Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes

Abstract

Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of 13 CO 2 and 15 NH 4 NO 3 . The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, 13 CO 2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous 13 CO 2 and 15 NH 4 NO 3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong 13 C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping.

In silico metabolic network analysis of Arabidopsis leaves

“…And thirdly, a powerful collection of modelling approaches, currently available to model and simulate stoichiometric metabolic networks, can be adapted to plant metabolic networks in a straightforward manner [19]. Among the available modelling approaches are in silico based analyses, such as elementary flux mode analysis [20, 21] and extreme pathway analysis [22, 23], as well as analyses, which rely on experimental data to deliver necessary constraints, such as 13 C-metabolic flux analysis ( 13 C-MFA) [24–26], high-throughput isotope based metabolic screening [27], flux balance analysis [16] and metabolic control analysis [28]. Among the approaches, in silico simulation appears particularly interesting due to its high speed, given e.g.…”

Preparation of uniformly labelled 13C- and 15N-plants using customised growth chambers

“…The increasing popularity of isotopically labelled plant material goes hand in hand with the development of complementary analytical tools e.g. NMR and high throughput GC-MS and LC-HRMS which enable differentiation between native and labelled metabolites (13,14) as well as the availability of bioinformatics (for automated data processing) and statistical tools (for differential metabolome analysis). Therefore, protocols for the reproducible production of globally labelled biological organisms including higher plants with target stable isotopes are of increasing interest.…”

“…Global 13 C-labelling is used to produce uniformly 13 C-labelled plants (here termed as 13 C 14 N plants) and requires a continuous supply of 13 CO 2 throughout the whole cultivation. The 13 CO 2 is applied as a substrate which is in planta further converted to all assimilates required to sustain plant metabolism.…”

“…Therefore, airtight cultivation chambers (labelboxes) with 13 CO 2 atmosphere become necessary. As the preparation of 13 C enriched plants affords long-term cultivations the technical equipment has to enable the regulation of growth parameters such as temperature, light and the atmospheric humidity as well as the levels of CO 2 and O 2 . Global 15 N-labelling is accomplished by applying 15 N-containing nutrient solution through the root system and 15 N distribution takes place by transport via xylem and the initial formation of 15 N-labelled glutamine in the plastids and further via transamination reactions.…”

“…Up to know, the stable isotopically labelling procedures are implemented in the labs of other research groups who were successful at producing different plants with different degrees of enrichment with 13 C (18-22), 15 N (23-26) and using both elements in form of dual labelling ( 13 C 15 N plants) or single labelling of plants cultivated in parallel ( 13 C 14 N and 12 C 15 N plants) (13,15,16,27). Here, we aim to present a fully automated and robust labelling equipment implemented to produce highly 13 C and 15 N enriched wheat, cultivated in parallel under the same conditions with high N and C metabolome coverage.…”