Combination of long-term 13CO2 labeling and isotopolog profiling allows turnover analysis of photosynthetic pigments in Arabidopsis leaves

Abstract

Background: Living cells maintain and adjust structural and functional integrity by continual synthesis and degradation of metabolites and macromolecules. The maintenance and adjustment of thylakoid membrane involve turnover of photosynthetic pigments along with subunits of protein complexes. Quantifying their turnover is essential to understand the mechanisms of homeostasis and long-term acclimation of photosynthetic apparatus. Here we report methods combining whole-plant long-term ¹³CO₂ labeling and liquid chromatography - mass spectrometry (LC-MS) analysis to determine the size of non-labeled population (NLP) of carotenoids and chlorophylls (Chl) in leaf pigment extracts of partially ¹³C-labeled plants.

Results: The labeling chamber enabled parallel ¹³CO₂ labeling of up to 15 plants of Arabidopsis thaliana with real-time environmental monitoring ([CO₂], light intensity, temperature, relative air humidity and pressure) and recording. No significant difference in growth or photosynthetic pigment composition was found in leaves after 7-d exposure to normal CO₂ (~ 400 ppm) or ¹³CO₂ in the labeling chamber, or in ambient air outside the labeling chamber (control). Following chromatographic separation of the pigments and mass peak assignment by high-resolution Fourier-transform ion cyclotron resonance MS, mass spectra of photosynthetic pigments were analyzed by triple quadrupole MS to calculate NLP. The size of NLP remaining after the 7-d ¹³CO₂ labeling was ~ 10.3% and ~ 11.5% for all-trans- and 9-cis-β-carotene, ~ 21.9% for lutein, ~ 18.8% for Chl a and 33.6% for Chl b, highlighting non-uniform turnover of these pigments in thylakoids. Comparable results were obtained in all replicate plants of the ¹³CO₂ labeling experiment except for three that were showing anthocyanin accumulation and growth impairment due to insufficient water supply (leading to stomatal closure and less ¹³C incorporation).

Conclusions: Our methods allow ¹³CO₂ labeling and estimation of NLP for photosynthetic pigments with high reproducibility despite potential variations in [¹³CO₂] between the experiments. The results indicate distinct turnover rates of carotenoids and Chls in thylakoid membrane, which can be investigated in the future by time course experiments. Since ¹³C enrichment can be measured in a range of compounds, long-term ¹³CO₂ labeling chamber, in combination with appropriate MS methods, facilitates turnover analysis of various metabolites and macromolecules in plants on a time scale of hours to days.

Keywords: 13CO2; Carotene; Carotenoids; Chlorophyll; Lutein; Pigment metabolism; Stable isotope labeling; Turnover.