Characterization of Two Arabidopsis L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis

Abstract

L-Ascorbic acid (AsA, vitamin C) is an essential antioxidant for plants and animals. There are four known ascorbate biosynthetic pathways in plants: the L-galactose, L-gulose, D-galacturonate, and myo-inositol routes. These pathways converge into two AsA precursors: L-galactono-1,4-lactone and L-gulono-1,4-lactone (L-GulL). This work focuses on the study of L-gulono-1,4-lactone oxidase (GulLO), the enzyme that works at the intersect of the gulose and inositol pathways. Previous studies have shown that feeding L-gulono-1,4-lactone to multiple plants leads to increased AsA. There are also reports showing GulLO activity in plants. We describe the first detailed characterization of a plant enzyme specific to oxidize L-GulL to AsA. We successfully purified a recombinant Arabidopsis GulLO enzyme (called AtGulLO5) in a transient expression system. The biochemical properties of this enzyme are similar to the ones of bacterial isozymes in terms of substrate specificity, subcellular localization, use of flavin adenine dinucleotide (FAD) as electron acceptor, and specific activity. AtGulLO5 is an exclusive dehydrogenase with an absolute specificity for L-GulL as substrate thus differing from the existing plant L-galactono-1,4-lactone dehydrogenases and mammalian GulLOs. Feeding L-GulL to N. benthamiana leaves expressing AtGulLO5 constructs led to increased foliar AsA content, but it was not different from that of controls, most likely due to the observed low catalytic efficiency of AtGulLO5. Similar results were also obtained with another member of the AtGulLO family (AtGulLO3) that appears to have a rapid protein turnover. We propose that AsA synthesis through L-GulL in plants is regulated at the post-transcriptional level by limiting GulLO enzyme availability.

Manipulation of Ascorbate Biosynthetic, Recycling, and Regulatory Pathways for Improved Abiotic Stress Tolerance in Plants

“…Increased expression of the rat (Rattus rattus L.) L-GulLO gene has increased ascorbate concentrations in a range of species, including 1.8-to 2.0-fold in Arabidopsis [56,57], 7.0-fold in lettuce [58], 2.4-fold in potato [59], 7.0-fold in tobacco [58], and 1.5-fold in tomato [60] (Table 1). However, increased expression of the Arabidopsis L-GulLOs genes in Arabidopsis did not change ascorbate concentrations [61] ( Table 1) and increased expression of the Arabidopsis L-GulLOs genes in tobacco cells only increased ascorbate concentrations when supplied with L-gulono-1,4-lactone, together suggesting that the L-gulose pathway is not a major contributor to ascorbate biosynthesis in plants [62]. Increased expression of the rat L-GulLO gene in plants has been associated with enhanced tolerance to salt, cold, heat, drought, and pyrene-and methyl viologen-induced stress [56,59,60] (Table 1).…”

The Myo‐inositol pathway does not contribute to ascorbic acid synthesis

“…Aboobucker et al . () also overexpressed the l ‐gulono‐1,4‐lactonase genes in Arabidopsis without any increase in AsA levels.…”

“…Only after feeding high concentrations of L-gulono-1,4-lactone (10 mM) did the BY2 cells have elevated levels of AsA. Aboobucker et al (2017) also overexpressed the L-gulono-1,4-lactonase genes in Arabidopsis without any increase in AsA levels.…”

“…These data do not confirm enzyme activity or physiological relevance. Aboobucker et al (2017) recently characterised two genes from Arabidopsis with Lgulono-1,4-lactone oxidase activity. The enzymes have a high K m (33 mM) and a very low k cat (0.005Ás À1 ).…”

Characterization of a L-Gulono-1,4-Lactone Oxidase Like Protein in the Floral Nectar of Mucuna sempervirens, Fabaceae

“…Three putative Arabidopsis GulLOs (AtGulLO2, 3, and 5) over-expressed in tobacco BY-2 cell cultures increased AsA after feeding with L -GulL ( Maruta et al, 2010 ). Aboobucker et al (2017) purified a recombinant Arabidopsis GulLO enzyme (AtGulLO5) in a transient expression system. They proved that AtGulLO5 is an exclusive dehydrogenase with an absolute specificity for L -GulL as substrate, thus differing from both existing plant GLDHs and mammalian GulLOs.…”