Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

Abstract

SummaryPhosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.

Virus-induced gene silencing and its application in plant functional genomics

“…In addition, genes involved in the regulatory functions of PCD have been recently identified using VIGS, for example, the mitochondrial-associated hexokinase Hxk1 gene [149], 20S proteasome, the 19S regulatory complex of the 26S proteasome [150] and a regulatory gene of PCD (CDC5 [151]). VIGS has also been used to characterize cellular functions of genes involved in chloroplasts and mitochondria biogenesis [129,150,152157], plastid biogenesis [138,150], peroxisome biogenesis [158], alkaloid biosynthesis [159][160][161], isoprenoid biosynthesis [162,163], ascorbic acid biosynthesis [164], sterol biosynthesis [36,165], and membrane biogenesis [166].…”

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

“…As a consequence, vtc1 plants contain only ϳ25% of wild type AsA levels (11,12). Very recently, evidence has been provided for the role of PMM in AsA biosynthesis in plants (3). Reduction of PMM expression levels through virus-induced gene silencing caused a substantial decrease in AsA content in Nicotiana benthamiana leaves, whereas raising PMM expression levels using viral vector-mediated ectopic expression resulted in an increase in AsA content.…”

“…However, no Arabidopsis T-DNA insertional knock-out mutants were reported, and attempts to produce knockdown plants using RNA interference technology were unsuccessful, suggesting that the PMM gene is essential. This inability to obtain plants with permanently decreased PMM levels impeded further studies into the possible involvement of PMM in other cellular processes (3).…”

“…Both prokaryotes and eukaryotes utilize GDP-mannose in the synthesis of complex structural carbohydrates. Accordingly, this sugar nucleotide contributes to the synthesis of different structural carbohydrates in plant cell walls (1) and fulfills a key role in the biosynthesis of ascorbic acid (AsA) in plants (2,3). GDPmannose is also essential for post-translational modifications, such as protein glycosylation and glycosylphosphatidylinositol (GPI) anchoring in eukaryotes, because mannose, derived from GDP-mannose, is a crucial building block of the core glycan chain attached to the modified proteins (4,5).…”

Cellular redox regulation, signaling, and stress response in plants

“…9,20,21) In this pathway, GDP-D-Man is synthesized from D-fructose-6-phosphate (Fru-6P) in three steps, which are catalyzed by phosphomannose isomerase (PMI), phosphomannomutase, and GDP-D-Man pyrophosphorylase (GMP). [30][31][32] Arabidopsis has two PMI genes, PMI1 and PMI2. Expressional and reverse genetic analyses revealed that only PMI1 was involved in the biosynthesis of AsA.…”