The Chromosome-Scale Assembly of the Curcuma alismatifolia Genome Provides Insight Into Anthocyanin and Terpenoid Biosynthesis

Abstract

Curcuma alismatifolia, a bulbous flower known for its showy bracts, is widely used around the world as a cut flower, potted, and garden plant. Besides its ornamental value, this species is rich in terpenoid metabolites and could serve as a resource for essential oils. Here, we report a chromosome-level genome assembly of C. alismatifolia and describe its biosynthetic pathways for anthocyanins and terpenoids. This high-quality, assembled genome size is 991.3 Mb with a scaffold N50 value of 56.7 Mb. Evolutionary analysis of the genome suggests that C. alismatifolia diverged from Zingiber officinale about 9.7 million years ago, after it underwent a whole-genome duplication. Transcriptome analysis was performed on bracts at five developmental stages. Nine highly expressed genes were identified, encoding for six enzymes downstream of the anthocyanin biosynthetic pathway. Of these, one gene encoding F3'5'H might be a key node in the regulation of bract color formation. Co-expression network analysis showed that MYB, bHLH, NAC, and ERF transcription factors collectively regulated color formation in the bracts. Characterization of terpenoid biosynthesis genes revealed their dispersal and tandem duplications, both of which contributed greatly to the increase in the number of terpene synthase genes in C. alismatifolia, especially to species-specific expansion of sesquiterpene synthase genes. This work facilitates understanding of genetic basis of anthocyanin and terpenoid biosynthesis and could accelerate the selective breeding of C. alismatifolia varieties with higher ornamental and medicinal value.

Keywords: C. alismatifolia; anthocyanin; evolution; genome; terpenoid.

Figure 1

Genome of Curcuma alismatifolia. (A) Plant, distal bract and rhizome of C. alismatifolia. (B) Chromosomal features: (i) chromosome, (ii) gene density, (iii) repeat sequence density, (iv) GC (guanine- cytosine) content, and (v) syntenic blocks.

Figure 2

Evolution of C. alismatifolia. (A) Phylogenetic tree showing the evolution of gene families and divergence times. The green and red numbers represent the numbers of expanded and contracted gene families, respectively. In the pie charts, green, red, and blue portions show expanded, contracted, and constant gene families, respectively. Numbers at nodes represent divergence times (million years ago). (B) Distribution of synonymous substitutions per synonymous site (Ks). Peaks of intra-species Ks distributions indicate ancient whole-genome duplication events, and peaks of inter-species Ks distributions indicate divergence events. The arrow shows the recent whole genome duplication peak in C. alismatifolia.

Figure 3

Biosynthesis of anthocyanin in C. alismatifolia. (A) Biosynthesis pathways of anthocyanin. Structural genes in anthocyanin biosynthetic pathway were divided into two groups, designated as “upstream structural genes” and “downstream structural genes” respectively. The upstream structural genes are implicated in the biosynthesis of precursors of flavonoids, while the downstream structural genes are specifically involved in anthocyanin biosynthesis. Malvidin, the major anthocyanin in bract in C. alismatifolia, is marked in red. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′ hydroxylase; DRF, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; 3GT, UDP-glucose flavonoid 3-glucosyltransferase; and OMT, O-methyltransferases. (B) Distal bracts from five developmental stages. From left to right, youngest to oldest (S1–S5). (C) Anthocyanin contents in the distal bracts collected from the five stages. (D) Expression heatmap of the 49 structural genes at different stages of bract development. The expression values at the column scale were z-score normalized. Low to high expression is indicated by a change in color from blue to red. The nine highly expressed downstream genes are marked with red lines. (E) Real-time PCR analysis of the nine downstream structural genes with high transcription levels. The expression levels were normalized to Actin1 (CUAL02G1340) and related to stage 1. Data are presented in mean ± SD (n = 3). (F) The co-expression network of the structural genes with transcription factors.

Figure 4

Biosynthesis of terpenoids in C. alismatifolia. (A) The backbone biosynthetic process of terpenoid. AACT, acetyl-CoA C-acetyltransferase; HMG-R, hydroxymethylglutaryl-CoA reductase; HMG-S, hydroxymethylglutaryl-CoA synthase; MK, mevalonate kinase; PMK, phosphor-mevalonate kinase; MPDC, diphosphomevalonate decarboxylase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl-diphosphate reductase; IPPI, isopentenyl-diphosphate △-isomerase; GPPS, dimethylallyltranstransferase/geranyl diphosphate synthase; FPPS, (2E,6E) farnesyl diphosphate synthase; and GGPPS, geranylgeranyl diphosphate synthase. Number of gene encoding these enzymes was compared among eight monocotyledonous plants, i.e., P. equestris, Cocos nucifera, A. comosus, O. sativa, M. acuminate, M. schizocarpa, Zingiber officinale, and C. alismatifolia. The notation “1-1-1-1-1-1-1” indicates one homologous gene was identified among each plant in the order listed in the previous sentence. (B) Expression heatmap of the 56 terpenoid backbone biosynthetic genes in the five tissues. The expression values at the row scale were z-score normalized. Low to high expression is indicated by a change in color from blue to brown. (C) The gene number of terpene synthases (TPS) in the eight plants. (D) The phylogenetic tree and expression heatmap of terpene synthase genes in C. alismatifolia. (i) phylogenetic tree and (ii) expression heatmap. (E) Gene clusters of TPS and P450 genes in C. alismatifolia. (F) Colinear analysis of TPS genes in C. alismatifolia (top) and Z. officinale (bottom).