13C labeling to determine intra-leaf photosynthetic heterogeneity dynamics during drought and rewatering.

The spatial-temporal dynamics of photosynthetic heterogeneity within leaves under environmental fluctuations are still not well understood, limiting accurate assessments of plant photosynthetic capacity. Here, we combined 13CO2 labeling with water status monitoring to quantify variations in intra-leaf photosynthetic rate (A13C) during a drought-rewatering cycle. Hydraulic properties and anatomical traits were further investigated in well-watered plants. Under well-watered conditions, both A13C and water use efficiency (WUE) increased progressively from the leaf base to the tip. However, severe drought followed by rewatering eliminated this longitudinal gradient in A13C, with leaf tips exhibiting significantly impaired photosynthetic recovery. This impairment was associated with tip-specific limitations: 1) reduced water storage capacity exacerbating dehydration, and 2) increased vulnerability of hydraulic conductance potentially leading to hydraulic failure. Importantly, the impaired leaf tips can reduce whole-leaf WUE. Our findings demonstrate the utility of 13CO2 labeling for measuring leaf photosynthetic heterogeneity, reveal the dynamics of photosynthetic heterogeneity in response to environmental fluctuations, and highlight the vulnerability of leaf tips.