Transcriptomic analysis of Litchi chinensis pericarp during maturation with a focus on chlorophyll degradation and flavonoid biosynthesis

Abstract

Background: The fruit of litchi (Litchi chinensis) comprises a white translucent edible aril surrounded by a pericarp. The pericarp of litchi has been the focus of studies associated with fruit size, coloration, cracking and shelf life. However, research at the molecular level has been limited by the lack of genomic and transcriptomic information. In this study, an analysis of the transcriptome of litchi pericarp was performed to obtain information regarding the molecular mechanisms underlying the physiological changes in the pericarp, including those leading to fruit surface coloration.

Results: Coincident with the rapid break down of chlorophyll, but substantial increase of anthocyanins in litchi pericarp as fruit developed, two major physiological changes, degreening and pigmentation were visually apparent. In this study, a cDNA library of litchi pericarp with three different coloration stages was constructed. A total of 4.7 Gb of raw RNA-Seq data was generated and this was then de novo assembled into 51,089 unigenes with a mean length of 737 bp. Approximately 70% of the unigenes (34,705) could be annotated based on public protein databases and, of these, 3,649 genes were significantly differentially expressed between any two coloration stages, while 156 genes were differentially expressed among all three stages. Genes encoding enzymes involved in chlorophyll degradation and flavonoid biosynthesis were identified in the transcriptome dataset. The transcript expression patterns of the Stay Green (SGR) protein suggested a key role in chlorophyll degradation in the litchi pericarp, and this conclusion was supported by the result of an assay over-expressing LcSGR protein in tobacco leaves. We also found that the expression levels of most genes especially late anthocyanin biosynthesis genes were co-ordinated up-regulated coincident with the accumulation of anthocyanins, and that candidate MYB transcription factors that likely regulate flavonoid biosynthesis were identified.

Conclusions: This study provides a large collection of transcripts and expression profiles associated with litchi fruit maturation processes, including coloration. Since most of the unigenes were annotated, they provide a platform for litchi functional genomic research within this species.

Figure 1

Images of litchi fruits and pigment contents in the pericarp. (A) Images of litchi fruits at different coloration stages. (B) Contents of total chlorophylls, total flavonoids, proanthocyanidins and anthocyanins in the pericarp of three coloration stages. The vertical bars represent the standard error of triplicate experiments.

Figure 2

Outcome of homology search of litchi unigenes against the nr database. (A) Venn diagram of unigene numbers annotated by BLASTx with an E-value threshold of 10⁻⁵ against protein databases. The numbers in the circles indicate unigenes numbers annotated by single or multiple databases. (B) E-value distribution of the top BLAST hits for each unique sequence. (C) Species distribution of the top BLAST hits for all homologous sequences.

Figure 3

Histogram of gene ontology (GO) classifications for litchi pericarp transcripts. The unigenes corresponded to three main categories: ‘biological process’, ‘cellular component’ and ‘molecular function’. The left and right-hand y-axes indicate the percentage and number of annotated unigenes, respectively.

Figure 4

Differential gene expression profiles based on the library of the three coloration stages. (A) The numbers of up- and down-regulated genes in comparisons of the red-VS-yellow, red-VS-green, and yellow-VS-green fruit samples. (B) Venn diagram showing the comparison of differentially expressed genes between any two stages of the litchi pericarp.

Figure 5

Clustering results of time-course data from RNA-Seq by STEM analysis. In the image each box corresponds to one of the model temporal expression profiles. The clusters with an asterisk had a statistically significant number of genes assigned. The number in the top right-hand corner of a profile box is the cluster number. The number before a semicolon at the bottom of a cluster box is the P-value for the genes assigned to a profile compared with what was expected. The number after the semicolon is the number of genes assigned to the profile.

Figure 6

Hierarchical clustering analysis of differentially-expressed genes (DEGs) during litchi pericarp maturation.

Figure 7

The pathway of chlorophyll breakdown and expressions of chlorophyll breakdown genes. (A) The PAO pathway of chlorophyll breakdown. (B) A heat map of the expressions of genes for chlorophyll breakdown in the pericarp during coloration. Chl, chlorophyll; CLH, chlorophyllase; HCAR, hydroxy-Chl a reductase; MCS, metal chelating substance; NCCs, nonfluorescent Chl catabolites; NOL, NYC1-like; NYC1, non-yellow coloring 1; PAO, pheide a oxygenase; pFCC, primary fluorescent Chl catabolite; Pheide, pheophorbide; PPH, pheophytinase; RCC, red Chl catabolite; RCCR, RCC reductase; SGR, stay-green protein.

Figure 8

Phylogenetic relationship of stay green (SGR) proteins and transient assays of LcSGR. A. Transient assays of LcSGR in Nicotiana benthamiana leaf show the induction of substantial chlorophyll loss. B. A Loss of chlorophyll fluorescence due to chlorophyll degradation using a chlorophyll fluorometer. C. Phylogenetic relationship between LcSGR and other SGRs proteins from other plant species.

Figure 9

Simplified scheme (A) and a heat map of the expression of genes (B) related to flavonoid biosynthesis. Enzyme names are abbreviated as follows; PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4 coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavanone 3′-hydroxylase; DFR, dihydroflavonol reductase; FLS, flavonol synthase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; UFGT, UDP-flavonoid glucosyltransferase; ANR, anthocyanidin reductase; and LAR, leucoanthocyanidin reductase. Enzyme names, unigene IDs and expression patterns are indicated on the right of each step.

Figure 10

Coefficient analysis of gene expression levels obtained from RNA-Seq and quantitative real-time PCR data. The real-time PCR log₂ values (x-axis) were plotted against coloration stages (y-axis). **indicates a significant difference at p ≤ 0.01.