Lessons from relatives: C4 photosynthesis enhances CO2assimilation during the low-light phase of fluctuations

Despite the global importance of species with C 4 photosynthesis, there is a lack of consensus regarding C 4 performance under fluctuating light. Contrasting hypotheses and experimental evidence suggest that C 4 photosynthesis is either less, or more efficient in fixing carbon under fluctuating light than the ancestral C 3 form. Two main issues were identified that may underly the lack of consensus: neglect of evolutionary distance between selected C 3 and C 4 species and use of contrasting fluctuating light treatments. To circumvent these issues, we compared photosynthetic responses to fluctuating light across three independent phylogenetically controlled comparisons between C 3 and C 4 species from Alloteropsis , Flaveria , and Cleome genera under 21% and 2% O 2 . Leaves were subjected to repetitive stepwise changes in light intensity (800 and 100 µmol m -2 s -1 PFD) with three contrasting durations: 6, 30 and 300 seconds. These experiments reconcile the opposing results found across previous studies showing that 1) stimulation of CO 2 assimilation in C 4 species during the low light phase was both stronger and more sustained than in C 3 species; 2) CO 2 assimilation patterns during the high light phase were genus-specific rather than impacted by photosynthetic pathway; and 3) the duration of each light step in the fluctuation regime can strongly influence experimental outcomes. One sentence significance statement Comparing photosynthesis in three pairs of closely related C 3 and C 4 species across three fluctuating light regimes showed that C 4 photosynthesis has a systematic advantage under the low light phase not related to suppression of photorespiration, while the comparative efficiency under the high light phase was not determined by photosynthetic pathway.