Comparative physiology and transcriptome analysis revealed the mechanisms underlying red leaf formation in Cymbidium ensifolium

• Abnormal chloroplast structure and reduced photosynthesis in red leaves. • Low gene expression levels of chlorophyll metabolism and photosynthesis in red leaves. • Levels of anthocyanin metabolites and related gene expression increase in red leaves. Colorful leaves, particularly red ones, represent an important ornamental trait of Cymbidium ensifolium . However, the mechanisms underlying red leaf formation in C. ensifolium are not well understood. In this study, we examined the phenotypic, physiological, and transcriptomic differences between red and green leaves at various developmental stages. We found that red leaves exhibited significantly lower levels of chlorophyll a , chlorophyll b , carotenoids, and total chlorophyll across all stages, while anthocyanin levels were notably higher. Ultrastructural analysis revealed abnormalities in the chloroplast structure of red leaves, including fewer chloroplasts, ruptured thylakoid membranes, an indistinct matrix layer, and the accumulation of osmiophilic particles, which led to reduced photosynthetic capacity. In addition, transcriptomic analysis showed significant differences in the expression of genes related to anthocyanin biosynthesis, chlorophyll metabolism, and photosynthesis between red and green leaves at different stages of growth. Therefore, we suggest that red leaf formation in C. ensifolium is driven by reduced expression of chlorophyll metabolism genes, resulting in impaired chloroplast development and chlorophyll synthesis, while upregulation of anthocyanin biosynthesis genes promotes anthocyanin accumulation, leading to the red coloration. These findings provide valuable insights into color formation mechanisms of red leaves in C. ensifolium .