GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis

Abstract

In roots of Arabidopsis (Arabidopsis thaliana), L-lactate is generated by the reduction of pyruvate via L-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative L-lactate-metabolizing enzymes based on their homology to CYB2, the L-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses L-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than L-lactate. The key factor making GOX3 more efficient with L-lactate than GOX1 and GOX2 is a 5-to 10-fold lower K m for the substrate. Consequently, only GOX3 can efficiently metabolize L-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-offunction and overexpressor plants indicate that L-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on L-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes L-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of L-lactate after its formation under normoxia.

Lack of GLYCOLATE OXIDASE1, but Not GLYCOLATE OXIDASE2, Attenuates the Photorespiratory Phenotype of CATALASE2-Deficient Arabidopsis

“…Our genetic analysis indicated that the lack of GOX3, HAOX1, and HAOX2 does not influence the photorespiratory phenotype of cat2-2, which is in line with their substrate preferences and tissue-specific expression (Hodges et al, 2013;Esser et al, 2014;Engqvist et al, 2015). GOX1 and GOX2, on the other hand, metabolize glycolate in vitro with similar kinetic parameters and have been assumed to equally support the photorespiratory pathway.…”

“…S7). Similarly, the lack of GOX3, which is predominantly expressed in roots and senescing leaves and uses glycolate and L-lactate with comparable efficiency (Engqvist et al, 2015), did not reduce the GOX activity (Supplemental Fig. S7).…”

“…GOX1 and GOX2 are the major isoforms found in leaf peroxisomes (Reumann et al, 2007;Foyer et al, 2009;Dellero et al, 2016). GOX3, on the other hand, is mainly expressed in roots where it converts glycolate and L-lactate with similar efficiency, making it an important player in lactate metabolism (Engqvist et al, 2015). HAOX1 and HAOX2 act predominantly on medium-and long-chain hydroxy acids, show a negligible activity toward glycolate (Esser et al, 2014), and are mainly expressed in seeds (Hodges et al, 2013).…”

Peroxisomes: versatile organelles with diverse roles in plants

“…Using dynamic light conditions that mimic the natural environment more closely, this study was able to reveal photosynthetic deficiencies in some peroxisomal mutants, such as gox1 (glycolate oxidase 1) and pxn1 (peroxisomal NAD + transporter), which otherwise show no apparent phenotypes in constant laboratory light conditions, suggesting the importance of these peroxisomal proteins in photosynthetic performance under high/ dynamic light conditions. Mutant and gene expression analyses in Arabidopsis showed that GOX1 and GOX2 function in photorespiration, whereas GOX3 is more involved in metabolizing l-lactate to sustain low concentrations of l-lactate in roots (Engqvist et al, 2015), and HAOX1 and HAOX2 are highly expressed in seeds (reviewed in Dellero et al, 2016). Li et al, 2019b).…”

The ancestors of diatoms evolved a unique mitochondrial dehydrogenase to oxidize photorespiratory glycolate

“…Enzymatic activity was assayed spectrophotometrically according to Engqvist et al (2015) using a Synergy HT Plate reader with Gen5 Analysis software (Biotek). The standard oxidase assay mixture consisted of 100 mM 3-Morpholinopropane-1-sulfonic acid (MOPS-NaOH, pH 7.0) or Tris-HCl (pH 8.5) containing 0.5 mM EDTA, 5 mM MgSO 4 , 4 mM phenylhydrazin, and either 2 mM FAD or 2 mM FMN as cofactors.…”