Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis

Abstract

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between D-fructose 6-phosphate and D-mannose 6-phosphate (Man-6P). The apparent K m and V max values for Man-6P of purified recombinant PMI1 were 41.3 ؎ 4.2 M and 1.89 mol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ؎ 13 M and 22.5 mol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn 2؉ , Cd 2؉ , and L-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants.

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.…”

“…Expressional and reverse genetic analyses revealed that only PMI1 was involved in the biosynthesis of AsA. 30) GDP-D-Man was shown to be successively converted…”

“…23,33) Although one of the candidates for a rate-limiting enzyme was GME because of its low V max , 9,29) the transient overexpression of this enzyme had no effect on the AsA content in Arabidopsis or tobacco plants. 38,39) Since GDP-D-Man and GDP-L-Gal are substrates involved in the synthesis of polysaccharides as well as AsA, 30,31,40) Arabidopsis VTC2 and VTC5 catalyze the first committed step in the D-Man/LGal pathway (Fig. 3).…”

“…49) Several lines of evidence have also been provided for the negative feedback regulation of AsA biosynthesis by AsA itself. 30,50,51) II. Enzymatic properties, subcellular distribution, regulation, and physiological roles of APX isoenzymes 1,53,54) Although higher plants possess GPX-like genes, the translational products of the genes use Trx, but not GSH as an electron donor for their peroxidase activity.…”

Increase in Ascorbate Content of Transgenic Tobacco Plants Overexpressing the Acerola (Malpighia glabra) Phosphomannomutase Gene

“…Furthermore, PMI has been used as an antibiotic-free positive selection marker in plant biotechnology ( Wenck and Hansen 2005 ). Recently, two putative PMI genes identifi ed in A. thaliana (PMI1, At3g02570; and PMI2, At1g67070) have been used to show that under normal growth conditions, PMI1 but not PMI2 is essential for the biosynthesis of AsA in A. thaliana leaves ( Maruta et al 2008 ). Since the AsA content of acerola is very high, we analyzed the plant for PMI activity in order to establish whether or not a relationship exists between the activity and AsA content.…”