Integration of C 1 and C 2 Metabolism in Trees

: C 1 metabolism in plants is known to be involved in photorespiration, nitrogen and amino acid metabolism, as well as methylation and biosynthesis of metabolites and biopolymers. Although the flux of carbon through the C 1 pathway is thought to be large, its intermediates are difficult to measure and relatively little is known about this potentially ubiquitous pathway. In this study, we evaluated the C 1 pathway and its integration with the central metabolism using aqueous solutions of 13 C-labeled C 1 and C 2 intermediates delivered to branches of the tropical species Inga edulis via the transpiration stream. Delivery of [ 13 C]methanol and [ 13 C]formaldehyde rapidly stimulated leaf emissions of [ 13 C]methanol, [ 13 C]formaldehyde, [ 13 C]formic acid, and 13 CO 2 , confirming the existence of the C1 pathway and rapid interconversion between methanol and formaldehyde. However, while [ 13 C]formate solutions stimulated emissions of 13 CO 2 , emissions of [ 13 C]methanol or [ 13 C]formaldehyde were not detected, suggesting that once oxidation to formate occurs it is rapidly oxidized to CO 2 within chloroplasts. 13 C-labeling of isoprene, a known photosynthetic product, was linearly related to 13 CO 2 across C 1 and C 2 ([ 13 C 2 ]acetate and [2- 13 C]glycine) substrates, consistent with reassimilation of C 1 , respiratory, and photorespiratory CO 2 . Moreover, [ 13 C]methanol and [ 13 C]formaldehyde induced a quantitative labeling of both carbon atoms of acetic acid emissions, possibly through the rapid turnover of the chloroplastic acetyl-CoA pool via glycolate oxidation. The results support a role of the C 1 pathway to provide an alternative carbon source for glycine methylation in photorespiration, enhance CO 2 concentrations within chloroplasts, and produce key C 2 intermediates (e.g., acetyl-CoA) central to anabolic and catabolic metabolism. 13 C 2 / 12 C] ratios reaching values up to 4–5% with a stronger 13 C-labeling of 13 C 2 acetate group relative to [ 13 C 1 ]acetate. Thus, [ 13 C 1 ]methyl acetate was the most abundant isotopologue emitted and [ 13 C 3 ]methyl acetate was more abundant than [ 13 C 2 ]methyl acetate. While the methoxy group was more strongly labeled than the acetate group under the [ 13 C]methanol, the [ 13 C 3 / 12 C] ratio of methyl acetate reached values up to 15%. Thus, a substantial fraction of methyl acetate was emitted as fully 13 C-labeled ([ 13 C 3 ]methyl acetate). The results show that under the pioneer species using aqueous solutions of 13 C-labeled C 1 (methanol, formaldehyde, formic acid) and C 2 (acetic acid, glycine) intermediates delivered via the transpiration stream. The results confirm that methanol initiates the complete C 1 pathway in plants (methanol, formaldehyde, formic acid, carbon dioxide) by providing real-time dynamic 13 C-labeling data showing their interdependence. Also evident is the rapid interconversion between methanol and formaldehyde, whereas once oxidation to formate occurs, it is quickly oxidized to CO 2 within chloroplasts where it can be re-assimilated by photosynthesis and therefore contribute to photosynthetic products like isoprene. We show that reassimilation of C 1 , respiratory, and photorespiratory CO 2 is a common mechanism for isoprene biosynthesis; a strong linear dependence of 13 C-labeling of isoprene on 13 C-labeling of CO 2 was observed across all C 1 and C 2 13 C-labeled substrates. Finally, we show, for the first time, that methanol and formaldehyde delivery to the transpiration stream leads to a rapid and quantitative conversion of carbon pools used in the biosynthesis of central C 2 compounds (acetic acid and acetyl CoA) and therefore represents a potentially new uncharacterized source of these key C 2 intermediates widely used as precursors for a diverse suite of anabolic (e.g., fatty acid biosynthesis) and catabolic (e.g., mitochondrial respiration) processes. in chloroplasts, our study presents the hypothesis that the integration of C 1 pathway into C 2/3 metabolism may boost carbon use efficiency and therefore represent an important mechanism by trees under photorespiratory conditions (e.g., high temperature stress). As agricultural crops are known to be high methanol producers, genetic manipulation of the C 1 pathway has the potential to improve yields and tolerance to environmental extremes, thereby providing a new tool to the agriculture, bioenergy, and biomanufacturing industries.