Plant NADPH-dependent thioredoxin reductases are crucial for the metabolism of sink leaves and plant acclimation to elevated CO2.

Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.

Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism.

BACKGROUND: Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state 13C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. However, the experimental system needs to be improved for continuous carbon enrichment from labelled sugars into metabolites for longer periods until complex secondary metabolism reaches steady state. RESULTS: We developed an in vitro plant culture strategy by using peppermint as a model plant with minimizing unlabelled carbon fixation where growing shoot tip was strongly dependent on labelled glucose for their carbon necessity. We optimized the light condition and detected the satisfactory phenotypical growth under the lower light intensity. Total volatile terpenes were also highest at the same light. Analysis of label incorporation into pulegone monoterpene after continuous U-13C6 glucose feeding revealed nearly 100% 13C, even at 15 days after first leaf visibility (DALV). Label enrichment was gradually scrambled with increasing light intensity and leaf age. This study was validated by showing similar levels of label enrichment in proteinogenic amino acids. The efficiency of this method was also verified in oregano. CONCLUSIONS: Our shoot tip culture depicted a method in achieving long term, stable and a high percentage of label accumulation in secondary metabolites within a fully functional growing plant system. It recommends the potential application for the investigations of various facets of plant metabolism by steady state 13C-MFA. The system also provides a greater potential to study sink leaf metabolism. Overall, we propose a system to accurately describe complex metabolic phenotypes in a growing plant.