| With the acceleration of industrialization and urbanization,the problem of soil heavy metal pollution has become increasingly prominent,and its impact on ecosystems,plant growth and human health has also attracted widespread attention.Rice(Oryza sativa L.)has a strong absorption of cadmium,which causes cadmium to enter the human body through the food chain and endanger human health.Plant metabolomics can study the changes of plant metabolites and their contents under different environmental conditions,explore the temporal and spatial accumulation patterns of plant metabolites,and provide a basis for further analysis of plant development and regulation mechanism in response to external environmental conditions.In order to understand the metabolites and major metabolic pathways of rice in response to cadmium stress,this study used the rice cultivar "Huanghuazhan" as experimental material,and performed targeted and non-targeted metabolomics studies of rice metabolites at seedling stage under cadmium stress by gas chromatography-mass spectrometry(GC-MS)and ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS),respectively.In addition,based on the preliminary work,high performance liquid chromatography(HPLC)combined with 11 compound standards were used to perform targeted metabolomics study on the changes of phenolic substances in response to cadmium stress during rice germination.The main findings are as follows:1.Cadmium stress caused yellowing of leaves and sheaths of rice seedlings,browning of roots,and height,root length,fresh weight and dry weight of rice seedlings decreased with the increase of cadmium stress concentration.The content of chlorophyll a,chlorophyll b and total chlorophyll of rice seedling increased first and then decreased with the increase of cadmium stress concentration.The contents of proline,soluble sugar and malondialdehyde increased,while the content of soluble protein decreased.The activity of superoxide dismutase(SOD)showed an increasing trend with the increase of cadmium stress concentration,and the activity of peroxidase(POD)increased significantly,while the activity of catalase(CAT)increased first and then decreased.2.A total of 31 primary metabolites of seedling rice,such as amino acids,organic acids and sugars were identified by GC-MS.Analysis of variance showed that L-serine decreased significantly with increasing cadmium stress concentration(p<0.05),L-threonine,4-aminobutyric acid,shikimic acid,citric acid,maltose,glucose and ?-Lactose increased significantly with the increase of cadmium stress concentration(p<0.05),and the other 23 metabolites increased first and then decreased.The heat map analysis showed that the content of most amino acids,organic acids and carbohydrates increased under low cadmium stress(50 ?M),the content of most the amino acids and organic decreased under the high concentration cadmium stress(500 ?M),but most of carbohydrates still showed an upward trend compared with the control.3.With the increase of cadmium stress concentration,the total phenolic content of seedling rice increased,the total phenolic content increased significantly under 500 ?M cadmium stress,and the total flavonoid content decreased first and then increased.UPLC-Q-TOF-MS was used to analyze the non-targeted metabolomics of rice metabolites under cadmium stress,and 34 metabolites were identified,including amino acids,organic acids,phenols,sugars and so on.Among them,except for L-phenylalanine,the contents of other amino acids were lower than those of the control group under cadmium stress,and the relative contents of leucine and aspartic acid were significantly lower in the cadmium stress group than in the control group(p <0.05).The content of 7 organic acids reached the maximum under high cadmium stress,and the contents of glyceric acid,malic acid,L-quinic acid and gluconic acid increased significantly(p<0.05).Except for caffeic acid,Caffeoylmalic acid,kaempferol and dihydromyricetin,the contents of the other eight phenolic increased with the increase of cadmium stress concentration,of which cinnamic acid,protocatechuic acid,ferulic acid and medicarpin increased significantly under high cadmium stress(p<0.05).The content of most carbohydrates increased with the increase of cadmium stress concentration,while the content of glycosides decreased.The results showed that amino acids,organic acids,phenols and sugars played an important role in the response of rice to cadmium stress,especially that the changes of phenolic substances involved in the phenylalanine metabolic pathway were more significant.4.Cadmium stress had inhibitory effects on the radicles and embryos of rice seeds,and the inhibitory effect on rice radicles was significantly greater than that on embryos.In the process of germination,the total content of 11 phenols increased significantly.With the germination process,low concentrations of cadmium(10 ?M)promoted the synthesis of protocatechuic acid,chlorogenic acid,caffeic acid and p-coumaric acid,and high concentrations of cadmium(500 ?M)significantly inhibited(p<0.05)the synthesis of rutin,resveratrol,cinnamic acid,quercetin and kaempferol.At the early stage of germination(2 days),rice seeds did not show any effects of cadmium toxicity,and the content of 6 phenolic compounds reached the highest under the treatment of the higher concentrations of cadmium(?200 ?M).However,rice seeds showed some degrees of cadmium toxicities in the middle(4 days)and late(6 days)stages of germination,the content of most phenols decreased with the increase of cadmium concentration,and rice seeds from the middle stage of germination was more sensitive than that from late stages.The results showed that cadmium stress had inhibitory effects on seed germination of rice,and the cadmium stress had a significant effect on the synthesis of phenolic compounds of rice.5.Metabolic analysis of rice compounds under the detected cadmium stress enriched these compounds into the biological pathways involved in the KEGG database,involving a total of 42 metabolic pathways.Among them,6 pathways including aminoacyl-tRNA biosynthesis,alanine-aspartate-glutamate metabolism,galactose metabolism,cyanogenic amino acid metabolism,nitrogen metabolism and glycine-serine-threonine metabolism were abundant in cadmium stress(p<0.05).These pathways were mainly involved in amino acid metabolism,organic acid metabolism and sugar metabolism processes,as well as involved in the phenylalanine metabolic pathway.In this paper,the metabolomic studies of rice under cadmium stress were carried out by GC-MS,UPLC-Q-TOF-MS and HPLC.A total of 67 metabolites were qualitatively analyzed and enriched in the corresponding metabolic pathways.The results indicated that under cadmium stress,rice might increase the content of metabolites involved in amino acid metabolism,organic acid metabolism and sugar metabolism,and then changed the glycolytic pathway,TCA and the phenylpropionic acid metabolism process derived from the shikimate pathway to improve self-resistant cadmium,but with the increase of cadmium stress concentration,rice might mainly improve cadmium resistance by increasing sugar content or synthesizing a large amount of phenolic substances to resist oxidative damage caused by cadmium poisoning. |
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