Biochemical and transcriptomic analyses reveal that critical genes involved in pigment biosynthesis influence leaf color changes in a new sweet osmanthus cultivar 'Qiannan Guifei'

Abstract

Background: Osmanthus fragrans (Oleaceae) is one of the most important ornamental plant species in China. Many cultivars with different leaf color phenotypes and good ornamental value have recently been developed. For example, a new cultivar 'Qiannan Guifei', presents a rich variety of leaf colors, which change from red to yellow-green and ultimately to green as leaves develop, making this cultivar valuable for landscaping. However, the biochemical characteristics and molecular mechanisms underlying leaf color changes of these phenotypes have not been elucidated. It has been hypothesized that the biosynthesis of different pigments in O. fragrans might change during leaf coloration. Here, we analyzed transcriptional changes in genes involved in chlorophyll (Chl), flavonoid, and carotenoid metabolic pathways and identified candidate genes responsible for leaf coloration in the new cultivar 'Qiannan Guifei'.

Methods: Leaf samples were collected from 'Qiannan Guifei' plants at the red (R), yellow-green (YG) and green (G) leaf stages. We compared the different-colored leaves via leaf pigment concentrations, chloroplast ultrastructure, and transcriptomic data. We further analyzed differentially expressed genes (DEGs) involved in the Chl, flavonoid, and carotenoid metabolic pathways. In addition, we used qRT-PCR to validate expression patterns of the DEGs at the three stages.

Results: We found that, compared with those at the G stage, chloroplasts at the R and YG stages were less abundant and presented abnormal morphologies. Pigment analyses revealed that the leaves had higher flavonoid and anthocyanin levels at the R stage but lower Chl and carotenoid concentrations. Similarly, Chl and carotenoid concentrations were lower at the YG stage than at the G stage. By using transcriptomic sequencing, we further identified 61 DEGs involved in the three pigment metabolic pathways. Among these DEGs, seven structural genes (OfCHS, OfCHI, OfF3H, OfDFR, OfANS, OfUGT andOf3AT) involved in the flavonoid biosynthesis pathway were expressed at the highest level at the R stage, thereby increasing the biosynthesis of flavonoids, especially anthocyanins. Six putativeOfMYB genes, including three flavonoid-related activators and three repressors, were also highly expressed at the R stage, suggesting that they might coordinately regulate the accumulation of flavonoids, including anthocyanins. Additionally, expressions of the Chl biosynthesis-related genes OfHEMA, OfCHLG and OfCAO and the carotenoid biosynthesis-related genes OfHYB and OfZEP were upregulated from the R stage to the G stage, which increased the accumulation of Chl and carotenoids throughout leaf development. In summary, we screened the candidate genes responsible for the leaf color changes of 'Qiannan Guifei', improved current understanding of the regulatory mechanisms underlying leaf coloration and provided potential targets for future leaf color improvement in O. fragrans.

Keywords: Anthocyanin; Carotenoid; Chlorophyll; Flavonoid; Leaf color; OfMYB genes; Osmanthus fragrans ‘Qiannan Guifei’.

Figure 1. (A–F) Leaf characteristics and chloroplast ultrastructures of ‘Qiannan Guifei’ at the three developmental stages. (G–H) Average number of chloroplasts per cell and pigment concentrations at the three stages.

Ch, Chloroplast; CW, Cell wall; Gr, Grana; P, Plastoglobuli; Th, Thylakoid. Bars represent the standard errors of three biological replicates. Different lowercase letters indicate a significant difference (p < 0.05) relative to the value at the G stage, as determined using ANOVA analysis, which is based on Duncan’s multiple range test.

Figure 2. (A) Repeatability evaluation of transcriptome data from ‘Qiannan Guifei’ after using Pearson’s correlation analysis. (B–D) Volcano plot of DEGs among the three developmental stages.

The upregulated and downregulated DEGs are shown in red and blue, respectively. The x-axis represents the fold changes on a log₂ scale. The y-axis represents the negative −log₁₀ transformed p-values (p < 0.05) of the t-test, which were used to identify differences among the three stages.

Figure 3. Expression profiles of the DEGs involved in chlorophyll metabolism at the three developmental stages.

The scale bar represents the changes of gene expression pattern: red rectangle indicates upregulated expression pattern, blue rectangle indicates downregulated expression pattern, and the white rectangle indicates gene whose expression did not change. The normalized signal intensity ranged from −1.0 to 1.0, which was consistent with color changes from blue to red.

Figure 4. Expression profiles of the DEGs involved in (A) carotenoid and (B) flavonoid biosynthesis pathways at the three developmental stages.

The colored rectangle indicates the gene expression pattern as defined in Fig. 3.

Figure 5. (A) Differentially expressed TFs. (B) Expression profiles of six differentially expressed OfMYBs at the three stages. (C) Phylogenic analysis of six OfMYBs with 35 flavonoid-related MYBs from other species.

Full-length amino acid sequences from theseMYBs were analyzed under maximum-likelihood (ML) phylogenetic methods. Numbers near branches indicate bootstrap values that were calculated from 1,000 replicates. OfMYBs are highlighted with solid black circles. MYBs phylogenetic tree contained five subgroups (SGs), SG4 (repressors of flavonoid biosynthesis), SG5 (activators of anthocyanin biosynthesis), SG6 (activators of anthocyanin biosynthesis), SG7 (activators of flavonol/flavone biosynthesis), and RH (activators of root hair growth). The subgroup of RH was included as an outgroup.

Figure 6. Validation of RNA-Seq results via qRT-PCR assays.

Nine DEGs were selected from the flavonoid, carotenoid, and chlorophyll metabolic pathways. (A–C) Scatter diagrams show the correlations among expression levels of the nine DEGs as measured by the qRT-PCR (x-axis) and RNA-Seq (y-axis) analyses. The fold changes for gene expression were transformed as log ₂(R/YG), log ₂ (R/G), and log ₂(YG/G) from the RNA-Seq and qRT-PCR data. (D–L) Expression profiles of the nine DEGs revealed by RNA-Seq (left y-axis) and qRT-PCR (right y-axis) at the three stages. Expression levels obtained from the qRT-PCR were normalized to the reference gene OfActin.