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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 microm and 1.89 micromol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372+/-13 microm and 22.5 micromol/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.

SUBMITTER: Maruta T

PROVIDER: S-EPMC2661998 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:YEAST STRAIN: A genetically modified Saccharomyces cerevisiae yeast strain with PMI40 knockout (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2 PMI40::HIS3). The parent strain was CEN.PK113-7A (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2) obtained from Euroscarf (9). CULTIVATION MEDIUM: The mineral medium used for the bioreactor cultivations was based on the composition described by Verduyn and co-workers (11). The medium consisted of 5 g/l (NH4)2SO4 (Sigma-Aldrich, USA), 3 g/l KH2PO4 (Fluka, Swizerland) and 0.5 g/l MgSO4 × 7 H2O (Sigma-Aldrich, USA), as well as 1 ml/l trace element and 1 ml/l vitamin solution. The composition of the trace element solution was 15 g/l EDTA, 4.5 g/l ZnSO4 × 7 H2O, 0.84 g/l MnCl2 × H2O, 0.3 g/l CuSO4 × 5 H2O, 3 g/l FeSO4 × 7 H2O (Riedel-de-Haën AG, Germany), 0.1 g/l KI, 0.3 g/l CoCl3 × 6 H2O, 1 g/l H3BO3, 4.5 g/l CaCl2 × 2 H2O (Merck, Germany), and 0.4 g/l Na2MoO4 × 2 H2O (BDH Laboratory Supplies, UK). The composition of the vitamin solution was 0.05 g/l D-Biotin, 1 g/l Ca-pantothenate, 1 g/l Nicotinic acid, 25 g/l myo-inositol, 1 g/l Thiamine-HCl, 1 g/l Pyridoxine HCl, and 0.2 g/l p-aminobenzoic acid (Sigma-Aldrich, USA). The media in each cultivation contained initially 15 g/L D-glucose and a varying amount of D-mannose (0.75 g/L, 1.0 g/L, 1.5 g/L, 3.0 g/L, or 5.0 g/L). CULTIVATIONS AND SAMPLING: Bioreactor cultivations were performed in 3.2 l Braun Biostat CT2-DCU 3 instruments (B. Braun Biotech International GmbH, Germany). The cultivation temperature was +30 °C, agitation speed 1000 rpm, initial cultivation volume 2.0 l, airflow 1.0 l/min, and pH 5.0. In each cultivation, samples for the measurement of gene expression levels were taken 6 hours after inoculation. MEASUREMENT OF GENE EXPRESSION LEVELS: 0.9 mg CDW of cells were quenched in cold (-40 °C) methanol (45%) and centrifuged (1 min, +4 °C, 16100 G). The pellets were suspended in 400 µl of breaking buffer (20 mM Tris-HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 2mM DTT), 400 µl 1:1 phenol-chloroform, and 5 µl 20% SDS. Equal volume of glass beads (diameter 0.40-0.60 mm, B.Braun Biotech International, Germany) were added and samples were shaken in a bead beater (Mini-BeadBeater-8, BioSpec Products, USA) three times 1 minute at +4 °C. The mixtures were centrifuged (15 min, +4 °C, 16100 G in 5415 R, Eppendorf AG, Germany) and supernatants were recovered. The total RNA was purified from the supernatants using the QIAGEN RNeasy Mini kit (Qiagen, USA) according to manufacturers instructions. After this step the double-stranded cDNA was synthesized from 15 µg of total RNA with SuperScript Double-Stranded cDNA synthesis kit (Invitrogen, USA) using a T7-oligo(dT) primer. Biotin-labeled cRNA was then synthesized employing an Enzo High Yield RNA Transcript Labeling Kit (Enzo Life Science, USA). The biotin-labeled cRNA was heat-fragmented and biotinylated cRNAs were hybridized to Affymetrix YG-S98 microarrays in Affymetrix Hybridization Oven 640. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000. Acquisition and quantification of array images were performed using the Affymetrix GeneChip Operating System 1.0. The signals were scaled into target intensity of 100. All samples were assayed in triplicates. Keywords: dose response

Project description:YEAST STRAIN:,A genetically modified Saccharomyces cerevisiae yeast strain with PMI40 knockout (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2 PMI40::HIS3). The parent strain was CEN.PK113-7A (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2) obtained from Euroscarf (9).,CULTIVATION MEDIUM:,The mineral medium used for the bioreactor cultivations was based on the composition described by Verduyn and co-workers (11). The medium consisted of 5 g/l (NH4)2SO4 (Sigma-Aldrich, USA), 3 g/l KH2PO4 (Fluka, Swizerland) and 0.5 g/l MgSO4 Ã 7 H2O (Sigma-Aldrich, USA), as well as 1 ml/l trace element and 1 ml/l vitamin solution. The composition of the trace element solution was 15 g/l EDTA, 4.5 g/l ZnSO4 Ã 7 H2O, 0.84 g/l MnCl2 Ã H2O, 0.3 g/l CuSO4 Ã 5 H2O, 3 g/l FeSO4 Ã 7 H2O (Riedel-de-HaÃ«n AG, Germany), 0.1 g/l KI, 0.3 g/l CoCl3 Ã 6 H2O, 1 g/l H3BO3, 4.5 g/l CaCl2 Ã 2 H2O (Merck, Germany), and 0.4 g/l Na2MoO4 Ã 2 H2O (BDH Laboratory Supplies, UK). The composition of the vitamin solution was 0.05 g/l D-Biotin, 1 g/l Ca-pantothenate, 1 g/l Nicotinic acid, 25 g/l myo-inositol, 1 g/l Thiamine-HCl, 1 g/l Pyridoxine HCl, and 0.2 g/l p-aminobenzoic acid (Sigma-Aldrich, USA).,The media in each cultivation contained initially 15 g/L D-glucose and a varying amount of D-mannose (0.75 g/L, 1.0 g/L, 1.5 g/L, 3.0 g/L, or 5.0 g/L).,CULTIVATIONS AND SAMPLING:,Bioreactor cultivations were performed in 3.2 l Braun Biostat CT2-DCU 3 instruments (B. Braun Biotech International GmbH, Germany). The cultivation temperature was +30 Â°C, agitation speed 1000 rpm, initial cultivation volume 2.0 l, airflow 1.0 l/min, and pH 5.0.,In each cultivation, samples for the measurement of gene expression levels were taken 6 hours after inoculation.,MEASUREMENT OF GENE EXPRESSION LEVELS:, ,0.9 mg CDW of cells were quenched in cold (-40 Â°C) methanol (45%) and centrifuged (1 min, +4 Â°C, 16100 G). The pellets were suspended in 400 Âµl of breaking buffer (20 mM Tris-HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 2mM DTT), 400 Âµl 1:1 phenol-chloroform, and 5 Âµl 20% SDS. Equal volume of glass beads (diameter 0.40-0.60 mm, B.Braun Biotech International, Germany) were added and samples were shaken in a bead beater (Mini-BeadBeater-8, BioSpec Products, USA) three times 1 minute at +4 Â°C. The mixtures were centrifuged (15 min, +4 Â°C, 16100 G in 5415 R, Eppendorf AG, Germany) and supernatants were recovered. The total RNA was purified from the supernatants using the QIAGEN RNeasy Mini kit (Qiagen, USA) according to manufacturers instructions. After this step the double-stranded cDNA was synthesized from 15 Âµg of total RNA with SuperScript Double-Stranded cDNA synthesis kit (Invitrogen, USA) using a T7-oligo(dT) primer. Biotin-labeled cRNA was then synthesized employing an Enzo High Yield RNA Transcript Labeling Kit (Enzo Life Science, USA). The biotin-labeled cRNA was heat-fragmented and biotinylated cRNAs were hybridized to Affymetrix YG-S98 microarrays in Affymetrix Hybridization Oven 640. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000. Acquisition and quantification of array images were performed using the Affymetrix GeneChip Operating System 1.0. The signals were scaled into target intensity of 100. All samples were assayed in triplicates.

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Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis.

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