Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain a-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P) + -to-NAD(P)H ratios. Additionally, increased rates of CO 2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO 2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency.

Photosynthetic carbon assimilation including photorespiration is dynamically regulated during the day/night cycle. This includes transcriptional regulation, such as the light induction of corresponding genes, but little is known about the contribution of photorespiratory metabolites to the regulation of gene expression. Here, we examined diurnal changes in the levels of photorespiratory metabolites, of enzymes of the photorespiratory carbon cycle, and of corresponding transcripts in wild-type plants of Arabidopsis (Arabidopsis thaliana) and in a mutant with altered photorespiratory flux due to the absence of the peroxisomal enzyme Hydroxypyruvate Reductase1 (HPR1). Metabolomics of the wild type showed that the relative amounts of most metabolites involved in photorespiration increased after the onset of light, exhibited maxima at the end of the day, and decreased during the night. In accordance with those findings, both the amounts of messenger RNAs encoding photorespiratory enzymes and the respective protein contents showed a comparable accumulation pattern. Deletion of HPR1 did not significantly alter most of the metabolite patterns relative to wild-type plants; only serine accumulated to a constitutively elevated amount in this mutant. In contrast, the hpr1 mutation resulted in considerable deregulation of the transcription of photorespiration-related genes. This transcriptional deregulation could also be induced by the external application of L-serine but not glycine to the Arabidopsis wild type, suggesting that serine acts as a metabolic signal for the transcriptional regulation of photorespiration, particularly in the glycine-to-serine interconversion reactions.

T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis and therefore, exerts little control over the photorespiratory pathway. T-protein is the aminomethyltransferase of the glycine cleavage multienzyme system (GCS), also known as the glycine decarboxylase complex, and essential for photorespiration and one-carbon metabolism. Here, we studied what effects varying levels of the GCS T-protein would have on GCS activity, the operation of the photorespiratory pathway, photosynthesis, and plant growth. To this end, we examined Arabidopsis thaliana T-protein overexpression lines with up to threefold higher amounts of leaf T-protein as well as one knockdown mutant with about 5% residual leaf T-protein and one knockout mutant. Overexpression did not alter photosynthetic CO uptake and plant growth, and the knockout mutation was lethal even in the non-photorespiratory environment of air enriched to 1% CO. Unexpectedly in light of this very low T-protein content, however, the knockdown mutant was able to grow and propagate in normal air and displayed only some minor changes, such as a moderate glycine accumulation in combination with somewhat delayed growth. Neither overexpression nor the knockdown of T-protein altered the amounts of the other three GCS proteins, suggesting that the biosynthesis of the GCS proteins is not synchronized at this level. We also observed that the knockdown causes less T-protein mostly in leaf mesophyll cells, but not so much in the vasculature, and discuss this phenomenon in light of the dual involvement of the GCS and hence T-protein in plant metabolism. Collectively, this work shows that T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis and therefore exerts little control over the photorespiratory pathway.

ABSTRACTThe multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one-carbon compound 5,10-methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organised as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry-based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. The molar ratios of the leaf GDC component proteins are 1L2-4P2-8T-26H and 1L2-4P2-8T-20H for pea and Arabidopsis, respectively, as determined by mass spectrometry. The minimum mass of the plant leaf GDC ranges from 1,550-1,650 kDa, which is larger than previously assumed. The Arabidopsis GDC contains four times more of the isoforms GCS-P1 and GCS-L1 in comparison with GCS-P2 and GCS-L2, respectively, whereas the H-isoproteins GCS-H1 and GCS-H3 are fully redundant as indicated by their about equal amounts. Isoform GCS-H2 is not present in leaf mitochondria. In the cyanobacterium Synechocystis sp. PCC 6803, GCS proteins are present at low concentration but above the complex formation threshold reported for pea leaf GDC. Indeed, formation of a cyanobacterial GDC from the individual recombinant GCS proteins in vitro could be demonstrated. Presence and metabolic significance of a Synechocystis GDC in vivo remain to be examined but could involve multimers of the GCS H-protein that dynamically crosslink the three GCS enzyme proteins, facilitating glycine metabolism by the formation of multienzyme metabolic complexes.

Summary The multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one‐carbon compound 5,10‐methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organised as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry‐based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. The molar ratios of the leaf GDC component proteins are 1L2‐4P2‐8T‐26H and 1L2‐4P2‐8T‐20H for pea and Arabidopsis, respectively, as determined by mass spectrometry. The minimum mass of the plant leaf GDC ranges from 1550 to 1650 kDa, which is larger than previously assumed. The Arabidopsis GDC contains four times more of the isoforms GCS‐P1 and GCS‐L1 in comparison with GCS‐P2 and GCS‐L2, respectively, whereas the H‐isoproteins GCS‐H1 and GCS‐H3 are fully redundant as indicated by their about equal amounts. Isoform GCS‐H2 is not present in leaf mitochondria. In the cyanobacterium Synechocystis sp. PCC 6803, GCS proteins concentrations are low but above the complex formation threshold reported for pea leaf GDC. Indeed, formation of a cyanobacterial GDC from the individual recombinant GCS proteins in vitro could be demonstrated. Presence and metabolic significance of a Synechocystis GDC in vivo remain to be examined but could involve multimers of the GCS H‐protein that dynamically crosslink the three GCS enzyme proteins, facilitating glycine metabolism by the formation of multienzyme metabolic complexes. Data are available via ProteomeXchange with identifier PXD018211.

No abstract