| Epimedium wushanense is a popular Chinese medicinal herb belongs to the family Berberidaceae. In this study, we introduced E. wushanense seed as experimental material and investigated the germination and embryo development behaviors responding to fluridone, gibberellin acid and temperature. we also surveyed the fatty acid metabolism and plant hormone homostatis under different stratification regimes. Based on the dormancy characteristic of E. wushanense we summarized, we not only established an efficient method for the seedling propagation, but elucidated the underlying mechanisms of dormancy in E. wushanense seed by inspecting the differentially expressed genes and proteins during physiological dormancy and/or morphological dormancy releasing with RNA-Seq and iTRAQ techniques. The main results are as follows:(1) E. wushanense seed has non-deep complex morphophysiological dormancy, of which physiological dormancy will be released at 20/10℃ stratification and morphological dormancy would be released at 4℃ stratification. There exist endogenous inhibitors in seed, whose systematically extracts with different organic solvents showed different inhibiting effects, as to root, the effect shows acetone>methanoll>water> ethyl acetate, as to shoot, the effect shows acetone> ethyl acetate >hexane>methanol.(2) We ascertained combination of 2.5 mg·L-1 fluridone and 400 mg·L-1 gibberellin acid is the most efficient combination not only in promoting dormancy releasing but in generating normal seedlings. As to seed at 20/10℃ stratification regime for 90 d, germination rate, germination energy and germination index increased by 33.7%,431.1% and 58.1% respectively, and mean germination time decreased by 13.6%. In addition, germination rate, germination energy, germination index increased by 103.3%,1675.0%,25.0% respectively, and mean germination time decreased by 19.2% in seed have experienced 150 d of 10/20℃ stratification.(3) Warm stratification at 10/20℃ accelerated the fatty acid metabolism in the subsequent cold stratification at 4℃. After 90 d of cold stratification, the oil content decreased by 1.9% and 2.8% respectively in seeds have been warm stratificationed for 0 d and 120 d respectively. Moreover, fluridone is capable of accelerating fatty acid metablolism too, oil content in seed pretreated with 3.0 mg·L-1,5.0 mg·L-1, 10mg·L-1 fluridone decreased by 3.0%,2.1% and 5.4% respectively.(4) 25.30 Gb clean data were generated by sequencing with Illumina Hiseq 2500 platform, after being processed,79107 non-redundant unigenes were achieved, with an average length of 644.25 bp. 32384 of those unigenes were annotated by NR, Swiss-Prot, Pfam, KOG, COG, GO and KEGG databases. Additionally, analysis of GO and KEGG shows the differentially expressed genes between physiological dormancy releasing and morphological dormancy releasing have similar enrichment behavior both in GO functions and KEEG pathways, however the expression pattern vanes.(5) Proteome analysis with iTRAQ from mophophysiological dormant seed, morphological dormant seed, non-dormant seed generated 558019 spectrums and derived 550 proteins after searching against the protein database builted with RNA-Seq data. Among the proteins,369 and 338 proteins were identified as differentially expressed during physiological dormancy releasing and morphological dormancy releasing respectively.(6) During physiological dormancy releasing, expression of 6-phosphogluconate dehydrogenase, key enzymes in pentose phosphate pathway, has been significantly up-regulated, enzymes in mitochondria and endoplasmic reticulum associated with fatty acid metabolism were up-regulated while enzymes in cytoplasm associated with fatty acid biosynthesis were un-changed. During morphological dormancy breaking, pectinesterase and 6-phosphogluconate dehydrogenase were significantly down-regulated may lead to a lower transformation efficiency from UDPG to pectin or pectin to pectate, genes associating with fatty acid metabolism in mitochondria and endoplasmic reticulum were down regulated while acetyl-CoA carboxylase in cytoplasm was up-regulated, indicating an activation in fatty acid biosynthesis rather than the metabolism. |
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