An integrated isotopic labeling and freeze sampling apparatus (ILSA) to support sampling leaf metabolomics at a centi-second scale

Abstract

Background: Photosynthesis close interacts with respiration and nitrogen assimilation, which determine the photosynthetic efficiency of a leaf. Accurately quantifying the metabolic fluxes in photosynthesis, respiration and nitrogen assimilation benefit the design of photosynthetic efficiency improvement. To accurately estimate metabolic fluxes, time-series data including leaf metabolism and isotopic abundance changes should be collected under precisely controlled environments. But for isotopic labelled leaves under defined environments the, time cost of manually sampling usually longer than the turnover time of several intermediates in photosynthetic metabolism. In this case, the metabolic or physiological status of leaf sample would change during the sampling, and the accuracy of metabolomics data could be compromised. Results: Here we developed an integrated isotopic labeling and freeze sampling apparatus (ILSA), which could finish freeze sampling automatically in 0.05 s. ILSA can not only be used for sampling of photosynthetic metabolism measurement, but also suit for leaf isotopic labeling experiments under controlled environments ([CO2] and light). Combined with HPLC–MS/MS as the metabolic measurement method, we demonstrated: (1) how pool-size of photosynthetic metabolites change in dark-accumulated rice leaf, and (2) variation in photosynthetic metabolic flux between rice and Arabidopsis thaliana. Conclusions: The development of ILSA supports the photosynthetic research on metabolism and metabolic flux analysis and provides a new tool for the study of leaf physiology.