5-Formyltetrahydrofolate Is an Inhibitory but Well Tolerated Metabolite in Arabidopsis Leaves

Abstract

5-Formyltetrahydrofolate (5-CHO-THF) is formed viaa second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 mol of CO 2 mol ؊1 ) and non-photorespiratory (3200 mol of CO 2 mol ؊1 ) conditions. The mutation had only mild visible effects at 370 mol of CO 2 mol ؊1 , reducing growth rate by ϳ20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 mol of CO 2 mol ؊1 , the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants. 5-Formyltetrahydrofolate (5-CHO-THF)1 is formed from 5,10-methenyltetrahydrofolate (5,10-CHϭTHF) by a hydrolytic reaction catalyzed by serine hydroxymethyltransferase (SHMT) in the presence of glycine (1, 2). Spontaneous chemical hydrolysis of 5,10-CHϭTHF may be a minor additional source (3). 5-CHO-THF is the most stable natural folate and the most enigmatic, for it is the only one that does not serve as a cofactor in one-carbon metabolism. Instead, 5-CHO-THF is a potent inhibitor of SHMT and most other folate-dependent enzymes in vitro (4, 5). 5-CHO-THF probably acts as a stable storage form of folate in seeds and fungal spores (5-7), but it is not clear what role, if any, it plays in metabolically active tissues (8). This question is particularly pertinent for leaves. Leaf mitochondria have very high levels of SHMT and, during photorespiration, receive a massive influx of glycine (which leads to a matching SHMT-mediated glycine 3 serine flux) (9). Conditions in leaf mitochondria therefore favor 5-CHO-THF formation (Fig. 1). Indeed, 5-CHO-THF can comprise 50% of the folate pool in leaf mitochondria (10, 11), which is far more than in mammalian mitochondria (12)(13)(14). Furthermore, 5-CHO-THF is reported to make up 14 -40% of the folate pool in leaves and o...

The Folylpolyglutamate Synthetase Plastidial Isoform Is Required for Postembryonic Root Development in Arabidopsis

“…S5D). This indicates that the exogenously supplied 5-CHO-THF was effectively taken up and converted into metabolic active folates by 5-formyl-THF cycloligase (Goyer et al, 2005; Fig. 1).…”

“…9D). Polyglutamylated 5-CHO-THF was found to be a major folate in leaf mitochondria (Goyer et al, 2005;Orsomando et al, 2005). Thus, it is possible that reduction of polyglutamylation in plastids induced a redirection of folate flux, which then caused an accumulation of this folate class in roots.…”

“…Shoots and roots from 15-d-old wild-type and mutant seedlings were analyzed for folates from 1 g of total tissue by HPLC as described previously (Goyer et al, 2005;Orsomando et al, 2005) with slight modifications: half of the folate extract was deglutamylated using a purified mixture of Arabidopsis recombinant g-glutamyl hydrolase isoforms 1 and 2 (Orsomando et al, 2005; 0.1 mg of each protein per g of tissue, with a 2-h incubation time). The rest of the extract was used for profiling folate polyglutamylation.…”

Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase

“…5-CHO-THF is metabolized back to 5,10-CH=THF by 5-formyltetrahydrofolate cycloligase, encoded in Arabidopsis by a single 5-FCL gene (Roje et al, 2002). However, the loss of 5-FCL activity does not dramatically reduce plant growth under photorespiratory conditions, despite the accumulation of 5-CHO-THF (Goyer et al, 2005). No compensatory increase in mitochondrial SHMT activity could be detected in the 5-FCL mutants, suggesting some other mechanism to tolerate elevated 5-CHO-THF (Goyer et al, 2005).…”

“…However, the loss of 5-FCL activity does not dramatically reduce plant growth under photorespiratory conditions, despite the accumulation of 5-CHO-THF (Goyer et al, 2005). No compensatory increase in mitochondrial SHMT activity could be detected in the 5-FCL mutants, suggesting some other mechanism to tolerate elevated 5-CHO-THF (Goyer et al, 2005). We speculate that the role of Fd-GOGAT bound to SHMT1 may be to reduce the sensitivity of SHMT activity to 5-CHO-THF or to oppose the accumulation of 5-CHO-THF, possibly by reducing the hydrolysis of of 5,10-CH=THF by SHMT.…”