Cytological and Transcriptomic Analysis Provide Insights into the Formation of Variegated Leaves in Ilex × altaclerensis 'Belgica Aurea'

Abstract

Ilex × altaclerensis 'Belgica Aurea' is an attractive ornamental plant bearing yellow-green variegated leaves. However, the mechanisms underlying the formation of leaf variegation in this species are still unclear. Here, the juvenile yellow leaves and mature variegated leaves of I. altaclerensis 'Belgica Aurea' were compared in terms of leaf structure, pigment content and transcriptomics. The results showed that no obvious differences in histology were noticed between yellow and variegated leaves, however, ruptured thylakoid membranes and altered ultrastructure of chloroplasts were found in yellow leaves (yellow) and yellow sectors of the variegated leaves (variegation). Moreover, the yellow leaves and the yellow sectors of variegated leaves had significantly lower chlorophyll compared to green sectors of the variegated leaves (green). In addition, transcriptomic sequencing identified 1675 differentially expressed genes (DEGs) among the three pairwise comparisons (yellow vs. green, variegation vs. green, yellow vs. variegation). Expression of magnesium-protoporphyrin IX monomethyl ester (MgPME) [oxidative] cyclase, monogalactosyldiacylglycerol (MGDG) synthase and digalactosyldiacylglycerol (DGDG) synthase were decreased in the yellow leaves. Altogether, chlorophyll deficiency might be the main factors driving the formation of leaf variegation in I.altaclerensis 'Belgica Aurea'.

Keywords: Ilex × altaclerensis ‘Belgica Aurea’; chlorophyll; leaf variegation; transcriptome; ultrastructure.

Figure 1

Comparative leaf structures between the full-yellow juvenile leaf (A) and mature variegated leaf (C) of I. altaclerensis ‘Belgica Aurea’. (B,D) were transverse sections of full-yellow and variegated leaf, respectively. Scale bar = 100 μm. y: full-yellow juvenile leaf; v: yellow sectors of variegated leaf; g: green sectors of variegated leaf.

Figure 2

Ultrastructure of mesophyll cells from green (A) and yellow (C) sectors of variegated leaf, and the full-yellow juvenile leaf (B). GR: grana; TH: thylakoid; PG: plastoglobule. Scale bar = 2 μm.

Figure 3

Tissue localization of chlorophyll, carotenoids, and anthocyanins in the leaves analyzed by confocal laser-scanning microscopy. p: palisade parenchyma; s: spongy parenchyma; DAPI: 4′,6-diamidino-2-phenylindole; DIC: differential interference contrast; y: full-yellow juvenile leaf; v: yellow sectors of variegated leaf; g: green sectors of variegated leaf. Scale bar = 100 μm.

Figure 4

GO classification of unigenes. All the unigenes annotated were classified into three main categories: biological process, cellular component and molecular function.

Figure 5

The KEGG pathway classification of assembled unigenes. The x-axis indicates the number of unigenes and y-axis indicates the function classes.

Figure 6

Histogram of KOG function classification of unigenes. The categories of the KOG are shown on y-axis and the x-axis represents the number of unigenes.

Figure 7

Statistical and clustering analysis of differentially expressed genes. (A) Statistic of differentially expressed genes. Upregulated genes were shown in red and downregulated genes were shown in blue. (B) Venn diagram of differentially expressed genes. Numbers in the overlapping regions indicate genes differentially expressed in more than one pairwise comparison. (C) Clustering heat map of differentially expressed genes. Red color in the figure represents the highly expressed genes and blue color represents the genes expressed at low levels.

Figure 8

GO enrichment of DEGs.

Figure 9

A bubble plot of KEGG pathway enrichment of DEGs showing the top 20 enriched pathways. The size of dots represents the number of genes. MAPK, mitogen-activated protein kinase.

Figure 10

Quantitative real-time PCR verification of RNA-Seq data. Seven DEGs were selected for the verification of the reliability of the transcriptome sequencing results. The expression of β-actin was used as an internal reference. Data represent means ± SEM of three independent experiments. Different lowercase letters (a, b and c) indicated a significant difference among yellow leaves (y), green sectors of the variegated leaves (g) and yellow sectors of the variegated leaves (v) at p  <  0.05 level. a: significant difference compared to yellow sectors of variegated leaves; b: significant difference compared to green sectors of variegated leaves; c: significant difference compared to juvenile yellow leaves.