Quantify the photochemical activity of variegated leaves by determining their light absorptance and maximum fluorescence yield under light

There is no consensus whether variegated leaves show superior photosynthesis performance than their congeneric or co-occurring green leaves, and the reason(s) remains ambiguous. Based on our literature review, when two main methods—CO₂ assimilation and chlorophyll fluorescence—were combined to assess the photosynthetic capacity of variegated leaves, the results were often inconsistent. We supposed this inconsistency is due to inaccurate estimations of the absorptance by photosynthetic pigments and the Fm' (maximum fluorescence when all Photosystem II reaction centers are closed), rendering inaccuracies of derived fluorescence metrics. To minimize these two uncertainties, we developed a simple two-layer model to quantify the absorptance by photosynthetic pigments, and applied the inverse relation between fluorescence intensity and saturating light pulse intensity to obtain the reliable Fm'. Results showed the actual chlorophyll absorptance of 8 variegated leaves’ species (cultivar) varied from 28.62 % to 80.31 % in white light. Fm′ of the 10 variegated leaves’ species was underestimated by 4.42–10.42 % by the conventional method. After corrections of absorptance and Fm′, we found that ETR2 (electron transport rate through PSII) for the 10 studied species was overestimated from 10.4 % to 81.64 % by the conventional method. At last, we validated this method by correlating Pn (net photosynthetic rate) and ETR2 under the condition of minimized alternative electron sinks. The slope between Pn and ETR2 was close to the theoretical value. The improved method was applied to compare the photosynthetic capacities between variegated and its congeneric green leaves, both gas exchange and fluorescence results showed a consistent pattern that variegated leaves tends to be more shade-acclimated with lower photosynthetic capacity. Overall, this method significantly improves the reliability of chlorophyll fluorescence measurements in variegated leaves, offering a robust tool to advance photosynthesis research across a broader range of plant taxa.