Analysis Of Metabolic During Seed Germination And Post-germination Of Astragalus Mongholicus

Astragalus mongholicus is a perennial herb belonging to legumes. Radix Astragali is prepared from the dried roots of Astragalus membranaceus Bge. var. mongholicus Hsiao or Astragalus membranaceus (Fisch.) Bge. Currently,the study of A.mongholicus mainly focused on ecology, breeding and pharmacology. But little is known about the metabolism during seed germination stage of Astragalus mongholicus. Seed germination is the transition of the quiescent embryo into a new photosynthetically active plant. Germination is a complex physiological and biochemical process during imbibed mature seed to seedlings in a rapid conversion and prepare for seedling growth. Because of its susceptibility to injury, disease and so on, germination is considered to be the most critical phase in the plant life cycle. Thus, It is one important issues of biochemical plant growth and quality of herbs to study biochemical and metabolism of seed germination stage. Analysis the variation and accumulation mode of metabolite during seed germination stage using metabolomics, provides a theoretical basis for the research of the seed germination and regulation of metabolic flux. Plant metabolomics is a non biased and essentially comprehensive high throughput analysis of complex metabolite mixtures which are typical part of plant extracts. Metabolic fingerprinting is an important part of plant metabolomics research. To investigate the developmental specificity accumulation of Astragalus active metabolite and differences in metabolic profile, we did study on Astragalus mongholicus from 4 aspects using ultra-pressure liquid chromatography with tandem mass spectrometry(UPLC-MS) and ultra performance liquid chromatography coupled with electrospray ionization quadrupole/time of flight mass spectrometry (UPLC-ESI-Q/TOF-MS) based phytometabonomics.(1) By studying the morphology of seed germination, seed germination stage is defined: A.mongholicus:from the imbibition day to the third day is the initial seed germination period, from third day to seventh day is the seed post-germination period. A.membranaceus:from the imbibition day to the fourth day is the initial seed germination period, from third day to eighth day is the seed post-germination period.(2) Established UPLC-MS method for simultaneous quantitative detection calycosin-7-O-β-D-glucoside, calycosin, formononetin and scutellarin. Chromatographic conditions:8% acetonitrile (0-1 min),8% -34% acetonitrile (1-1.5 min),34% acetonitrile (1.5-4min),34%-60% acetonitrile (4-6 min),60% acetonitrile (6-7 min),60% -8% acetonitrile (7-7.5 min),8% acetonitrile (7.5-9 min). MS conditions:calycosin-7-O-β-D-glucoside m/z 446.9â†'284.2; calycosin m/z 285.0â†'269.9; Formononetin m/z 269.0â†'196.2; Scutellarin (IS) m/z 463.0â†' 287.0. The chromatographic retention time of calycosin-7-O-β-D-glucoside, calycosin, formononetin and scutellarin(IS) was 2.23min,2.80min,4.75min and 2.06min respectively. The results showed the target compounds have changes in the content of both the initial seed germination period and the seed post-germination period. Combined with the results of morphological study of seed germination, A.membranaceus and the expression levels of related enzymes help prove that accumulation of flavonoids secondary metabolites regulated by seed growth and development.(3) In this research, a method of metabolic fingerprints was established for the medicinal plant A. mongholicus. An ultra performance liquid chromatography coupled with electrospray ionization quadrupole/time of flight mass spectrometry (UPLC-ESI-Q/TOF MS) was employed. Base on the non-targeted metabolomics MS/MS spectrum and data from the literatures, ten metabolic compounds were identified in positive ion, including four flavonoids, three fatty acids, one amino acid, one glycoside, one saponin and one other metabolite. To investigate the accumulation of biomarkers during germination, samples from two different groups were collected, including seed imbibition and seed germination. Visualization of the 10 biomarkers profile was performed by hierarchical cluster analysis (HCA). Accumulation of biomarkers displayed a clear variation in terms of their abundance in different stage. Compared with germination, calycosin increase up 2 times in imbibition stage. Surprisingly, the accumulation of ononin was 158 times. These phenomena suggest that a variety of metabolites jointly regulate from seed imbibition to germination stage of A. mongholicus. Amino acids, fatty acids and glycosides were mainly storage in seed imbibition period to provide energy. However, saponins compound was reduced in seed germination, this were caused by reason that the synthetic pathway of certain key metabolites was impeded or some other synthesis inhibitors lead to synthesis reduced.By targeting metabolomics technology we identified eight synthetic precursors of flavonoids and 13 saponins synthetic precursors, and was drawn a metabolites-to-gene network to further understanding of precursor compounds change in the early seed germination. Through the content of metabolites change in the initial seed germination, suggesting that the main impact of the synthesis of flavonoids precursor compounds were p-Coumaroyl-CoA and liquiritigenin. Therefore, by increasing the amount of these two synthetic precursor compounds to achieve the accumulation of flavonoids, which protect seed germination and environment adapt. Moreover, four saponins synthetic precursors showed significant accumulation in initial seed germination, including squalene, cycloartenol, farnesyl diphosphate and mevalonic acid. Perhaps it was the accumulation of these metabolites to inhibit the synthesis of other metabolits, thus affecting the saponins (Astramembranoside A) synthesis.(4) An integrated and tegrated approach of UPLC/Q-TOF coupled with multivariate statistical analysis was developed to discriminate the lipid profiles between different development groups and discover the lipid biomarkers in response to various development stages. Pattern recognition by OSC-PLS showed that samples between different groups could be separated by their different lipid profiles. Fifteen lipid biomarkers were selected, which contains amino acid, flavonoids, hormone, fatty acid and other metabolites. This implied that these biomarkers were mainly accumulated in the period after the completion of seed germination, for seedling form completion and provide energy basis to plant growth. In addition, the flavonoids as significant biomarker, which may indicate that the content of flavonoids gradual change with the plant growth and development, suggesting that they were a key role for development and growth of plants.For the period after seed germination the flavonoids metabolic regulatory network was constructed. It implied that mainly affected flavonoids synthesis two major precursor compounds were coumaroyl-CoA and isoliquiritigenin. Interestingly, the content of Isoliquiritigenin continued to increase after seed germination, which mean that coumaroyl-CoA with Isoliquiritigenin have a positively correlation. In addition, the sapnonis metabolic netawork show that significantly accumulated wherein the compound (R)-5-Diphosphomevalonate and isopentenyl diphosphate during after seed germination stage. Finally, seed germination and initial period of gene-to-metabolites network combined, we could get such a conclusion, in the entire development process of A. mongholicus, the same metabolites have presented different variation in different stages, This inconsistency was caused by the relevant synthetic pathway gene expression levels change during the plant development stage.