Pigment Diversity in Leaves of Caladium × hortulanum Birdsey and Transcriptomic and Metabolic Comparisons between Red and White Leaves

Abstract

Leaf color is a key ornamental characteristic of cultivated caladium (Caladium × hortulanum Birdsey), a plant with diverse leaf colors. However, the genetic improvement of leaf color in cultivated caladium is hindered by the limited understanding of leaf color diversity and regulation. In this study, the chlorophyll and anthocyanin content of 137 germplasm resources were measured to explore the diversity and mechanism of leaf color formation in cultivated caladium. Association analysis of EST-SSR markers and pigment traits was performed, as well as metabolomics and transcriptomics analysis of a red leaf variety and its white leaf mutant. We found significant differences in chlorophyll and anthocyanin content among different color groups of cultivated caladium, and identified three, eight, three, and seven EST-SSR loci significantly associated with chlorophyll-a, chlorophyll-b, total chlorophyll and total anthocyanins content, respectively. The results further revealed that the white leaf mutation was caused by the down-regulation of various anthocyanins (such as cyanidin-3-O-rutinoside, quercetin-3-O-glucoside, and others). This change in concentration is likely due to the down-regulation of key genes (four PAL, four CHS, six CHI, eight F3H, one F3'H, one FLS, one LAR, four DFR, one ANS and two UFGT) involved in anthocyanin biosynthesis. Concurrently, the up-regulation of certain genes (one FLS and one LAR) that divert the anthocyanin precursors to other pathways was noted. Additionally, a significant change in the expression of numerous transcription factors (12 NAC, 12 bZIP, 23 ERF, 23 bHLH, 19 MYB_related, etc.) was observed. These results revealed the genetic and metabolic basis of leaf color diversity and change in cultivated caladium, and provided valuable information for molecular marker-assisted selection and breeding of leaf color in this ornamental plant.

Keywords: Caladium × hortulanum; anthocyanin; chlorophyll; leaf color; metabolome; transcriptome.

Figure 1

Diversity analysis of chlorophyll and anthocyanin in cultivated caladium germplasm resources. (A) Chlorophyll-a content. (B) Chlorophyll-b content. (C) Total chlorophyll content. (D) Total anthocyanins content.

Figure 2

Comparison of chlorophyll and anthocyanin in cultivated caladium germplasm resources of different color groups. (A) Chlorophyll-a content. (B) Chlorophyll-b content. (C) Total chlorophyll content. (D) Total anthocyanins content. Groups exhibiting significant differences were denoted by distinct letters (a, b) at a significance level of p < 0.05.

Figure 3

Association analysis of chlorophyll and anthocyanin based on 26 EST-SSRs. (A) QQ plot of chlorophyll-a content. (B) QQ plot of chlorophyll-b content. (C) QQ plot of total chlorophyll content. (D) QQ plot of total anthocyanins content. (E) Box chart analysis of chlorophyll-b content at CAL15 locus. (F) Box chart analysis of total chlorophyll content at CAL15 locus. (G) Box chart analysis of total anthocyanins content at CAL162 locus. Values above the blue dashed line indicate p < 0.05, and values above the red dashed line indicate p < 0.01. Groups exhibiting significant differences were denoted by distinct letters (a, b) at a significance level of p < 0.05. The “**” in (G) indicates p < 0.01.

Figure 4

Anthocyanin metabolome analysis of ‘Lieyanxiongxin’ (HH) and its white-leaf mutant (HB). (A) Leaf images of red-leaf ‘Liyanxiongxin’ and its white-leaf mutant. (B) Comparison of total anthocyanin content between HH and HB. (C) Number of common and unique anthocyanin metabolites between HH and HB. (D) Heatmap. (E) OPLS-DA score plot. (F) VIP and log₂FC values of significantly different anthocyanin metabolites between HH and HB leaves. The “***” in (B) indicates p < 0.001. The “*” in (F) indicates the anthocyanin metabolites unique to HH.

Figure 5

Transcriptome analysis of ‘Lieyanxiongxin’ (HH) and its white-leaf mutant (HB). (A) PCA score plot. (B) Volcano plot of differentially expressed genes. (C) GO enrichment analysis of differentially expressed genes. (D) KEGG enrichment analysis of differentially expressed genes. The items with red bracket are related to anthocyanins biosynthesis.

Figure 6

Differential expression analysis of anthocyanin biosynthesis pathway genes in ‘Lieyanxiongxin’ (HH) and its white-leaf mutant (HB). Red gene family names indicate that up-regulated genes are predominant. Blue gene family names indicate that down-regulated genes are dominant. Black gene family names indicate that the number of up-regulated and down-regulated genes are comparable. PAL: phenylalanine ammonia-lyase; C4H: cinnamate 4-hydroxylase; 4CL: 4-coumarate-CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavonone 3-hydroxylase; F3′H: flavonoid 3′-hydroxylase; FLS: flavonol synthase; F3′5′H: flavonoid 3′5′-hydroxylase; DFR: dihydroflavonol reductase; LAR: leucoanthocyanidin reductase; ANS: anthocyanidin synthase; UFGT: UDP-glycose flavonoid glycosyltransferase. ANR: anthocyanidin reductase.

Figure 7

Differential expression analysis of transcription factors in ‘Lieyanxiongxin’ (HH) and its white-leaf mutant (HB). (A) Up-regulated differentially expressed transcription factors. (B) Down-regulated differentially expressed transcription factors. (C) Heatmap of gene expression of top five up-regulated transcription factor families. (D) Heatmap of gene expression of top five down-regulated transcription factor families.

Figure 8

qRT-PCR verification of 13 key genes. HH stands for ‘Lieyanxiongxin’. HB stands for its white-leaf mutant.

Figure 9

Schematic diagram of the potential metabolic and molecular regulation of the loss of red color in the leaves of the white-leaf mutant of ‘Lieyanxiongxin’. Red font indicates higher gene expression or metabolite content. Blue font indicates lower gene expression or metabolite content. Red-blue gradient of ERF, bHLH and MYB_related indicates a large number of both up-regulated and down-regulated genes in these gene families. The red numbers indicate the number of up-regulated genes, and the blue numbers indicate the number of down-regulated genes. The information on these key differentially expressed genes is listed in Tables S8, S10 and S11.