Study On The Physiological And Biochemical Changes And Molecular Mechanisms In Cercis Chinensis Seeds During Dormancy Releasing

Cercis chinensis Bunge is an important landscape plant with great economic and ecological value,it also has important medicinal uses.C.chinensis is propagated mostly from seeds,but the strong seed dormancy is a major restriction to its propagation.In this study,we systematically studied the mechanisms of C.chinensis seeds by investigating the dynamic changes of storage materials,endogenous inhibitors,plant hormones and key enzyme activities of respiratory metabolism during seed dormancy releasing.In order to understanding the molecular mechanisms of switches from dormancy to germination in C.chinensis seeds,we preformed transcriptome and proteome analyses among dormant seeds as control and dormancy-released seeds treated by cold stratification.These results would provide us with a thorough understanding of the specific mechanisms that led to the seed dormancy releasing.The main results were as follows:1.The content of soluble sugar decreased slightly at the beginning of stratification,then increased as the time prolongs,but after radicle breaking though testa,the content of soluble sugar decreased again.The content of soluble protein showed a"low-high-low"curve.The activity of amylase increased gradually with the extension of stratification time,and the content of fat and starch decreased gradually.These results showed that the macromolecular storage materials were degraded and transformed into soluble small molecular substances,which provided material and energy for the growth and development of embryos during seeds dormancy releasing.2.The activities of G6PDH and MDH increased gradually with the extension of stratification time,while the activity of GPI increased at the beginning of stratification(15 d),then decreased as the stratification time prolongs.The results showed that the enzyme activity of EMP pathway decreased and the enzyme activity of TCA and PPP pathways increased during dormancy releasing eventually led to the breakage of C.chinensis seeds.3.The inhibitory activities of extracts from seed coat and endosperm at 0 d,15 d,30 d,45 d and 60 d were examined.The germination rate of Chinese cabbage seeds treated with the extracts of seed coat and endosperm of C.chinensis were only 17.3%and 37.2%,significantly lower than the control(91.3%),indicating that some strong inhibitory ingredients existed in the seed coat and endosperm of C.chinensis seeds.By GC-MS analysis,the endosperm and seed coat of C.chinensis were mainly composed of acids,esters,phenols and alcohols.By testing the 50%inhibitory concentration(IC₅₀)and quantitative analysis by GC-MS/HPLC,we found that the content of pyrogallol in seed coat decreased gradually with the extension of stratification time,and the content of pyrogallol significantly lower than the IC₅₀ when the seeds germinated.Therefore,we cinfirmed that pyrogallol is an endogenous inhibitor in C.chinensis seeds.4.The content of ABA in C.chinensis seeds decreased gradually with the extension of stratification time,while the contents of GA,IAA and JA increased gradually.The ratios of GA/ABA,IAA/ABA,JA/ABA and(GA+IAA)/ABA increased gradually during dormancy releasing.These results showed that ABA inhibited the dormancy releasing and germination of C.chinensis seeds,while GA,IAA,JA and ABA had antagonistic effects and promoted seed dormancy releasing and germination.5.The dormant seeds(stratified for 0 d)and dormancy-released seeds(stratified for 45 d)were sequenced using high throughput sequencing technology Illumina Hiseq TM 2000 platform.209,359 transcripts with larger than 200 bp and 166,087 unigenes were obtained.By comparative transcriptomics,a total of 54,970 differentially expressed genes with at least 2-fold change were identified,among which 38,316 unigenes were up regulated and 16,654 unigenes were down regulated.Based on the results of GO function and KEGG pathway enrichment analysis of these DEGs,we found that the dormancy release of C.chinensis seeds is mainly related to significant changes of DEGs,such as GA20ox3,GID,DELLA,NCED6,CYP701,AUX1 and TIR1 genes in the plant hormone signal transduction pathway,the key enzyme like G6PDH,MDH and GPI in starch and sucrose metabolism and glycolysis pathway.6.By label-free protein sequencing,we obtained 1,031 differentially expressed proteins(DEPs)with at least 2-fold change from dormant seeds(stratification 0 d)and dormancy-released seeds(stratification 45 d).Among them,779 proteins were up regulated and 252 were down regulated.Based on the results of GO function and KEGG pathway enrichment analysis of these DEPs,we found that the dormancy release of C.chinensis seeds is mainly related to splicing,ribosome,starch and sucrose metabolism,protein process in endoplasmic reticulum,glycolysis/gluconeogenesis,amino sugar and nucleotide sugar metabolism process.7.In this study,the data of transcriptome and proteome of C.chinensis seeds were comprehensively analyzed.The results showed that the dormancy breakage of C.chinensis seeds was mainly related to phenylpropanoid biosynthesis,cysteine and methionine metabolism,glycolysis/gluconeogenesis,protein processing of endoplasmic reticulum,oxidative phosphorylation,pyruvate metabolism,?-linolenic acid metabolism,vitamin B6 metabolism and flavonoids significant changes in genes involved in biosynthesis,galactose metabolism,citric acid cycle(TCA cycle),alanine,aspartic acid and glutamate metabolism,pentose phosphate pathway and glucosinolates biosynthesis.