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.

Control of histone demethylation by nuclear-localized α-ketoglutarate dehydrogenase

“…Measuring of primary metabolites in seedlings by liquid chromatography–tandem mass spectrometry (LC-MS/MS) were conducted as previously described with minor modifications ( 57 , 58 ). Briefly, seedlings were harvested and ground into fine powder in liquid nitrogen, followed by metabolite extraction.…”

“…The LC gradient was set with eluent A (10 mM ammonium acetate and 5% v/v acetonitrile solution, adjusted to pH 9.5 with ammonia water) and eluent B (acetonitrile) as follows: 0 to 1 min, 15% A; 1 to 8 min, 15 to 70% A; 8 to 20 min, 70 to 95% A; 20 to 22 min, 95% A; and 22 to 25 min, 15% A. For the MS analysis, QTRAP 6500+ (AB Sciex) was used in the MRM model, and all parameters were derived from previous studies with optimization ( 57 , 58 ). The concentration of each metabolite was calculated using a “concentration-peak area” curve of standard samples and presented as nanomoles per gram fresh weight.…”