Overexpression of PSY1 increases fruit skin and flesh carotenoid content and reveals associated transcription factors in apple (Malus × domestica)

Abstract

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis.

Keywords: carotenoid biosynthesis; gene expression; phytoene synthase; plastids; transcription factors; transcriptome; transgenic apple.

FIGURE 1

PSY1 expression increased carotenoid accumulation in ‘Royal Gala’ apple fruit. (A) Fruit of WT and PSY transgenic lines (OE-1, OE-5, and OE-7) at different developmental stages. (B) Bar graphs of carotenoid content measured by HPLC as beta-carotene equivalents (left column) and chlorophyll (right column) in fruit skin (top panel) and fruit flesh (bottom panel) in wild type and transgenic lines OE-1, OE-5, and OE-7. The error bars represent the standard errors of the mean of three biological replicates, with each replicate a pool of 5–7 fruit. Bar graph with asterisk show significant difference from WT at the same fruit stage using Dunnett’s test (*P < 0.05; **P < 0.01).

FIGURE 2

Visualization of chlorophyll and carotenoid autofluorescence in wild type (WT) and PSY ‘Royal Gala’ fruit. Fresh fruit tissues, at 120, 135, and 150 D, were analyzed by confocal microscopy to show plastids containing chlorophyll (red), carotenoid (green) and both pigments (yellow). Graph represents fluorescence emission spectra of WT and PSY transgenic tissue. Fluorescence emission between 500 and 600 nm represents carotenoids and 650–750, chlorophyll pigments.

FIGURE 3

Comparison of carotenoid gene expression in wild-type (WT) and PSY ‘Royal Gala’ apple lines, OE-5 and OE-7, as determined by qPCR, relative to expression of housekeeping genes (MdActin and MdEF1A) in fruit skin (A) and fruit flesh (B). Bars represent means and SE of four biological replicates. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, zeta carotene desaturase; CRTISO, carotene isomerase; LCB, lycopene beta-cyclase; BCH, beta-carotene hydroxylase. Bar graph with asterisk shows significant difference from WT at the same fruit stage using Dunnett’s test (*P < 0.05; **P < 0.01).

FIGURE 4

Differentially expressed genes in PSY transgenic apple fruit. Venn diagram of number of differentially expressed genes (log₂ fold change ≥ 2, P < 0.05) between WT and OE-7 fruit at four developmental stages (90, 120, 135, 150 D). Upregulated (A), downregulated (B) in PSY fruit flesh.

FIGURE 5

Analysis of carotenoid-associated transcription factor genes. (A) Heat map of differentially expressed transcription factor genes (Supplementary Table S4) in apple fruit flesh during development. The average gene expression of biological replicates at each of the four stages: 90, 120, 135, and 150 D, from WT and PSY transgenic line OE-7, were clustered using Euclidean distance relationships. Green to red color gradient indicates low to high relative gene expression. (B) Correlation matrix of gene transcripts and carotenoid metabolites (β-carotene, BCAR; total carotenoid content, TOTCAR) in transgenic PSY fruit. Values represent correlation coefficients with color gradient from green to red indicating weak to strong correlations at P < 0.05.

FIGURE 6

PSY1 increased fruit carotenoid content in apple in the absence of light. (A) Representative bagged and non-bagged fruit of WT and PSY line OE-3 at 150 D. (B) Graphs showing carotenoid content in fruit skin (left) and flesh (right) of different PSY lines (OE-2, OE-3, OE-4) and WT. Error bars represent standard error of total carotenoid contents of three biological replicates. Bar graph with asterisk show significant difference from the non-bagged fruit of the same line using Dunnett’s test (*P < 0.05; **P < 0.01). (C) Stained fruit sections of WT and OE-3 displaying plastids (arrowed) in the cells.

FIGURE 7

Differentially expressed genes (DEGs) in bagged apple fruit. (A) Go Plot R of DEGs in bagged fruit of WT and OE-3 fruit. The scatter plots show log₂ fold change (logFC) for each gene under the gene ontology (GO) number. Red dots indicate upregulated genes and blue dots show downregulated genes. The z-score bars are a measure of the extent to which each identified process is upregulated or downregulated. (B) Heat map of differentially expressed transcription factor genes in bagged fruit. Clustering done using the average expression values of three biological replicates of bagged (BG) and non-bagged (NBG) fruit of WT and OE-3 transgenic line. Green to red color gradients indicate low to high relative gene expression. The highlighted TFs are common to the bagging and fruit development (Figure 5A) data sets.

FIGURE 8

Correlations between carotenoid content and TF gene expression as determined by qRT-PCR in (A) fruit skin and (B) fruit flesh of WT and PSY fruit. R² values indicate coefficient of determination.