KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Abstract

Humans are unable to synthesize L-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.

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

“…Increased co-expression of the GMP + GME + GGP + GPP genes in tomato also increased ascorbate concentrations 2.0-fold; however, this was the same fold-change for increased expression of the GGP gene alone, suggesting that GMP is not a major rate-limiting step in the L-galactose pathway in tomato [26]. This conclusion is also supported by other studies increasing the expression of the GMP gene in Arabidopsis and tobacco and increasing the co-expression of the GMP + GME genes in tobacco that did not report significant changes in ascorbate concentrations [29,30] (Table 1).…”

“…Two nucleotide sugar phosphorylase-like proteins, KONJAC 1 and 2 (KJC1 and KJC2), were identified based on their phylogenetic relationship to GMP [30]. Loss-of-function mutations in the KJC1 and KJC2 genes in Arabidopsis reduced ascorbate to 40% and 70% of wild-type levels, respectively, due to reducing GMP enzymatic activity to 10% and 70% of wild-type levels, respectively [30].…”

The C2H2 zinc‐finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B

“…The COP9 signalosome subunit CSN5B promotes the degradation of VTC1 (the Arabidopsis ortholog of GMP) through the 26S proteasome in the dark (Wang et al, 2013). The KONJAC proteins promote the accumulation of GMP and act as positive regulators of AsA biosynthesis (Sawake et al, 2015). There are four additional proteins that contribute to the regulation of AsA.…”

“…CSN5B, a component of the photomorphogenic COP9 signalosome (CSN), can affect AsA biosynthesis and subsequently modulate plant responses to abiotic stress by binding to VTC1 and promoting its degradation in the dark (Wang et al, 2013). GMP activity is stimulated by KONJAC proteins (Sawake et al, 2015). AMR1 negatively modulates the expression of a few genes in the AsA biosynthetic pathway, but the underlying regulatory mechanism remains unknown.…”

Ascorbate concentration inArabidopsis thalianaand expression of ascorbate related genes using RNAseq in response to light and the diurnal cycle

“…11. These are KONJAC (Sawake et al, 2015) Table S6. 295 Response to a step change in PFD: Generally, regulatory genes showed a less than two fold 296 change in gene expression upon increasing the PFD and in two cases the initial trend was a decrease in gene expression compared to the higher steady state expression of plants grown 298 constantly at the higher PFD (Table S7).…”