Beyond the limits of photoperception: constitutively active PHYTOCHROME B2 overexpression as a means of improving fruit nutritional quality in tomato

Abstract

Photoreceptor engineering has recently emerged as a means for improving agronomically beneficial traits in crop species. Despite the central role played by the red/far-red photoreceptor phytochromes (PHYs) in controlling fruit physiology, the applicability of PHY engineering for increasing fleshy fruit nutritional content remains poorly exploited. In this study, we demonstrated that the fruit-specific overexpression of a constitutively active GAF domain Tyr²⁵² -to-His PHYB2 mutant version (PHYB2^Y252H ) significantly enhances the accumulation of multiple health-promoting antioxidants in tomato fruits, without negative collateral consequences on vegetative development. Compared with the native PHYB2 overexpression, PHYB2^Y252H -overexpressing lines exhibited more extensive increments in transcript abundance of genes associated with fruit plastid development, chlorophyll biosynthesis and metabolic pathways responsible for the accumulation of antioxidant compounds. Accordingly, PHYB2^Y252H -overexpressing fruits developed more chloroplasts containing voluminous grana at the green stage and overaccumulated carotenoids, tocopherols, flavonoids and ascorbate in ripe fruits compared with both wild-type and PHYB2-overexpressing lines. The impacts of PHYB2 or PHYB2^Y252H overexpression on fruit primary metabolism were limited to a slight promotion in lipid biosynthesis and reduction in sugar accumulation. Altogether, these findings indicate that mutation-based adjustments in PHY properties represent a valuable photobiotechnological tool for tomato biofortification, highlighting the potential of photoreceptor engineering for improving quality traits in fleshy fruits.

Keywords: Solanum lycopersicum; antioxidants; biofortification; carotenoids; flavonoids; photobiotechnology; vitamin C; vitamin E.

Figure 1

PHYB2 and PHYB2^Y252H overexpression modify fruit pigmentation and global transcriptomic profile. (a) PHYB2 or PHYB2^Y252H mRNA levels throughout fruit development of the tomato (Micro‐Tom cultivar) transgenic lines. Transcript abundance was normalized against PHYB2 mRNA levels detected in wild‐type (WT) at each stage. Data are mean ± SE, and dots represent individual values. Statistical differences within each stage are given by asterisks (Dunnett’s test with WT as control group, α = 0.05). (b) Visual phenotype of transgenic fruits in different development stages. (c) Venn's diagram analysis and Gene Set Enrichment Analysis (GSEA) of exclusive differentially expressed genes (DEGs) of PPC2::PHYB2 (PPC::B2) and PPC2::PHYB2^Y252H (PPC::B2^Y252H ) BK fruits compared with WT counterparts. Up‐ and down‐regulated genes are indicated in red and blue colours, respectively. Numbers represent overlapping changes among DEGs (FDR < 0.05). CPM values and GO terms are detailed in Tables S2 and S3, respectively. IMG, immature green; MG, mature green; BK, breaker; RR, red ripe.

Figure 2

Fruit‐specific PHYB2 and PHYB2^Y252H overexpression promote chloroplast biogenesis and differentiation and chlorophyll biosynthesis. (a) Representative optical microscopy (upper panels) and transmission electron microscopy images (bottom panels) of plastids at the pedicel region of immature green (IMG) fruits of wild‐type (WT), PPC2::PHYB2 (PPC::B2) and PPC2::PHYB2^Y252H (PPC::B2^Y252H ) plants of Micro‐Tom cultivar. Black asterisks indicate stacked thylakoids and white asterisks indicate plastoglobules. (b) Plastid number per pericarp cell of WT, PPC::B2 and PPC::B2^Y252H fruits at mature green (MG) stage. (c) Heatmap representation of the statistically significant differences in mRNA abundance of plastid‐related genes normalized against WT transcript levels at IMG stage (Dunnett's test, α = 0.05) via RT‐qPCR. Gene abbreviations and relative transcript values are detailed in Table S4. (d) Total chlorophyll content of WT, PPC::B2 and PPC::B2^Y252H fruits. (e) Gene Set Enrichment Analysis (GSEA) of exclusive differentially expressed genes (DEGs) of PPC::B2^Y252H MG fruits compared with WT counterparts. GO terms are detailed in Table S3. (f) Simplified chlorophyll biosynthetic pathway. Intermediate reactions are omitted. Up‐regulated chlorophyll biosynthesis‐related genes in PPC::B2^Y252H MG fruits according to RNA‐seq analysis are highlighted in red. Gene abbreviations, as well as logFC and FDR values, are detailed in Table S6. In (b) and (d), data are mean ± SE, dots represent individual values, and statistical differences within each stage are given by different letters (Tukey's Test, α = 0.05). BK, breaker; ALA, 5‐aminolevulinic acid.

Figure 3

Fruit‐specific PHYB2^Y252H overexpression promotes isoprenoid metabolism. (a) Carotenoid (lycopene, phytoene, phytofluene, lutein and β‐carotene) and tocopherol (α, β, δ and γ forms) content in wild‐type (WT), PPC2::PHYB2 (PPC::B2) and PPC2::PHYB2^Y252H (PPC::B2^Y252H ) red ripe (RR) fruits of Micro‐Tom cultivar. Data are mean ± SE, and dots represent individual values. Statistical differences are given by different letters (Tukey's test, α = 0.05). (b) Schematic representation of isoprenoid metabolism interconnecting carotenoid (orange), tocopherol (blue) and chlorophyll (green) pathways. Intermediate reactions are omitted. Heatmap representation of the statistically significant differences in mRNA abundance of isoprenoid‐related genes normalized against WT transcript levels at each stage (Dunnett's test, α = 0.05). Gene abbreviations and relative transcript values are detailed in Table S7. IMG, immature green; MG, mature green; BK, breaker; MEP, methylerythritol phosphate; GGDP, geranylgeranyl diphosphate; HGA, homogentisic acid; MBPQ, 2‐methyl‐6‐phytyl‐1,4‐benzoquinone; DMBPQ, 2,3‐dimethyl‐6‐phytyl‐1,4‐benzoquinone.

Figure 4

Flavonoid and ascorbate levels are increased in PHYB2^Y252H ‐overexpressing fruits. (a) Flavonoid content in wild‐type (WT), PPC2::PHYB2 (PPC::B2) and PPC2::PHYB2^Y252H (PPC::B2^Y252H ) red ripe (RR) fruits of Micro‐Tom cultivar. (b) Total ascorbate pool in RR fruits. (c,d) FLS and GME1 mRNA levels throughout fruit development. Transcript abundance was normalized against wild‐type (WT) samples at the mature green (MG) stage. (e) Total antioxidant activity of polar extracts expressed as Trolox Equivalent Antioxidant Capacity (TEAC) in RR fruits. Data are mean ± SE, and dots represent individual values. Metabolites, gene abbreviations and relative transcript values are detailed in Tables S8 and S9. Statistical differences within each stage are given by asterisks (Dunnett's test with WT as control group, α = 0.05) or different letters (Tukey's test, α = 0.05). DHA, dehydroascorbate; AsA, ascorbic acid; BK, breaker.

Figure 5

Fruit‐specific PHYB2 and PHYB2^Y252H overexpression impacts on primary metabolism are limited to changes in lipid and sugar contents. (a,b) Heatmap representation of the relative abundance of polar (a) and apolar (b) compounds in PPC2::PHYB2 (PPC::B2) and PPC2::PHYB2^Y252H (PPC::B2^Y252H ) red ripe (RR) transgenic fruits of Micro‐Tom cultivar. Only statistically different results are displayed in generalized logarithm (glog) normalized against the wild‐type (WT) (Dunnett's test, α = 0.05). Relative metabolite abundances are detailed in Table S10. Dendrograms indicate hierarchical clustering relationships between lines. (c) Soluble sugars (sucrose, glucose and fructose) content in RR fruits. (d) Starch content in immature green (IMG) fruits. (e) AGPaseL1 mRNA levels in IMG fruits. In (d,e), data are mean ± SE and dots represent individual values. Metabolites, gene abbreviations and relative transcript values are detailed in Tables S11 and S13. Statistical differences are given by different letters (Tukey's test, α = 0.05).

Figure 6

PHYB2^Y252H overexpression also promotes fruit nutritional content in commercial tomato cultivar. (a) Visual phenotype of mature green (MG) and red ripe (RR) fruits of wild‐type (WT), PPC2::PHYB2 (PPC::B2) and PPC2::PHYB2^Y252H (PPC::B2^Y252H ) plants of Ailsa Craig cultivar growing under greenhouse conditions. (b) PHYB2 or PHYB2^Y252H mRNA levels in MG fruits. (c) Total chlorophyll content and POR1 mRNA levels MG fruits. (d) Carotenoid (lycopene, phytoene, phytofluene, lutein and β‐carotene) and tocopherol (α, β, δ and γ forms) content in RR fruits. (d) GGDR and VTE3b mRNA levels in RR fruits. Transcript abundance was normalized against the WT samples. Data are mean ± SE, and dots represent individual values. Statistical differences are given by different letters (Tukey's test, α = 0.05). POR1, protochlorophyllide oxidoreductase; GGDR, geranylgeranyl diphosphate reductase; VTE3b, 2,3‐dimethyl‐5‐phytylquinol methyltransferase.