| Seed germination initiates with imbibition and terminates with radicle breaking through testa or endosperm.1/2of crop food supply and association with plant survive lead to seed germination as the hottest topics in plant physiology research. To investigate the metabonome change and effect of afterripening to short-afterripening-period seeds during germination without light, we did quantitative study on mungbean (Vigna radiata) from3aspects using nuclear magnetic resonance (NMR) and hyphenated reversed phase liquid chromatography-diode array detector-mass spectrum (RPLC-DAD-MS) based phytometabonomics.Firstly, we optimized the method for aqueous phytometabonome extraction and quantitative NMR research. The rusults showed that:(1)50%methanol was the best among the six solvents used in our research;(2)3times extraction when tissue-to-solvent ratio between1:10to1:15(mg:μL) were sufficient;(3) Combination of tissuelyser homogenization and ultrasonication will break plant cells more efficiently;(4) Appropriate extract concentration was around6.7mg·mL-1for NMR analysis;(5) Complete relaxed spectra were better for quantitative NMR analysis.Secondly, we established a rapid and sensitive quantiative method based on RPLC-DAD-MS for aromatic carboxylic acids (ACAs) involved in shikimate metabolism pathway, and provided exhaustive information about MS fragmentation, ultraviolet absorptive wavelengths λ and molar absorptive coefficient ε for them. The results showed that:(1) Proper usage of acetonitrile as postcolumn derivation regent will narrow half peak width and ehance signal-to-noise ratio (SNR) of MS signal;(2) This approach showed good linearities, enhanced sensitivity, high repeatability and reliable accuracy.(3) With the method, we detected12carboxylic acids associated with shikimate metabolism, and quantified2/3of them in rice stem extracts after fed by brown planthopper for4days and the controls. Their changes were consistent with the results derivated from references, which prove the feasibility of our method in practical application.Thirdly, we investigated the metabonome variation during fresh and afterripening mungbean (Elii No.4) germiantion under dark.(1) During fresh mungbean germination:(A) Testa colour fades from black to green slowly; seeds germination began at9hours; After imbibition for18hours, the germination rate has been as high as90%, the moisture reached65%, grain wet weight achieved2.5times of seed weight, but seed dry weight changed less obviously.(B) Seed germination contained5phases:imbibition preparation (phase I,0to3hours, imbibed slowly), early germination (phase II,3to9hours, imbibed fast), medium germination (phase III,9to14hours, imbibed slowly), germination preparation (phase Ⅳ,14to16hours, imbibed fast) and postgermination (phase V,16hours later, imbibed fast). During phas I, few metabolisms initiated. During phase Ⅱ, metabolites related to anaerobic respiration like lactate, ethanol, alanine and y-aminobutyrate (GABA) increased; Tartarate associated with antioxydant dropped; Nicotinamide involved in electron transport chain, sucrose, monosaccharides and raffinose family oligosaccharides (RFOS) connected with carbohydrate metabolism, ACAs involved in SMP and amino acids varied notably; Nucleotide synthesized showing as uridine decrease and uridine monophosphate (UMP) and cytidine monophosphate (CMP) increase; Water introduction induced inositols and cholines in seed extracts and culture medium dramatical change to maintain the osmotic balance. During phase Ⅲ, metabolisms in mungbean cells tended to be slow characterized by the decrease of metabolites change rate; Cells reabsorbed the small moleculars from culture medium. During phase Ⅳ, rapid imbibition was observed again, all metabolites’concentraion in culture medium arrived highest. During phase V, second reabsorption of metabolites from culture medium to cell happened; Metabolites involved in carbohydrates, amino acids, cholines, TCA cycle and uracil metabolism altered remarkablely.(C) Urethane, asparate, GABA, alanine,2-amino-4-oxopentanate and tryptophan played important roles during fresh mungbean germination, and the process may involve glycolic acid metabolism.(2) Comparing with germinated seeds, ungerminated seeds showed less metabolites and slower change in culture medium; RFOS degradation, nucleotides, cholines, amino acids, TCA cycle, SMP and formic acid metabolism were disordered.(3) Afterripening affects mungbean germination observably:(A) Afterripening accelarated the germination and made seeds more synchronization, showing as quicker achieving maximum germination rate and smaller standard deviation of germination rate and moisture.(B) Afterripening changed the concentration of urethane, O-methyl.scyllo-inositol, choline, N1-methyl nicotinate, uracil, inositol, asparate and methionine.(C) Metabolites in afterripening seed extracts and culture medium changed similar but much quicker than fresh mungbean during germination. And compared with fresh mungbean, aerobic respiration was more active in cells of afterripening seeds while anaerobic respiration was less active. |
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