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Photorespiration is the second largest carbon flux in most leaves and is integrated into metabolism broadly including one-carbon (C1) metabolism. Photorespiratory intermediates such as serine and others may serve as sources of C1 units, but it is unclear to what degree this happens in vivo, whether altered photorespiration changes flux to C1 metabolism, and if so through which intermediates. To clarify these questions, we quantified carbon flux from photorespiration to C1 metabolism using 13CO2 labelling and isotopically non-stationary metabolic flux analysis in Arabidopsis thaliana under different O2 concentrations which modulate photorespiration. The results revealed that ~5.8% of assimilated carbon passes to C1 metabolism under ambient photorespiratory conditions, but this flux greatly decreases under limited photorespiration. Furthermore, the primary carbon flux from photorespiration to C1 metabolism is through serine. Our results provide fundamental insight into how photorespiration is integrated into C1 metabolism, with possible implications for C1 metabolic response to climate change. 

SUMMARY The metabolism of tetrahydrofolate (H₄PteGlu_n)‐bound one‐carbon (C₁) units (C₁metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C₁units in specific organelles and tissues. One possible source of C₁units is via formate‐tetrahydrofolate ligase, which catalyzes the reversible ATP‐driven production of 10‐formyltetrahydrofolate (10‐formyl‐H₄PteGlu_n) from formate and tetrahydrofolate (H₄PteGlu_n). Here, we report biochemical and functional characterization of the enzyme fromArabidopsis thaliana(AtFTHFL). We show that the recombinant AtFTHFL has lowerK_mandk_catvalues with pentaglutamyl tetrahydrofolate (H₄PteGlu₅) as compared to monoglutamyl tetrahydrofolate (H₄PteGlu₁), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation ofArabidopsisplants with the EGFP‐tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T‐DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H₄PteGlu_n + 5,10‐methylene‐H₄PteGlu_nand serine, accompanied with the depletion of formate and glycolate, in roots of the transgenicArabidopsisplants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C₁network in roots with C₁units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5‐methyl‐H₄PteGlu_n, methionine, andS‐adenosylmethionine. This finding has implications for any future attempts to engineer one‐carbon unit‐requiring products through manipulation of the one‐carbon metabolic network in non‐photosynthetic organs.