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.
Phosphomannomutase (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.
Qian
Weiqiang
W
The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Yu
Chunmei
C
Qin
Huanju
H
Liu
Xin
X
Zhang
Aimin
A
Johansen
Ida Elisabeth
IE
Wang
Daowen
D
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001205:Ascorbic Acid / Q000096:biosynthesis*; D015870:Gene Expression; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010269:Paraquat; D017875:Phosphotransferases (Phosphomutases) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D017875:Phosphotransferases (Phosphomutases); C052799:phosphomannomutase; D010269:Paraquat; D001205:Ascorbic Acid