Toxicity Of Different Forms Of Antimony On Rice Plants And Its Metabolic Mechanism

Antimony（Sb）is carcinogenic for humans,and will pose a potential health threat to humans via the food chain.Flooding condition is conducive to the dissolution and release of Sb from soil particles,resulting in a enhanced enrichment of Sb by rice plants.Excessive Sb is harmful for plant growth.Previous studies demostrated that excess antimonite[Sb（III）]can inhibit plant growth,induce oxidative stress,inhibit photosynthesis and restrict uptake of essential nutrients in plants.However,few studies were conducted to investigate the following mechanisms,including 1)the toxic of antimonate[Sb（V）]to plants;and 2)the differences in their toxicity to plants between Sb（III）and Sb（V）,especially for the induced oxidative stress by them,photosynthetic electron transport disorder,photosynthetic efficiency reduction and disturbance of photosynthetic product synthesis.Based on the above reasons,this study set up a series of hydroponic experiments using a rice variety（Yangdao 6）to systematically explore the toxic differences of Sb（III）and Sb（V）in 1)the plant growth and the uptake of essential elements;2)the production of reactive oxygen species（ROS）and the responses of antioxidant system;3)the responses of photosynthetic efficiency;4)the photosynthetic carbon assimilation（including soluble sugar and starch content）and carbohydrate metabolism;5)In addition,we used a metabolomics technology to compare the differences in the effects of Sb（III）and Sb（V）on synthesis of metabolites in rice leaves.Further,we analyzed the flow directions of glycosyls produced from photosynthesis in rice leaves under Sb（III）and Sb（V）stress.The main results are as follows.Oxidative damages and element uptake.20 mg·L^-1 Sb（III）significantly reduced the biomass of shoots and roots.Both Sb（III）and Sb（V）produced oxidative stress in this rice plant.Most of Sb treatments significantly increased shoot and root malondialdehyde（MDA）contents.Compared to the control,20 mg·L^-1Sb（Ⅲ）significantly increased the shoot Ca concentration,but decreased the shoot concentrations of K and Cu.Sb（V）treatments significantly increased the concentrations of K,Ca,Mg and Cu.Compared to the control,Sb（Ⅲ）treatments significantly increased the shoot O₂^-·concentration,and 20 mg·L^-1 Sb（Ⅲ）significantly increased the shoot H₂O₂ concentration.However,Sb（V）significantly increased shoot O₂^-·and H₂O₂ concentration.Under Sb（Ⅲ）stress,the ratios decreased from 25.53(5 mg·L^-1)to 13.73(20 mg·L^-1),while under Sb（V）stress,the corresponding ratios decreased from 15.47 to 11.57.GSH-PX enzyme activity was significantly enhanced by Sb treatments,except for 20 mg·L^-1Sb（V）treatment.It suggested that Sb（Ⅲ）interfered the disproportionation of O2^-·to H₂O₂,which may be related to the loss of more essential elements caused by Sb（Ⅲ）.GSH PX enzyme activity in rice antioxidant system increased significantly under most sb treatments,which means that the enzyme plays a major role in H₂O₂ clearance under Sb stress.Photosynthetic gas exchange parameters and pigment contents.Sb（Ⅲ）treatments significantly reduced the leaf water content,increased water deficit（Vpd）,stomatal conductance（Gs）,net photosynthetic efficiency（Pn）,transpiration rate（Tr）and water use efficiency（WUE）.The addition of Sb（V）had no significant effects on the above indexes.Compared to the control,only 5 mg·L^-1 Sb（Ⅲ）significantly increased the content of chlorophyll a,and 5 mg·L^-1Sb（Ⅴ）significantly increased the content of chlorophyll b.Photosynthetic electron transfer.Sb（Ⅲ）treatments significantly reduced the values of the following chlorophyll fluorescence parameters in PSII complex,includingΨ_O,φ_Eo,φ_Ro,ET_o/RC,PI_ABS and RC/CS;increased that of ABS/RC,DI_o/RC and TR_o/RC;but did not significantly affect the values of F_v/F_m andφ_Do.The addition of Sb（V）did not significantly the values of the above parameters.However,the addition of Sb（V）significantly increased Y（ND）(5 mg·L^-1)and Y（NA）(10 mg·L^-1)in PS I.Different forms of Sb significantly reduced the values of P_m,Y（I）and ETR（I）.The addition of Sb（Ⅲ）significantly increased the value of Y（ND）,while 10 mg·L^-1Sb（Ⅲ）significantly decreased the value of Y（NA）.Changes of chloroplast microstructure.The shapes of starch granules under Sb（Ⅲ）and Sb（V）treatments became narrow and long,especially under Sb（Ⅲ）treatment.Plastoglobule（PG）also appeared under Sb（Ⅲ）treatment.The stacking degree of grana thylakoids and the thickness of cell wall increased under Sb（Ⅲ）stress,especially under the 10 mg·L^-1 Sb（Ⅲ）treatment.Enzyme activities correlated with Calvin cycle and contents of soluble sugar and starch.Sb（Ⅲ）and 5 mg·L^-1Sb（Ⅴ）significantly inhibited FBP enzyme activity.The 10 mg·L^-1Sb（Ⅲ）treatment significantly increased Rubisco enzyme activity(it was non-significantly enhanced by 5 mg·L^-1Sb（Ⅲ）).5 mg·L^-1 Sb（Ⅲ）significantly increased TPI enzyme activity.The addition of Sb（Ⅲ）and 5 mg·L^-1 Sb（V）significantly increased the contents of soluble sugar and starch in rice leaves.Enzyme activities correlated with synthesis and decomposition of sucrose and starch.Sb（III）addition significantly reduced the activity of sucrose phosphate synthase（SPS）,but Sb（V）did not significantly affect its activity.The activity of insoluble acid invertase（B-AI）in cell wall was significantly enhanced by Sb（III）and Sb（V）.Only 5 mg·L^-1 Sb（V）significantly increased the activity of soluble acid invertase（S-AI）.Compared to the control,Sb（III）significantly increased the activity of adenosine diphosphate glucose pyrophosphorylase（AGP）,but Sb（V）did not significantly affect its activity.Different forms of Sb did not significantly affect the activity ofα-amylase（α-al）.Metabolomic analysis.Based on UHPLC-MS/MS technology,645 differential metabolites were identified in rice leaves treated with Sb（III）and Sb（V）.A total of153 differential metabolites were annotated in 14 KEGG secondary functional pathways,of which the first three categories with the largest numbers of metabolites include:amino acid metabolism（48 substances,41.38%）,biosynthesis and secondary metabolism（36 substances,accounting for 31.03%）,and lipid metabolism（24substances,accounting for 20.69%）.Sb（III）stress mainly affected the changes of the following metabolic pathways in rice leaves:galactose metabolic pathway,plant hormone signal transduction pathway,glutathione metabolic pathway,carbon sequestration of photosynthetic organisms,sphingolipid metabolic pathway,phenylalanine metabolic pathway,phenylpropane biosynthesis pathway and a variety of amino acid synthesis and metabolic pathways（glycine,serine,threonine,tryptophan,phenylalanine,arginine and proline）.Sb（V）stress mainly affected the following metabolic pathways in rice leaves:galactose metabolic pathway,plant hormone signal transduction pathway,glutathione metabolic pathway,flavone and flavonol biosynthesis pathway,phenylpropane biosynthesis pathway,linoleic acid metabolism pathway Phenylalanine metabolic pathway and various amino acid synthesis and metabolic pathways（glycine,serine,threonine,tryptophan,phenylalanine,arginine,cyano amino acids）.Flow directions of glycosyls.We screened 160 different metabolites（DMs）containing glycosyls.These 160 DMs were classified into carbohydrate and carbohydrate conjugates（38,29.23%）,flavonoid glycosides（28,21.54%）,aliphaloside（11;8.46%）and terpene indican（13.7%;17.8%）.Further,we carried out a KEGG enrichment analysis using these 160 DMs.The results showed that only 29DMs were enriched into different metabolic pathways,including 1)purine metabolism;2)pentose phosphate pathway;3)galactose metabolism pathway;4)amino sugar and nucleotide pathway;5)anthocyanin biosynthesis pathway;and 6)flavone and flavonol biosynthesis pathway.The glycosyl products of photosynthesis（especially Sb（III））under Sb stress may mainly flow into:（1）the synthesis pathway of antioxidant substances,such as anthocyanin,salicylic acid,flavonoids and flavonoid alcohols;（2）oligosaccharide synthesis pathways,such as sucrose,stachyose and sucralose;（3）synthesis pathway of cell wall components（especially lignin synthesis）and lipids.The results of this study can not only fill the knowledge gaps of the relevant research fields,but also provide theoretical supports for the development of technologies for the remediation of Sb contaminated soils in future.