Developmental stages of delayed-greening leaves inferred from measurements of chlorophyll content and leaf growth

Chlorophyll (Chl) accumulation and leaf growth were analysed in delayed-greening leaves of Theobroma cacao (L.) to examine whether these parameters are correlated during leaf development and can be used as non-destructive indicators of leaf developmental stages. There was a clear correlation between Chl content and leaf relative growth rate (RGR) and between Chl content and percentage of full leaf expansion (%FLE) under different growth conditions. Five distinct developmental phases were defined according to the correlation between these parameters and corroborated by data from the analyses of leaf growth (epidermal cell size and specific leaf area) or photosynthetic properties (maximal PSII efficiency, CO2 assimilation and non-structural carbohydrate contents). The five phases were characterised by rapid leaf expansion by cell division (I), pronounced cell expansion (II), development of photosynthetic capacity concomitant with reinforcement of leaf structure (III), and maturation (IV and V). The transition from cell division to cell expansion happened uniformly across the leaf lamina between phase I and II; the sink-to-source transition was found between phase III and IV. These results demonstrate coordinated development of photosynthetic machinery and leaf structure in delayed-greening leaves and provide a simple and non-invasive method for estimation of leaf developmental stages in T. cacao.