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

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’.