Study On Differences Of Metabolites In Rice Under Cadmium Stress Conditions

Rice is the most important cereal crop capable of absorbing Cd.Cd enters rice through the root-xylem-phloem-grain transportation route and enters the human body through the food chain,causing some harm to human health.Therefore,understanding the molecular mechanism of low-Cd rice varieties can reduce the content of Cd in rice and help solve the problem of rice quality and safety in China.Based on the above issues,this study eatablished a method for the analysis of metabolic profiles of rice leaves based on hydrophilic interaction chromatography and reversed-phase chromatography quadrupole-time of flight mass spectrometry.The ecological indicators of low-Cd rice mutants and wild-type were determined through hydroponic experiments,and screened out the phenotypic differences of Cd concentration and treatment time.Differential metabolites were identified using metabolomics techniques,and metabolic pathways involved in differential metabolites were summol/Larized,hoping to provide theoretical evidence for further studies on the mechanism of low Cd accumulation in rice varieties.The followings are main results:(1)This study is aiming to establish the metabolic analysis method of rice based on RPLC and HILIC combined with Q-TOF/MS by comparing the influences of different extraction methods in rice metabolites.The extraction and separation of rice leaf metabolites using three different methods including methanol-chloroform-water,methanol-chloroform-ammol/Lonia,methanol-MTBE-water and different chromatographic systems were compared by the numbers of peaks,identified metabolites and the metabolic pathways.The results showed that the method of methanol-chloroform-water reached the highest coverage rate of metabolites in rice leaves,and the maximum number of unique metabolites including prephenic acid,luteolin,?-linolenic acid,aconitic acid,gibberellin A12 aldehyde,isovitexin,L-Glutamate were detected.Metabolites with different polarity in rice leaf could be detected by using HSS T3 and XBridge Amide.A total of 16 kinds of organic acids,17 kinds of nucleotides,21 kinds of amino acids,66 kinds of fatty acids,11 kinds of phospholipids and 7 kinds of sphingolipids were identified.XBridge Amide had an absolute advantage in detecting phospholipids and sphingolipids.The metabolic pathways involved purine metabolism,pyrimidine metabolism,tricarboxylic acid cycle,arginine metabolism,fatty acid metabolism,phospholipid metabolism,sphingolipid metabolism,phenylalanine metabolism and vitamin B2 synthesis.It showed certain complementarity between the two columns in identifying metabolites and involved the metabolic pathways.The established method is expected to be useful for the metabolomic studies of rice.(2)Low-Cd rice mutants and wild-type were used as experimental materials for hydroponic experiments.Cd concentration gradients of 0,0.1,and 5 ?mol/L were set,and dynamic sampling was performed for 2 h,1 d,3 d,7 d,and 15 d.Effect of Cd concentration and sampling time on rice growth.The root length,plant height,root dry weight and shoot dry weight of low Cd rice mutants and wild-type all increased with the prolongation of sampling time;when the Cd concentration(0.1 ?mol/L and 5 ?mol/L)was added,the growth of low-Cd rice mutants was not inhibited.The growth of the corresponding wild type was inhibited to varying degrees with the increase of Cd concentration.The wild type was not significantly inhibited compared with CK under 0.1 ?mol/L.The growth of the wild type was significantly inhibited under 5?mol/L.There was no significant difference in root length,plant height,root dry weight and shoot dry weight between low Cd rice mutants and wild type at different sampling times under 0.1 ?mol/L.After 5 ?mol/L Cd concentration treatment for 15 days,low Cd rice mutants and wild type showed significant differences;when Cd concentration(0.1 ?mol/L and 5 ?mol/L)was added,The Cd content in roots and shoots was significantly lower than that wild-type in different sampling time,probably some compounds may be resistant to Cd stress.(3)Metabolic analysis of rice phenotypes using low-Cd rice mutants and wild-types treated with 5 ?mol/L Cd for 15 d using a rice metabolite analysis method based on hydrophilic interaction chromatography and reversed-phase chromatography quadrupole time-of-flight mass spectrometry.The results showed that there were 25 differential metabolites in the roots of the low Cd rice mutants and the wild type,amino acids such as alanine,aminobutyric acid,valine,aspartic acid,valine,lipids such as oleic acid,phytosphingosine,citric acid,PG(20:5(5Z,8Z,11Z)(14Z,17Z)/0:0),PA(19:3(10Z,13 Z,16Z)/0:0),PA(18:2(9Z,12Z)/0:0),which showed greater resistance to heavy metal Cd on plants;A total of 22 differential metabolites in shoots,organic acids such as malic acid,citric acid,fumaric acid,vitamin Bs such as riboflavin(vitamin B2),pyridoxal(vitamin B6)and pantothenic acid,amino acids such as hydroxyproline significant changed,but other amino acids need further research and exploration.The metabolic pathways of amino acid metabolism,organic acid metabolism,vitamin B family synthesis,lipid metabolism,and the tricarboxylic acid cycle may be involved in the differential metabolites of low-Cd rice mutants and wild-type.The strong Cd resistance of low Cd rice mutants was more related to these altered metabolic pathways.