Genome-wide analysis of flavonoid biosynthetic genes in Musaceae (Ensete, Musella, and Musa species) reveals amplification of flavonoid 3',5'-hydroxylase

Abstract

Flavonoids in Musaceae are involved in pigmentation and stress responses, including cold resistance, and are a component of the healthy human diet. Identification and analysis of the sequence and copy number of flavonoid biosynthetic genes are valuable for understanding the nature and diversity of flavonoid evolution in Musaceae species. In this study, we identified 71-80 flavonoid biosynthetic genes in chromosome-scale genome sequence assemblies of Musaceae, including those of Ensete glaucum, Musella lasiocarpa, Musa beccarii, M. acuminata, M. balbisiana and M. schizocarpa, checking annotations with BLAST and determining the presence of conserved domains. The number of genes increased through segmental duplication and tandem duplication. Orthologues of both structural and regulatory genes in the flavonoid biosynthetic pathway are highly conserved across Musaceae. The flavonoid 3',5'-hydroxylase gene F3'5'H was amplified in Musaceae and ginger compared with grasses (rice, Brachypodium, Avena longiglumis, and sorghum). One group of genes from this gene family amplified near the centromere of chromosome 2 in the x = 11 Musaceae species. Flavonoid biosynthetic genes displayed few consistent responses in the yellow and red bracts of Musella lasiocarpa when subjected to low temperatures. The expression levels of MlDFR2/3 (dihydroflavonol reductase) increased while MlLAR (leucoanthocyanidin reductase) was reduced by half. Overall, the results establish the range of diversity in both sequence and copy number of flavonoid biosynthetic genes during evolution of Musaceae. The combination of allelic variants of genes, changes in their copy numbers, and variation in transcription factors with the modulation of expression under cold treatments and between genotypes with contrasting bract-colours suggests the variation may be exploited in plant breeding programmes, particularly for improvement of stress-resistance in the banana crop.

Keywords: Anthocyanins; F3ʹ5 ʹH; F3ʹH; Musaceae; banana; diversification; flavonoids; genetics; genomics; ginger; monocotyledons.

Figure 1.

The part of the flavonoid biosynthetic pathway analysed here, with regulators of the genes, in plant cells (based on maize, Arabidopsis, Brassica, Musa, kiwifruit and soybean). Structural genes encoding enzymes are: PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase; IFS, isoflavone synthase; F3H, flavanone 3-hydroxylase; F3’H, flavanone 3’-hydroxylase; F3’5’H, flavanone 3’5’-hydroxylase; DFR, dihydroflavonol reductase; FLS, flavonol synthase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; UGP/UFGT, UDP-flavonoid glucosyl transferase; ANR, anthocyanidin reductase; LAR, leucoanthocyanidin reductase. EBGs: early biosynthetic genes; LBG: late biosynthetic genes. Regulatory transcription factors MYB (P1/PL1/C1 or PL1/C1) and bHLH (R/B) are from maize; WD40 (TTG1), MYB (TT2/PAP1/PAP2), and bHLH (TT8/GL3/EGL3) are from Arabidopsis; MYBC1 and WRKY44 are from kiwifruit; and MYB29 is from soybean.

Figure 2.

Phylogenetic tree and chromosomal location of F3ʹ5ʹHs. (A) Phylogenetic tree of the F3ʹ5ʹH gene family of Musaceae plus rice, Brachypodium, Avena longiglumis, sorghum, ginger and Arabidopsis as outgroups. (B) One clade of the F3ʹ5ʹH phylogenetic tree terminal with genus (initial letter) and species (six letter) abbreviations and (C) Chromosome 2 gene location maps in Musaceae. Genes from different families are shown in different colours (F3ʹ5ʹH in purple). Horizontal bars represent the gene locations (Mb, megabases) on syntenic chromosome 2 in Fig. 2C. Genes with tandem duplications are framed by black boxes; F3ʹ5ʹH clusters near the chromosome 2 centromere are indicated by arrows. MBE, Mbeccar, M. beccarii; MLA, Mlasioc, M. lasiocarpa; EGL, Eglaucu, E. glaucum; MAC, Macumin, M. acuminata; MBA, Mbalbis, M. balbisiana; MSC, Mschizo, M. schizocarpa. A colour version of this figure appears in the online version of this article.

Figure 3.

Circos plot of flavonoid biosynthetic gene paralogue locations on the chromosomes of six Musaceae species. (A) E. glaucum (green). (B) M. lasiocarpa (yellow). (C) M. beccarii (purple). (D) M. acuminata (red). (E) M. balbisiana (orange). (F) M. schizocarpa (blue). Different gene families are represented by different coloured lines. A colour version of this figure appears in the online version of this article.

Figure 4.

Synteny analysis and chromosomal location of ANRs. (A) Synteny analysis of flavonoid biosynthetic genes identified between MLA (M. lasiocarpa) and MAC (M. acuminata). The grey lines indicate all collinear gene blocks, while the red lines highlight the syntenic flavonoid biosynthetic gene pairs. The brown and blue lines indicate the ANR and UGT syntenic blocks, respectively. (B) Chromosome localization of flavonoid biosynthetic genes identified in M. lasiocarpa and M. acuminata. Genes from different families are shown in different colours. The chromosome number is shown at the top of each bar. The horizontal bars represent the gene locations on each chromosome (Mb). ANR genes with tandem duplications are framed by black boxes. A colour version of this figure appears in the online version of this article.

Figure 5.

The expression of flavonoid biosynthetic genes in Musella lasiocarpa var. lasiocarpa (circles labelled Y) and M. lasiocarpa var. rubribracteata (labelled R); 15 °C-treated groups labelled with ‘cold’, and control groups marked with ‘normal’. The flavonoid biosynthetic gene expression levels were calculated as transcripts per million (TPM) values. Different log₂(TPM + 1) values labelled with different colours, red to blue colours indicate expression levels from high to low. A colour version of this figure appears in the online version of this article.