A Preliminary Study On The Effect And Its Mechanism Of Carotenoid Metabolism On Seed Germination In Rice

Seed germination,which is the beginning of the plant growth and development,directly affects the survival and reproduction of plants.Seed germination can be inhibited by abscisic acid(ABA),and its main biosynthetic pathway is the carotenoid metabolism.In this study,wild type rice(WT)and golden rice(GR)were selected as experimental materials to explore the influence of carotenoid metabolism on rice seed germination from aspects of the characteristics of seed germination,the changes of endogenous hormones contents,carotenoid metabolism,and the analysis of transcriptome sequencing.This study will provide a theoretical basis for revealing the influence of carotenoid metabolism for seed germination in the future.The main research results are as follows:1.The results of seed germination test showed that both the seeds of two varieties were germinated after 48 h,and the germination of WT were significantly faster than GR.The germination vigor and the germination index(length of embryo and radicle,fresh weight)of WT were also significantly higher than that of GR.The analysis of water absorption showed that the water absorption of GR and WT was at the rapid stage before 12 h,and then turn to a slow water absorption stage between 12 h and 48 h.After 48 h,water absorption of WT was faster than that of GR,which may be caused by the extension of radicle in WT seeds.2.The main nutrient contents in seeds of WT and GR were determined and analyzed.It was found that the contents of moisture,starch,amylose and free fatty acid were significantly lower in GR seeds than WT seeds,but the contents of fat and protein were significantly higher in GR.The b* value and the total carotenoid contents of GR seeds were significant higher than WT,which may affect the accumulation of ABA in the process of seed germination.The over accumulation of ABA in GR seeds could inhibit its germination,leading to slower germination in GR than WT.3.The contents of abscisic acid(ABA),gibberellin(GA)and auxin(IAA)at three time points(12 h,24 h,48 h)during seed germination were determined by high performance liquid chromatography.The results showed that during seed germinating,the ABA contents in two varieties were all decreasing first and then rising,and the ABA contents in GR were higher than WT during the whole stages of seed germinating.The GA3 contents in seeds of GR showed an increasing trend overall,while that in WT showed a decreasing trend.Especially at 12 h of germination,the contents of GA3 in GR was significantly lower than WT,and the ratio of GA3 to ABA was also lower.GA3 can promote the germination of seeds,and when the ratio of GA3 to ABA is low,it can delay the germination of seeds.Therefore,it is speculated that this may one reason for slow germination in GR.The contents of ABA in GR showed an increasing trend in general,while it showed a decreasing trend in WT.These may also affect the germination of GR.4.The carotenoid composition and its contents in seeds of two varieties at three time points during the germination were qualitatively and quantitatively analyzed by liquid chromatography tandem mass spectrometry(LC-MC/MS).A total of 13 carotenoids were detected in GR during seed germination,and the main components were phytoene,lutein,zeaxanthin,β-carotene,α-carotene,α-cryptoxanthin,β-cryptoxanthin,and neoxanthin.A total of 8 carotenoids were detected in WT during seed germination,and the main components were phytoene,lutein,antheraxanthin,neoxanthin and violaxanthin.It is worthy that the contents of phytoene,lycopene,β-carotene,zeaxanthin,antheraxanthin and violaxanthin in GR were significantly higher than in WT,and all showed a trend of decreasing first and then increasing,which was consistent with the trend of ABA contents.5.The differentially expressed genes of the transcriptome data in seeds of two varieties at three time points during the germination were analyzed,28 candidate differentially expressed genes were selected,of which,there were 6 differentially expressed genes related to carotenoid metabolism,5 differentially expressed genes related to ABA,8 differentially expressed genes related to IAA,and 5 differentially expressed genes related to GA.6.The expression patterns of 8 differentially expressed candidate genes during seed germination of WT and GR were analyzed by real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR).It was found that their expression trend was consistent with the results in the transcriptome,and the transcriptome sequencing data was highly correlated with the fluorescence quantitative expression results.In conclusion,during seed germination in rice,the ABA content of GR with rich carotenoids was higher than WT,and the content changes of components related to carotenoid metabolism in GR were consistent with the content changes of ABA.In addition,at 48 h of germination,the contents of components related to carotenoid metabolism were increased in GR seeds,and the increase of violaxanthin was more significant.Moreover,the expression levels of differentially expressed genes Os NCED5,Os NCED3 and Os NCED4 in GR were all up-regulated,which leaded to the increase of ABA content.These results suggested that GR may increased the accumulation of ABA through the carotenoid metabolism pathway,thus inhibited its seed germination,and leaded to its slower germination than WT.