Spatial and widely targeted metabolomics and anatomical analysis reveal the mechanisms associated with petal bicolor patterning of two Dendrobium species

Orchid flowers possess high ornamental value owing to their delicate structure and color. The color patterns are a result of unbalanced pigment distribution among the petal segments, with bicolor being the most common and important for pollination. However, little is known about the relationship between petal bicolor patterns and metabolites and vein structure. Here, we focused on Dendrobium nobile Lindl., with pigmentation mainly at the tip of the flower petal, and D. anosmum ‘A Touch of Class’ Lindl, with pigmentation along the entire petal edge. We combined spatial and widely targeted metabolomics with venation anatomical analysis to identify metabolite and structural differences between the two flower bicolor patterns. In our study, we discovered that the concentration of energy-related substances, such as sugars, did not exhibit significant variance across different color regions of the flower. However, defensive metabolites like alkaloids, phenolic acids, and polyamines were found to be more concentrated in areas with more vibrant pigmentation. Specifically, in D. nobile, a higher accumulation of these metabolites was observed in the vein regions, as identified through spatial metabolic analysis. The study also revealed that different vein types had distinct distribution patterns, yet no significant difference was observed in the density of longitudinal and transverse veins between the two examined species. Notably, D. nobile showed a significantly higher density of open veins in the distal part (pigmented part) of the petal compared to the proximal part. In contrast, D. anosmum 'A Touch of Class' exhibited no significant difference in open vein density between these two regions. Our findings contribute to a deeper understanding of color patterning in orchid flowers and provide a theoretical foundation for the breeding of Dendrobium species.