Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development

Abstract

SUMMARYCellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.

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

“…The lack of any obvious embryonic and shoot defects in atdfb supports recent reverse genetic studies suggesting that AtDFC or AtDFD is able to compensate for the absence of AtDFB during embryo and aboveground vegetative development (Mehrshahi et al, 2010). However, a novel finding from our studies is that these other FPGS isoforms do not always function redundantly, at least within a specific window of postembryonic root development.…”

“…In a recent reverse genetics study, various double mutant combinations of these Arabidopsis FPGS isoforms resulted in dramatic developmental phenotypes, providing compelling evidence for the importance of polyglutamylated folates in plant development (Mehrshahi et al, 2010). However, because single FPGS mutants did not appear to have any obvious defects in the aboveground organs of mature plants that were grown directly from soil, it was concluded that redundancy in compartmentalized FPGS activity is likely operating during plant development (Mehrshahi et al, 2010). In this study, we show that disruption of a gene encoding a single FPGS isoform (AtDFB) resulted in a dramatic reduction in primary root elongation.…”

“…5-CHO-THF has been used to rescue the inability of cultured globular arrest1 embryos, which are defective in the gene encoding AtDFA, to form calli and partially rescue the developmental defects of Arabidopsis FPGS double mutants (Mehrshahi et al, 2010). When all three mutant alleles of AtDFB were planted on medium supplemented with 500 mM 5-CHO-THF, their primary roots were restored to wild-type lengths (Fig.…”

“…The mutant contained a T-DNA insertion in the At5g05980 gene, which encodes the plastidial FPGS isoform designated as AtDFB for Arabidopsis DHS-FPGS isoform B (Ravanel et al, 2001) and recently renamed FPGS1 by Mehrshahi et al (2010). The short primary root of atdfb was associated with a disorganized quiescent center (QC), reduced cell division and expansion, an extensively bundled actin cytoskeleton, and dissipation of the auxin gradient in the root cap.…”

“…In a recent reverse genetics study, different double mutant combinations to these Arabidopsis FPGS isoforms resulted in dramatic developmental phenotypes including embryo lethality, seedling lethality, late flowering, and dwarf plants with various reproductive defects (Mehrshahi et al, 2010). However, phenotypes were only reported in double FPGS mutants but not in single mutants, leading to the conclusion of redundancy in compartmentalized FPGS activity (Mehrshahi et al, 2010). Also in Arabidopsis, double mutants to genes encoding two 10-formyl-tetrahydrofolate (10-CHO-THF) deformylases (PurU) that metabolize 10-CHO-THF to formate and THF were smaller and paler compared with wild-type plants.…”