Combined Analysis of the Fruit Metabolome and Transcriptome Reveals Candidate Genes Involved in Flavonoid Biosynthesis in Actinidia arguta

Abstract

To assess the interrelation between the change of metabolites and the change of fruit color, we performed a combined metabolome and transcriptome analysis of the flesh in two different Actinidia arguta cultivars: "HB" ("Hongbaoshixing") and "YF" ("Yongfengyihao") at two different fruit developmental stages: 70d (days after full bloom) and 100d (days after full bloom). Metabolite and transcript profiling was obtained by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer and high-throughput RNA sequencing, respectively. The identification and quantification results of metabolites showed that a total of 28,837 metabolites had been obtained, of which 13,715 were annotated. In comparison of HB100 vs. HB70, 41 metabolites were identified as being flavonoids, 7 of which, with significant difference, were identified as bracteatin, luteolin, dihydromyricetin, cyanidin, pelargonidin, delphinidin and (-)-epigallocatechin. Association analysis between metabolome and transcriptome revealed that there were two metabolic pathways presenting significant differences during fruit development, one of which was flavonoid biosynthesis, in which 14 structural genes were selected to conduct expression analysis, as well as 5 transcription factor genes obtained by transcriptome analysis. RT-qPCR results and cluster analysis revealed that AaF3H, AaLDOX, AaUFGT, AaMYB, AabHLH, and AaHB2 showed the best possibility of being candidate genes. A regulatory network of flavonoid biosynthesis was established to illustrate differentially expressed candidate genes involved in accumulation of metabolites with significant differences, inducing red coloring during fruit development. Such a regulatory network linking genes and flavonoids revealed a system involved in the pigmentation of all-red-fleshed and all-green-fleshed A. arguta, suggesting this conjunct analysis approach is not only useful in understanding the relationship between genotype and phenotype, but is also a powerful tool for providing more valuable information for breeding.

Keywords: Actinidia arguta; candidate genes; flavonoid biosynthesis; fruit coloring; metabolites.

Figure 1

The phenotype of Actinidia arguta cv. “HB” and “YF” at two different sampling stages 70 DAFB and 100 DAFB, respectively. (A) Green fruit “HB” at 70 DAFB, HB70; (B) Red fruit “HB” at 100 DAFB, HB100; (C) Green fruit “YF” at 70 DAFB, YF70; (D) Green fruit “YF” at 100 DAFB, YF100.

Figure 2

KEGG pathway classification: metabolites detected and annotated. (A) POS model; (B) NEG model. The x-axis represents level-2 terms of the KEGG pathway and the y-axis represents the number of metabolites identified.

Figure 3

Identified metabolites classified into the top 50 KEGG pathways. (A) POS model; (B) NEG model. The x-axis represents the top 50 KEGG pathways and the y-axis represents number of identified metabolites involved in this pathway.

Figure 4

Identified metabolites from level-two identification were classified into 11 HMDB super classes. (A) POS model; (B) NEG model. The x-axis represents HMDB super classes, and the y-axis represents the number of identified metabolites.

Figure 5

The numbers of transcription factors involved in the top 20 transcription factor families. The x-axis represents the numbers of transcription factors and the y-axis represents the top 20 transcription factor families.

Figure 6

Identification of candidate DEGs. (A) Venn Diagram of pathways. (i) indicates Venn Diagram result between three groups, HB100 vs. HB70, HB100 vs. YF100, and YF100 vs. YF70. (ii) indicates the two specific pathways; (B) Venn Diagram of DEGs involved in flavonoid biosynthesis. (i) indicates Venn Diagram result between three groups, HB100 vs. HB70, HB100 vs. YF100, and YF100 vs. YF70. (ii) indicates specific 8 DEGs.

Figure 7

Expression profiles of structural and regulatory genes involved in flavonoid biosynthesis in the flesh of “HB” and “YF”. (A) 14 structural genes; (B) 5 regulatory genes. The x-axis represents two different development stages of “HB” and “YF”, and the y-axis represents relative expression. Data were analyzed using the t-test. * p < 0.05, ** p < 0.01.

Figure 8

Cluster analysis for structural and regulatory genes. Blue boxes indicate high expression levels and red boxed indicate low expression levels.

Figure 9

Phylogenetic analysis of 40 MYB transcription factor genes in different species. The green frame indicates that the two genes AaMYB and CsMYB5a are clustered together.

Figure 10

Regulatory network of flavonoid biosynthesis in two development stages “HB100” and “HB70”. HB100: “HB” kiwifruit at 100 DAFB; HB70: ”HB” kiwifruit at 70 DAFB. Color scale from dark blue to bright red represents relative expression levels 0–5. Small pink cells represent fold change of metabolites between HB100 and HB70. The solid frame arrow represents only one step of process, and the dotted frame arrow represents more than one step of process. PAL, phenylalanine ammonia-lyase; C4H, trans-cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; C3’H, coumaroylquinate 3’-monooxygenase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavonoid 3’-hydroxylase; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP glucose-flavonoid 3-O-glcosyl-transferase; ANR, anthocyanidin reductase.