Transcriptomic and Structural Insights into Leaf Variegation Development in Ilex × 'Solar Flare'

Abstract

The mechanisms underlying leaf variegation in the ornamental Ilex × 'Solar Flare' remain poorly understood. To investigate this phenomenon, we conducted a comprehensive characterization of its variegated leaves. Compared to green sectors, yellow sectors exhibited severe chloroplast structural abnormalities, including swollen chloroplasts, damaged thylakoid membranes, and reduced chloroplast numbers. These yellow sectors also showed significantly lower chlorophyll and carotenoid levels, along with a depletion of key chlorophyll precursors-protoporphyrin IX (Proto IX), magnesium protoporphyrin IX (Mg-Proto IX), and protochlorophyllide (Pchlide). Photosynthetic efficiency was significantly impaired. Comparative transcriptome analysis identified 3510 differentially expressed genes (DEGs) between yellow and green sectors. Key disruptions in chlorophyll biosynthesis included upregulated CHLD expression and downregulated CHLH and CHLG expression, leading to impaired chlorophyll synthesis. Additionally, chlorophyll degradation was accelerated by PAO upregulation. Defective chloroplast development in yellow sectors was associated with the downregulation of GLK1, GLK2, and thylakoid membrane-related genes (PsbC, PsbO, PsbR, PsaD, and PsaH). These molecular alterations likely drive the variegated phenotype of I. × 'Solar Flare'. These observations advance our understanding of the genetic and physiological mechanisms regulating leaf variegation in this cultivar.

Keywords: chloroplast development; leaf variegation; photosynthesis; pigment metabolism; transcriptome.

Figure 1

Leaf traits, pigment profiles, and chlorophyll precursors content in VY and VG. (A) Ilex × ‘Solar Flare’ plant. (B) Leaf traits of VY and VG. Scale bar = 1 cm. (C) Pigments content of VY and VG. (D) Pigments ratio of VY and VG. (E) SPAD value of VY and VG. (F) Chlorophyll precursors content of VY and VG. (G) Flavonoid content of VY and VG. The data are expressed as mean ± SD, with t-test statistical significance represented by: ns (not significant), *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Abbreviations: ALA (5-aminolevulinic acid), Car (carotenoid), Chl (chlorophyll), Coprogen III (coproporphyrinogen), DW (dry weight), FW (fresh weight), Mg-Proto IX (Mg-protoporphyrin IX), PBG (porphobilinogen), Pchlide (protochlorophyllide), Proto IX (protoporphyrin IX), SD (standard deviation), SPAD (soil and plant analyzer development), Urogen III (uroporphyrinogen III), VG (the green sector of Ilex × ‘Solar Flare’), VY (the yellow sector of I. × ‘Solar Flare’).

Figure 2

Comparative leaf anatomy and epidermal characteristics of VY and VG. Images (A–D) correspond to VY samples. Images (E–H) correspond to VG samples. (A) Anatomical structure of VY. (B) Upper epidermis of VY. (C,D) Lower epidermis of VY. (E) Anatomical structure of VG. (F) Upper epidermis of VG. (G,H) Lower epidermis of VG. Scale bars: (B,F) = 500 μm, (C,G) = 100 μm, (A,E) = 50 μm, (D,H) = 30 μm. Abbreviations: AC (arch cover), GC (guard cell), LE (lower epidermis), PT (palisade tissue), R (ridge), SA (stomatal apparatus), S (stoma), ST (spongy tissue), UP (upper epidermis), VG (the green sector of Ilex × ‘Solar Flare’), VY (the yellow sector of I. × ‘Solar Flare’).

Figure 3

Chloroplast ultrastructure in VY and VG. Images (A–C) correspond to VY samples. Images (D–F) correspond to VG samples. Scale bars: (D) = 10 μm, (A) = 5 μm, (B,C,E) = 2 μm, (F) = 1 μm. Abbreviations: CP (chloroplast), CW (cell wall), GL (grana lamella), P (Plastoglobuli), SG (starch granule), SL (stroma lamella), VE (vesicle), VG (the green sector of Ilex × ‘Solar Flare’), VY (the yellow sector of I. × ‘Solar Flare’).

Figure 4

GO and KEGG enrichment analysis between VY and VG. (A) Pearson correlations. (B) Volcano plot distribution of DEGs. Log₂ (fold change) plotted against −log₁₀Padj, with upregulated transcripts highlighted in red and downregulated transcripts in green. (C) Top 30 enriched GO terms. (D) Top 20 enriched KEGG terms. The size of each bubble represents the number of associated DEGs, while color gradient (purple → blue → green → red) indicates increasing statistical significance (−log₁₀Padj). Enrichment score is displayed on the horizontal axis. Note: VG (the green sector of Ilex × ‘Solar Flare’), VY (the yellow sector of I. × ‘Solar Flare’).

Figure 5

Chlorophyll metabolism pathway associated with DEGs between VY and VG. (A) Chlorophyll metabolism pathway. Upregulated genes are shown in red and downregulated genes in blue. (B) Heatmap of DEGs in chlorophyll metabolism pathway. Heatmap displays mean log₂ (fold change) values calculated from three biological replicates per type with upregulated genes shown in red and downregulated genes in blue. Note: VG (the green sector of Ilex × ‘Solar Flare’), VY (the yellow sector of I. × ‘Solar Flare’).

Figure 6

Carotenoid metabolism pathway DEGs between VY and VG. (A) Carotenoid metabolism pathway. Upregulated genes are shown in red and downregulated genes in blue. (B) Heatmap of DEGs in carotenoid metabolism pathway. Heatmap displays mean log₂ (fold change) values calculated from three biological replicates per type with upregulated genes shown in red and downregulated genes in blue. Note: VG (the green sector of Ilex × ‘Solar Flare’), VY (the yellow sector of I. × ‘Solar Flare’).

Figure 7

Transcriptional changes in photosynthetic machinery between VY and VG. (A) Schematic representation of the photosynthesis pathway. (B) Schematic representation of plant light-harvesting chlorophyll–protein complexes. (C) Expression heatmap of photosynthesis-related DEGs. Values represent mean log₂ (fold change) calculated from triplicate samples of VY vs. VG. Upregulated genes appear in red; downregulated genes in blue. Note: VG (the green sector of Ilex × ‘Solar Flare’), VY (the yellow sector of I. × ‘Solar Flare’). Pathway diagrams in panels A and B were sourced from the KEGG database (https://www.kegg.jp, accessed on 10 January 2025).

Figure 8

Diagram illustrating potential molecular leaf variegation in Ilex × ‘Solar Flare.’ A vertical red arrow denotes gene upregulation, while a vertical blue arrow indicates downregulation. The variegated leaf originates from the mutant (I. × ‘Solar Flare’), whereas the green leaf is from its wild-type counterpart (I. × ‘Conaf’).