Chlorophyll depletion and anthocyanin compositional shifts distinguish spotted leaf sectors in Tricyrtis macropoda

Sharply delimited leaf spots may reflect local changes in chlorophyll background, anthocyanin composition and gene expression, but these components are rarely resolved between adjacent green and spotted tissues. Three spatially defined leaf tissue types were sampled from one wild T. macropoda individual: green tissue from an upper non-spotted leaf (GGreen), and adjacent green (SGreen) and dark spotted (SSpot) tissues from the same lower spotted leaf. Three independently processed spatial subsamples were collected from each tissue type, yielding nine analytical samples. Chlorophyll content and targeted anthocyanin profiles were measured, and RNA sequencing was performed, followed by candidate transcript-anthocyanin association analysis and qRT-PCR of pooled samples. SSpot retained approximately half the chlorophyll measured in adjacent SGreen, whereas their summed abundance of detected anthocyanin derivatives did not differ significantly. Seven derivatives met the fold-change criterion in the SSpot-SGreen comparison, indicating a compositional shift rather than broad anthocyanin accumulation. RNA-seq separated all three tissue types; the dominant variation distinguished GGreen from both tissues of the spotted leaf, while the local SSpot-SGreen contrast contained 1,459 differentially expressed genes. An SGR-like chlorophyll-degradation candidate and transcripts assigned to anthocyanin biosynthesis, modification and transport showed SSpot-biased expression, and selected transcript abundances covaried with the seven fold-change-selected anthocyanins. The data define SSpot as a chlorophyll-depleted, compositionally distinct sector within the sampled individual and prioritize candidates for validation across plants and leaves.