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

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-ß-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.

In Vivo Imaging and Quantification of Carbon Tracer Dynamics in Nodulated Root Systems of Pea Plants.

Legumes associate with root colonizing rhizobia that provide fixed nitrogen to its plant host in exchange for recently fixed carbon. There is a lack of understanding of how individual plants modulate carbon allocation to a nodulated root system as a dynamic response to abiotic stimuli. One reason is that most approaches are based on destructive sampling, making quantification of localised carbon allocation dynamics in the root system difficult. We established an experimental workflow for routinely using non-invasive Positron Emission Tomography (PET) to follow the allocation of leaf-supplied 11C tracer towards individual nodules in a three-dimensional (3D) root system of pea (Pisum sativum). Nitrate was used for triggering a reduction of biological nitrogen fixation (BNF), which was expected to rapidly affect carbon allocation dynamics in the root-nodule system. The nitrate treatment led to a decrease in 11C tracer allocation to nodules by 40% to 47% in 5 treated plants while the variation in control plants was less than 11%. The established experimental pipeline enabled for the first time that several plants could consistently be labelled and measured using 11C tracers in a PET approach to quantify C-allocation to individual nodules following a BNF reduction. Our study demonstrates the strength of using 11C tracers in a PET approach for non-invasive quantification of dynamic carbon allocation in several growing plants over several days. A major advantage of the approach is the possibility to investigate carbon dynamics in small regions of interest in a 3D system such as nodules in comparison to whole plant development.

Non-invasive determination of plant biomass with microwave resonators.

Non-invasive and rapid determination of plant biomass would be beneficial for a number of research aims. Here, we present a novel device to non-invasively determine plant water content as a proxy for plant biomass. It is based on changes of dielectric properties inside a microwave cavity resonator induced by inserted plant material. The water content of inserted shoots leads to a discrete shift in the centre frequency of the resonator. Calibration measurements with pure water showed good spatial homogeneity in the detection volume of the microwave resonators and clear correlations between water content and centre frequency shift. For cut tomato and tobacco shoots, linear correlations between fresh weight and centre frequency shift were established. These correlations were used to continuously monitor diel growth patterns of intact plants and to determine biomass increase over several days. Interferences from soil and root water were excluded by shielding pots with copper. The presented proof of principle shows that microwave resonators are promising tools to quantitatively detect the water content of plants and to determine plant biomass. As the method is non-invasive, integrative and fast, it provides the opportunity for detailed, dynamic analyses of plant growth, water status and phenotype.