Mechanisms for leaf color changes in Osmanthus fragrans 'Ziyan Gongzhu' using physiology, transcriptomics and metabolomics

Abstract

Background: Color-leaved O. fragrans is a variety of Osmanthus fragrans, which has both the fragrance of Osmanthus and the color of color-leaved plants. However, the molecular mechanism of color change of color-leaved O. fragrans is not clear. In this study, we analyzed the regulatory mechanism of four different color leaves of 'Ziyan Gongzhu' through physiological, transcriptome and metabolome levels.

Results: Firstly, we measured the leaf pigments content and leaf chromatic parameters for correlation analysis, indicating a significant correlation between them. Overall, the content of chlorophyll a + b is low and the content of anthocyanin is high in T1 and T2 leaves, along with low expression of chlorophyll synthesis genes (HEMA, CHLG, and CAO, etc.) and high expression of anthocyanin synthesis genes (F3H, F3'H, DFR and ANS, etc.), resulting purple red and light purple in T1 and T2 leaves, respectively. It was also found that the pigment closely related to the color leaves of 'Ziyan Gongzhu' was cyanidin. The content anthocyanins, may be regulated by two putative MYB activators (OfMYB3 and OfMYB4) and two putative MYB repressors (OfMYB1 and OfMYB2). In contrast, the content of chlorophyll a + b is high and the content of anthocyanin is low in T3 and T4 leaves, along with high expression of chlorophyll synthesis genes and low expression of anthocyanin synthesis genes, resulting yellow green and dark green in T3 and T4 leaves, respectively. And abnormal chloroplast development affects chlorophyll content in T1, T2, and T3 leaves. Although the content of carotenoids first dropped in T2 leaves, it then rapidly accumulated in T4 leaves, in sync with the increase in the expression of genes related to carotenoid biosynthesis (ZDS, LHYB, and ZEP, for example). Analysis of photosynthetic, carbohydrate and hormone-related differentially abundant metabolites (DAMs) and DEGs found that they may participate in the regulation of leaf color change of 'Ziyan Gongzhu' by affecting pigment synthesis.

Conclusion: Our results pave the way for a comprehensive knowledge of the regulatory processes governing leaf color in 'Ziyan Gongzhu' and identify possible genes for application regarding molecular colored-leaf cultivar breeding.

Keywords: Anthocyanin; Carotenoid; Chlorophyll; Metabolomics; OfMYB genes; Osmanthus fragrans ‘Ziyan Gongzhu’; RNA-seq.

Fig. 1

Leaf characteristics of ‘Ziyan Gongzhu’ at the four developmental stages. (T1: purple red leaf (RHS 67A), T: stage, T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 2

Percentage of pigments content in leaves of ‘Ziyan Gongzhu’ at the four developmental stages. Chl a, Chlorophyll a content; Chl b, Chlorophyll b content; Car, Carotenoid content; Ant, Anthocyanin content. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 3

CIE Lab value (L, a*, b*) (A) and correlation analysis results between leaf chromatic parameters and pigment content and ratio (B) of ‘Ziyan Gongzhu’ leaves at the four developmental stages. Different lowercase letters indicate a significant difference (p < 0.05) relative to the value at the T4, as determined using ANOVA analysis, which is based on Duncan’s multiple range test. Chl a + b, Chlorophyll a + b content; Car, Carotenoid content; Ant, Anthocyanin content. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 4

The pH of ‘Ziyan Gongzhu’ leaves at the four developmental stages. Different lowercase letters indicate a significant difference (p < 0.05) relative to the value at the T4, as determined using ANOVA analysis, which is based on Duncan’s multiple range test. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 5

Chloroplast ultrastructures in leaves of ‘Ziyan Gongzhu’ at the four developmental stages. A T1, purple red leaf (RHS 67A); B T2 light purple leaf (RHS 65A); C T3, yellow green leaf (RHS 1C); D T4, dark green leaf (RHS N137A). CW: Cell wall; Gr: Grana; Pg: Plastoglobuli; SG: starch grain; V: vacuole. Bar = 2 µm

Fig. 6

Average number of chloroplasts per cell (A), chloroplasts length (B) and width (C) in leaves of ‘Ziyan Gongzhu’ at the four developmental stages. 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 T4, as determined using ANOVA analysis, which is based on Duncan’s multiple range test. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 7

Chlorophyll fluorescence parameter Y(II) (reflects the actual photosynthetic efficiency, A), ETR (electron transfer rate, B) and NPQ (non-photochemical quenching, C) in leaves of ‘Ziyan Gongzhu’ at the four developmental stages. 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 T4, as determined using ANOVA analysis, which is based on Duncan’s multiple range test. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 8

Upregulated and downregulated genes (A) and venn analysis of DEGs (B) identified in T1 vs T2, T1 vs T3, T1 vs T4, T2 vs T3, T2 vs T4 and T3 vs T4 of ‘Ziyan Gongzhu’. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 9

A Expression profiles of four differentially expressed OfMYBs at the four stages. B Phylogenic analysis of four OfMYBs with 26 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. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 10

Correlation analysis of RNA-seq and RT-PCR data. DEGs were selected from the flavonoid and chlorophyll metabolic pathways. Expression levels obtained from the RT-PCR were normalized to the reference gene Actin. FC, Fold Change

Fig. 11

Upregulated and downregulated metabolites in T1 vs T2, T1 vs T3, T1 vs T4, T2 vs T3, T2 vs T4 and T3 vs T4 of ‘Ziyan Gongzhu’. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 12

Venn analysis of DAMs identified in T1 vs T2, T1 vs T3, T1 vs T4, T2 vs T3, T2 vs T4 and T3 vs T4 of ‘Ziyan Gongzhu’ in POS (A) and NEG (B). (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 13

Two-way Orthogonal Partial Least Square (O2PLS) loading plots for DAMs (A) and DEGs (B). The top 10 DEGs and DAMs that had greater influence on the other omics were indicated by red dots

Fig. 14

Expression profiles of the DEGs and accumulation of the DAMs involved in flavonoid biosynthesis pathway in leaves of ‘Ziyan Gongzhu’ at the four developmental stages. The scale bar represents the changes of gene expression pattern/metabolite accumulation: red/yellow rectangle indicates upregulated expression pattern, blue rectangle indicates downregulated expression pattern, and the white rectangle indicates gene or metabolite whose expression and accumulation did not change. The normalized signal intensity ranged from -1.0 to 1.0, which was consistent with color changes from blue to red/yellow. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 15

Expression profiles of the DEGs and accumulation of the DAMs involved in chlorophyll and heme metabolism pathway in leaves of ‘Ziyan Gongzhu’ at the four developmental stages. The scale bar represents the changes of gene expression pattern/metabolite accumulation: red/yellow rectangle indicates upregulated expression pattern, blue rectangle indicates downregulated expression pattern, and the white rectangle indicates gene or metabolite whose expression and accumulation did not change. The normalized signal intensity ranged from -1.0 to 1.0, which was consistent with color changes from blue to red/yellow. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 16

Expression profiles of the DEGs and accumulation of the DAMs involved in carotenoid metabolic pathway in leaves of ‘Ziyan Gongzhu’ at the four developmental stages. The scale bar represents the changes of gene expression pattern/metabolite accumulation: red/yellow rectangle indicates upregulated expression pattern, blue rectangle indicates downregulated expression pattern, and the white rectangle indicates gene or metabolite whose expression and accumulation did not change. The normalized signal intensity ranged from -1.0 to 1.0, which was consistent with color changes from blue to red/yellow. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)

Fig. 17

Schematic of changes in the regulatory genes and metabolites in leaf color-transition of ‘Ziyan Gongzhu’. ↑, upregulation of gene expression; ↓, downregulation of gene expression. (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A)