| Rice(Oryza sativa L.)belongs to the Oryza of Poaceae in herbaceous plant.It is one of the oldest and most important crops in the world.It is widely distributed all over the world and is the staple food crop for more than half of the world’s population.Most varieties of rice are very sensitive to low temperature.In particular,chilling damage at the seedling stage can cause leaf water loss,chlorosis,involution,and even plant death,which seriously affects rice yield.It is of positive scientific significance to study the metabolic changes and cold resistance mechanisms of rice seedlings in low temperature environments.In this study,two rice varieties,Qiutianxiaoting(japonica rice)and 93-11(indica rice)were used as experimental materials.Seedlings cultivated for 15 days(3 leaves and 1 heart stage)were treated with 4℃ cold stress for 0,12 and 36 h,respectively,and recovered for 48 h.Based on the comprehensive analysis of the phenotypic changes after cold stress,the metabolic regulation mechanism of rice seedlings in response to cold stress was analyzed based on metabolomics and transcriptomics.The main results are as follows.(1)Low temperature conditions significantly affected the phenotypic changes of Qiutianxiaoting and 93-11.As the stress time increased,Qiutianxiaoting was able to maintain the phenotypic stability of the relative conductivity,leaf color and shape,which had higher antioxidant enzyme activity and soluble protein content,but the growth indicators showed a downward trend,such as fresh weight,dry weight,shoot length and root length.The growth index took on an increasing trend in 93-11,such as relative conductivity,total phenols,total flavonoids,total chlorophyll and total anthocyanins,while chlorosis and involution of leaves occurred.At the recovery stage,Qiutianxiaoting was able to maintain a stable life activity,while 93-11 suffered a large number of deaths.Results showed that Qiutianxiaoting has a high tolerance to low temperature,while 93-11 is more sensitive.(2)Targeted metabolomics analysis of rice seedlings was performed by high performance liquid chromatography(HPLC)using phenolic acid standard compounds.Results showed that 10 phenolic acids were detected in the two varieties,such as gallic acid,protocatechuic acid,chlorogenic acid,caffeic acid,p-coumaric acid,rutin,ferulic acid,salicylic acid,cinnamic acid and kaempferol.The concentrations of total phenolic compounds decreased first in Qiutianxiaoting after chilling stress,then returned to normal levels when recovered for 48 h.Whereas the corresponding levels in 93-11 showed an upward trend as chilling time increased,reaching the highest level at 36 h.The concentrations of gallic acid,protocatechuic acid,and p-coumaric acid in Qiutianxiaoting increased first then decreased,and levels of chlorogenic acid and caffeic acid decreased first then increased,and levels of rutin,ferulic acid,and salicylic acid increased slightly.The major phenolic acids in 93-11 are gallic acid and caffeic acid,whose concentration reached the maximum value at 36 h.Levels of protocatechuic acid and kaempferol decreased first then increased after chilling treatment.The levels of phenolic compounds with a lower concentration decreased and changed little during chilling stress,namely chlorogenic acid,p-coumaric acid and cinnamic.Levels of ferulic acid and salicylic acid increased slightly after cold stress.(3)Non-targeted metabolomics analysis of rice seedlings was performed by UPLC-Q-TOF-MS using the Progeneis QI data processing platform.Results showed that the differential metabolites in Qiutianxiaoting and 93-11 were mainly amino acids,lipids,organic acids and carbohydrates,etc.During the recovery period,large-scale amino acid accumulated in 93-11.These results showed that amino acids could be considered as important markers of chilling injury.PCA and OPLS-DA all showed that the metabolome of the two rice varieties changed under cold stress.The metabolome changed little in Qiutianxiaoting,while it changed greatly in 93-11,especially during the recovery period.(4)Metabolomics analysis was performed of two rice varieties under cold stress by GC-MS combined with derivatization.The identified metabolites mainly belong to many types,such as carbohydrates,amino acids,fatty acids,nucleotides,organic acids and alcohols.As chilling time increased,the number of metabolites in Qiutianxiaoting increased first then decreased,and it returned to normal levels at the recovery period;while 93-11 took on an upward trend and it increased rapidly when returned to normal growth.Heat map analysis showed that the metabolites of the two rice varieties changed significantly after cold treatment.Levels of metabolites in Qiutianxiaoting had changed drastically and could return to normal levels during the recovery period;while levels of metabolites in 93-11 gradually increased,especially during the recovery period.PCA and PLS-DA analysis all showed that metabolome in Qiutianxiaoting had a little change during chilling treatment;the metabolome changed little in 93-11 during the chilling period,however,it had a great change during the recovery period.The analysis of VIP value based on PLS-DA showed that the different metabolites were mainly carbohydrates and amino acids.(5)Electrospray extraction ionization mass spectrometry(EESI-MS)technology was used to carry out non-targeted metabolomics analysis in rice seedlings,which was used to obtain the primary and secondary fingerprint data of O.sativa samples.In total,34 compounds were identified including 13 phenolic acids,11 amino acids,3polyamines,1 alkaloid,1 vitamin,2 hormones,and 3 other compounds.The contents of methyl jasmonate(Me JA)had an increasing trend in Qiutianxiaoting,and RT-PCR showed that the level of Me JA biosynthesis-related gene expression improved significantly during chilling treatment.The contents of amino acids and phenolic acids in 93-11 showed an increasing trend.The contents of polyamines(triethanolamine and tryptamine)and betaine in the two varieties of rice increased slightly.PCA and OPLSDA analysis all showed that Qiutianxiaoting,not 93-11,could be separated with each other under chilling stress.(6)The change of transcription level was analyzed in the two rice varieties under low temperature stress,using the Illumina platform to perform transcriptome sequencing of samples,combined with bioinformatics methods including multivariate analysis of differentially expressed genes and enrichment analysis.Under low temperature conditions,the number of differentially expressed genes in Qiutianxiaoting increased first then decreased,and returned to normal levels during the recovery period;however,it showed an upward trend in 93-11,especially during the recovery period.PCA analysis showed that the transcriptome of Qiutianxiaoting returned to normal levels with the duration of cold stress,however,the difference of transcript levels in93-11 increased greatly during the recovery period.GO and KEGG enrichment analysis all showed that the significantly enriched pathways in Qiutianxiaoting are mainly carbohydrate metabolism,amino acid metabolism and antioxidant metabolism,while significantly enriched pathway in 93-11 contains a large amount of photosynthesisrelated metabolism.(7)Based on the phenotypic changes of rice under low temperature conditions,metabolomics and transcriptomics are combined to analyze the metabolic response mechanism in rice seedlings.Phenotypic analysis showed that Qiutianxiaoting could maintain the stability of phenotype and metabolic level under cold stress,while its growth index showed a downward trend,which took on an increasing trend in 93-11,but its life system collapsed finally.Metabolomics analysis showed that the significantly enrichment pathways in Qiutianxiaoting mainly included energy metabolism and antioxidant metabolism,while antioxidant-related metabolism was significantly enrichment in 93-11.To a certain extent,the changes of metabolites might be used to explain the reason for the higher cold tolerance of Qiutianxiaoting than 93-11.Transcriptome analysis showed that carbohydrate metabolism,amino acid metabolism and antioxidant metabolism were significantly enriched in Qiutianxiaoting,while photosynthesis-related metabolism was significantly enriched in 93-11.In order to maintain the stability of the metabolome and transcriptome under low temperature,energy metabolism might be used in Qiutianxiaoting to produce ATP,and amino acid metabolism was used to maintain the stability of the internal environment,and antioxidant-related metabolism was utilized for removing excess reactive oxygen species;while 93-11 continued to grow and develop under chilling stress,which might cause disorders of the metabolome and transcriptome,and caused a large number of deaths.Based on systematic metabolome and transcriptome data,this research reveals the different chilling response mechanism in two rice varieties.Qiutianxiaoting stopped growth and development,and it could maintain the stability of the life system through energy metabolism,amino acid metabolism and antioxidant-related metabolism,which achieved the stability of the metabolome and the recovery of the transcriptome at the recovery period.Photosynthesis-related metabolism in 93-11 was activated to continue growth and development,and amino acid metabolism and antioxidant-related metabolism were used to resist cold stress,but a large number of deaths occurred during recovery growth.The results could lay the foundation for the selection and breeding of rice varieties,and provide references for the research on cold-resistant mechanisms of other plants. |
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