Effects Of Light On Quality Of Tartary Buckwheat Sprouts And Anthocyanin Biosynthesis

The buckwheat is a dicotyledonous plant of Fagopyrum of Polygonaceae,which is a minor coarse cereal crop with homology of medicine and food in the southwest of China.It has high nutritional and health-care value.Compared with the buckwheat grains,the buckwheat sprouts have richer nutrient substance types and qualities,the sprouts have different morphological characteristics under the light condition and the dark condition,the light can obviously affect the nutrient substance content of the buckwheat sprouts and the synthesis of anthocyanins.But the nutrients of Tartary buckwheat sprouts under different light conditions have not been deeply studied,and the synthetic pathway of anthocyanin in Tartary buckwheat under the light regulation is not clear.In order to clarify the difference of nutritional composition of Tartary buckwheat sprouts under different light conditions and the effect of light on anthocyanin biosynthesis pathway.Taking sprouts obtained from seed germination of Fagopyrum esculentum variety ‘Jinqiao 2’(‘JQ2’)under dark and light treatments as materials in this study.The basic nutritional components of two groups of sprouts were detected to explore their changing rules.The metabonomics and transcriptome analysis of the two groups of sprouts were carried out to further analyze the differences of metabolites and genes in the two groups of sprouts,and the mechanism of anthocyanin biosynthesis in Tartary buckwheat sprouts was studied.the related genes in anthocyanin biosynthesis in Tartary buckwheat sprouts were screened in order to explore the mechanism of anthocyanin biosynthesis in Tartary buckwheat.In order to improve the nutritional level of the Tartary buckwheat sprouts and provide molecular biological basis for the biosynthesis research of anthocyanin in the Tartary buckwheat,and then enhance the effective utilization of Tartary buckwheat resources,and lay a good foundation for the industrialization of buckwheat sprouts.The main conclusion of this study are as follows:1.Tartary buckwheat sprouts under dark treatment were etiolated,with yellow cotyledons and white hypocotyls,and the overall length of the sprouts was significantly higher than that of the control group.The Tartary buckwheat sprouts under light were green sprouts,and the cotyledons were green,and the hypocotyls were short and red.The weight,stem length and total length of sprouts in dark treatment group were larger than those in light treatment group,but the length of main roots was smaller than that in light treatment group,which indicated that the growth and development of Tartary buckwheat sprouts were greatly affected by light.Elemental determination(elements N,P,K,Ca,Mg and S and trace elements Fe,Mn,B,Zn,Mo and Cu)showed that Tartary buckwheat sprouts were rich in elements.The contents of N,P,K and Fe,Mn,B,Mo in sprouts grown under light conditions are higher than those in dark conditions,and there are significant differences in the contents of each element.In the analysis of main nutrients(soluble protein,soluble sugar)and secondary metabolites(flavonoids,chlorophyll,carotenoids and anthocyanins),the soluble protein content and soluble sugar content of Tartary buckwheat sprouts in the light treatment group were higher than those in the dark treatment group,and there was a very significant difference in the main nutrients between the two groups.In the analysis of secondary metabolites,the flavonoid,chlorophyll a,chlorophyll b,carotenoid and anthocyanin contents of Tartary buckwheat sprouts in the light treatment group were better than those in the dark treatment group.2.The differences of metabolites of F.esculentum sprouts under the two treatments were analyzed by gas chromatography-mass spectrometry.The results showed that the metabolites of buckwheat sprouts were significantly different under the two light treatments.A total of 383 metabolites were detected from the experimental materials,among which137 metabolites were significantly different under different light treatments,which were mainly concentrated in amino acids and their derivatives,carbohydrates and their derivatives,aromatic compounds and fatty acids,etc.The cluster analysis of heat map showed that the content of most metabolites in Tartary buckwheat sprouts cultured under light condition was increased,and the metabolite content was higher than that in dark condition.The production and accumulation of most metabolites were significantly reduced under dark treatment.The KEGG metabolic pathway enrichment results showed that the differential metabolites enriched 136 pathway pathways,most of which were related to substance metabolism,and the most abundant pathways were involved in protein degradation and absorption,biosynthesis and metabolism of secondary metabolites,which were basically corresponding to the types of differential metabolites,indicating that the differential metabolites were mainly related to substance metabolism under light conditions.Different light conditions led to changes in the accumulation of metabolites of multiple species in buckwheat sprouts,which might be due to the effect of light on the morphogenesis and nutritional value of buckwheat sprouts by disturbing these related metabolic pathways.Illumination could promote the generation and accumulation of most nutrient metabolites in buckwheat sprouts.The nutrient components were more abundant in dark culture and had higher nutritional value.3.By transcriptome analysis of light and dark treated Tartary buckwheat sprouts,a total of 2,774 differential genes were identified in two groups of Tartary buckwheat sprouts,including 2,578 known genes,996 genes with up-regulated expression(high expression under dark condition)and 1,582 genes with down-regulated expression(high expression under light condition);There were 196 new genes,114 up-regulated genes and82 down-regulated genes.A total of 209 differentially expressed transcription factors were obtained,which can be divided into 34 categories.The top five categories with the largest number are MYB,AP2-EREBP,b HLH,NAC and HB.The differential genes were compared with the GO and KEGG databases,and the co-enrichment ratios of the differentially expressed genes reached 3,227 GO term and 109 KEGG Pathway.There were 54 KEGG metabolic pathways co-enriched by the transcriptome and metabolome,involving 614 genes and the fluctuation of204 metabolites.The co-enriched metabolic pathways included three branches of environmental information processing,genetic information processing,and metabolism,which could be divided into 12 secondary KEGG Pathway metabolic pathways.Pathways related to anthocyanin metabolism in Tartary buckwheat sprouts included the ko00940 Phenylpropanoid biosynthesis pathway,the ko00941 Flavonoid biosynthesis pathway,and the ko00942 Anthocyanin biosynthesis pathway,in which the phenylpropanoid biosynthesis pathway was enriched in both the transcriptome and the metabolome.There were 12 genes directly involved in flavonoid metabolism that were annotated in the above pathway.These are Ft C4H-1(Ft Pin G0009241400.01)、Ft C4H-2(Ft Pin G0005329600.01)、Ft CHS-1(Ft Pin G0001351300.01)、Ft CHS-2(Ft Pin G0002106500.01)、 Ft CHS-3(Ft Pin G0003701500.01)、Ft CHS-4(Ft Pin G0003710800.01)、Ft CHS-5(Ft Pin G0006832300.01)、Ft FLS(Ft Pin G0006907100.01)、Ft ANR(Ft Pin G0007896600.01)、Ft UFGT(Ft Pin G0003140500.01)、Ft DFR(Ft Pin G0008631700.01)、Ft GST(Ft Pin G0001546300.01),and the expressions of Ft C4H-1,Ft C4H-2 and Ft CHS-2 were up-regulated,while the expressions of the remaining nine genes were down-regulated.Which respectively regulated the synthesis of related enzymes in the metabolism of flavonoid compounds in Tartary buckwheat sprouts,included eight genes directly related to anthocyanin metabolism.Through transcriptome data analysis,we screened out 10 COP1 regulatory factors,1 HY5,13 b HLH and 22 MYB transcription factors,of which 17 were up-regulated and 29 were down-regulated.4.After screening and analysis,three candidate regulatory factors that might regulate anthocyanin synthesis in Tartary buckwheat sprouts were successfully cloned,namely,Ft COP1(Ft Pin G0004259300.01),which was annotated as a regulatory factor involved in the metabolism of anthocyanin compounds;MYB transcription factor Ft MYB(Ft Pin G0003544400.01),which was annotated as being homologous to corn and related to anthocyanin synthesis;and Ft HY5(Ft Pin G0002935500.01),which was annotated as having transcriptional activation activity homologous to arabidopsis thaliana.The regulatory factor Ft COP1 is 144 bp longer than the reference sequence in the genome,and the rest is consistent with the reference sequence.The cloned base length is 2,112 bp,which indicates that this regulatory factor is likely to have alternative splicing phenomenon;The reference sequences of Ft HY5 and Ft MYB are completely consistent with those in genome,with lengths of 426 bp and 825 bp,respectively.Analysis of its base sequence and amino acid sequence revealed that Ft COP1-2 had 14 exons and non-coding regions at both ends of the gene3’ and 5’,encoding 703 amino acids.There are two exons in Ft HY5,and there are non-coding regions at both ends of 3’ and 5’,which encode 141 amino acids.Ft MYB have three exons,and only the 5’ end of the gene has a non-coding region,encodimg 274 amino acids.The three proteins contain conserved domains of their respective families,all of which are hydrophobic proteins,and all of them are predicted to be located in the nucleus,which is consistent with their characteristics of regulating the expression of downstream genes as regulatory factors.Yeast one-hybrid experiment showed that there was only a weak interaction between Ft MYB transcription factor and MRE cis-acting element in the upstream promoter sequence of Ft DFR,probably because Ft MYB needed to combine with other transcription factors to form a multimer before it could effectively play the role of transcription factor.