methyltransferase activity,etc.3.The results through symplastic pathway and apoplastic transport pathways analysis showed that the increase of the total Cd in rice was accompanied with the increase in symplastic and apoplastic pathways.Either symplastic or apoplastic pathways,the order in Cd concentration is Lsi1-RNAi line>WT>Lsi1-OE line,and most of the Cd content((80%)absorbed by rice was distributed on the symplast pathway.Si addition significantly decreased the Cd concentration of symplastic pathway and apoplastic pathways,Lsi1-OE line owed the most effect of Si reducing Cd concentration in symplastic pathway and apoplastic pathways due to Si treatment,which accounted for 44.62%.It indicated that Lsil overexpression could regulate and promote the reducing of total Cd in the symplastic pathway and apoplastic pathways.4.In hydroponics,uptake kinetics of Cd in three rice lines and the effect of Si on the Cd uptake were observed.The results showed that there was no significant difference between WT and Lsi1-OE line in Km under Cd stress without Si,but Lsi1-RNAi line was significantly higher than that in WT.The Vmax was shown the highest in Lsi1-RNAi line and the lowest in Lsi1-OE line when compared to WT.Si application reduced Km and Vmax and there existed significant difference in Km and Vmax among three lines,which suggested that both Lsi1 overexpression and Si addition could reduce Cd uptake in roots.Further,the changes in chlorophyll content,dry weight and five types of plant hormones were observed under 50?M Cd with or not Si addition.It was found that chlorophyll content and dry weight as well as IAA,GA3 and JA were declined,the contrary trend was demonstrated in SA and ABA.Chlorophyll content,dry weight,IAA,GA3 and JA were increased under Cd stress in accompanied with Si,whereas SA and ABA were decreased.Cd caused less inhibitory effect on Lsi1-OE line than WT,which contains the highest IAA,GA3 and JA,the lowest SA and ABA,however,it showed the contrary trend in Lsi1-RNAi line.Therefore,we deduced that Lsil involved in regulation in Cd tolerance.5.The plants were exposed to combinations of three concentrations of Cd(0,50,and 100 ?M)and three concentrations of Si(0,1.5,and 2.0 mM).The results showed that Cd absorption capacity for each rice lines:Lsi1-RNAi line was the highest,followed by WT and Lsi1-OE line.The Cd-accumulations in the rice parts increased with increasing Cd in solution in the order of roots>shoots>leaves.Further study on the treatment of 50 ?M Cd without Si-addition showed that either 1.5 or 2.0 mM Si could significantly decrease Cd in the roots,shoots,and leaves of the rice,and that Lsi1-OE line exhibited a greater decrease in Cd than WT did,while-Lsi1-RNAi line had the least decline with a same Si-addition.It suggested that Si can restrain root absorption of cadmium and limit Cd transportation from the gound part to the above part.Without Si addition,the difference was not significant in three lines,but Si concentration in root,shoot and leaf in Lsi1-OE line was higher than WT and Lsi1-RNAi line,this further confirmed the silicon absorption by Lsi1 gene expression.When Lsi1 was overexpressed,Si absorbtion in Lsi1-OE line was enhanced,but Lsi1 was RNAi,it was decreased in Lsi1-RNAi line.In both Cd and Si treatment,high concentration of Cd influenced Si absorption.High concentration of Cd limited plant absorption to Si.Cd exposure easily causes oxidative stress in plants.Lsi1-OE line had higher SOD?POD?CAT activity and lower MDA content.Si addition further increased SOD?POD?CAT activity and reduced MDA content,these foundings suggested that Lsi1 overexpression and Si addition could protect antioxidase system from damage so as to alleviate Cd toxicity in rice plants.6.In order to futher understand Si alleviates Cd toxicity to rice in the whole growth period mediated by Si supplement,soil pot experiments with three of Cd dosages concentration and with three of Si concentrations were conducted to examine the differences in chlorophyll content,the photosynthetic rate(Pn)in the grain-filling period and grain yield and Cd resistence at harvest.At harvest,Si significantly reduced the Cd-content in rice under Cd-stress.The effect was most significant in Lsi1-OE line under high Cd-stress.Lsi1-OE line contained more Si and less Cd than WT,whereas the Lsi1-RNAi line showed an opposite result.Meanwhile,Cd in soil lowered the Si-uptake of the plants indicating a significant interaction between the two elements.During the grain-filling period,Cd-stress greatly reduced the chlorophyll content and Pn of the rice resulting in a diminished grain output.However,Lsi1-OE line with a higher chlorophyll content and Pn than either WT or Lis1-RNAi line maintained a high photo-assimilate transportation for high yield potential.No significant difference on the grain yields existed between WT and Lsi1-RNAi line.This might be due to a compensation effect generated by Lsi1-RNAi line on the whole growth period.It therefore suggested that Si available in the soil,and inducing Lsi1 expression as well as enhancing resistance to Cd-toxicity in plants,could be a plausible approach for production improvement.In conclution,Si mediated by Lsi1 plays an important role in reducing Cd toxicity to rice plans,this was involved in a complicate molecular physiological process that Lsi1 mediates the metabolism process between plants and heavy metal related genes/protein system,then generats a series of physiological and biochemical reaction and signal transduction to activate plant defense genes and induced the expression of plant activity against heavy metal toxicity,thus restrained the toxicity of heavy metal Cd.