Spatial patterning of chloroplasts and stomata in developing cacao leaves.

Leaf development and the coordinated formation of its key components is a fundamental process driving plant growth and adaptation. In tropical species like cacao, flush growth, a period of rapid leaf expansion, is particularly dependent on the optimized spatial patterns of chloroplasts and stomata. In this study, we investigated the patterns in cacao leaves during growth Stage C, a phase marked by rapid chlorophyll accumulation. Microscopic image data revealed significant acropetal variations in the size and density of chloroplast clusters and stomata, with the largest values found near the leaf base, mirroring the leaf greenness gradient. These findings suggest a coordinated developmental sequence between chloroplasts, stomata, and leaf ontogeny. A Support Vector Machine (SVM) model successfully classified distinct leaf regions based on these morphological features (>80% accuracy), highlighting the potential of machine learning applications in this area. Our results provide novel insights into the spatial coordination of chloroplast and stomatal development during cacao leaf maturation, offering a foundation for future research on flush growth optimization. To the best of our knowledge, this is the first report that combines microscopic data and machine learning analysis to investigate the leaf developmental process at stage C in cacao.