Improving folate (vitamin B9) stability in biofortified rice through metabolic engineering.

Biofortification of staple crops could help to alleviate micronutrient deficiencies in humans. We show that folates in stored rice grains are unstable, which reduces the potential benefits of folate biofortification. We obtain folate concentrations that are up to 150 fold higher than those of wild-type rice by complexing folate to folate-binding proteins to improve folate stability, thereby enabling long-term storage of biofortified high-folate rice grains.

A folate independent role for cytosolic HPPK/DHPS upon stress in Arabidopsis thaliana.

Cytosolic HPPK/DHPS (cytHPPK/DHPS) in Arabidopsis is a functional enzyme with activity similar to its mitochondrial isoform. Genomic complementation of the cytHPPK/DHPS knockout mutant with the wild type gene led to a complete rescue of the stress sensitive mutant phenotype in seed germination tests under abiotic stress conditions. Moreover, over-expression of the gene resulted in higher germination rate under stress as compared to the wild-type, confirming its role in stress resistance.

Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the sensitive determination of folates in rice.

High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC-MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC-MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC-MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection.

Optimisation and validation of a liquid chromatography-tandem mass spectrometry method for folates in rice.

An LC-MS/MS method has been developed and validated for the analysis of six monoglutamate folates in rice. Accurate determination of folates in rice seeds required the optimisation of an extraction procedure encompassing homogenisation, heat treatment, tri-enzyme treatment (alpha-amylase, protease and deconjugase), centrifugation and ultrafiltration. The resulting extract was loaded onto a Polaris C 18-A column, followed by gradient elution and detection by tandem mass spectrometry.

Folate biofortification in food plants.

Folate deficiency is a global health problem affecting many people in the developing and developed world. Current interventions (industrial food fortification and supplementation by folic acid pills) are effective if they can be used but might not be possible in less developed countries. Recent advances demonstrate that folate biofortification of food crops is now a feasible complementary strategy to fight folate deficiency worldwide.

Folate fortification of rice by metabolic engineering.

Rice, the world's major staple crop, is a poor source of essential micronutrients, including folates (vitamin B9). We report folate biofortification of rice seeds achieved by overexpressing two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. We obtained a maximal enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.

pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates.

The stability and eventual interconversion of nine mono-glutamate folates (5-methyl-tetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, 5,10-methylenetetrahydrofolate, dihydrofolate, 10-formylfolic acid, 10-formyltetrahydrofolate and folic acid) during the typical sample preparation steps (heat treatment for 10 min at 100 degrees C and incubation for 2 h at 37 degrees C) at different pH values have been investigated by LC-MS/MS. An LC-MS/MS method with isotopically labelled [(13)C(5)]5-methyltetrahydrofolate and [(13)C(5)] folic acid as internal standards has been developed with enhanced sensitivity using a Chromolith RP-18 column. 5-Methyltetrahydrofolate, folic acid and 10-formylfolic acid are relatively stable at different pHs (from 2 to 10) with and without heat treatment.

Cytosolic hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase from Arabidopsis thaliana: a specific role in early development and stress response.

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction.

Investigation of the extraction behavior of the main monoglutamate folates from spinach by liquid chromatography-electrospray ionization tandem mass spectrometry.

An LC-MS/MS method has been developed for the determination of main monoglutamate folates in spinach with folic acid as an internal standard. A sample preparation with ultrafiltration (molecular weight cut-off membrane, 5 kDa) was followed by a chromatographic run of 14.2 min, rendering the method very simple and fast.

Free and total para-aminobenzoic acid analysis in plants with high-performance liquid chromatography/tandem mass spectrometry.

para-Aminobenzoic acid (PABA), a precursor in the synthesis of folates in plants, is determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In plants PABA can be converted into its beta-D-glucopyranosyl ester (PABA-Glc) and can also exist in its free form. In this work, we developed and validated a quantitative method to study free and total PABA in plants.

Determination of total folate in plant material by chemical conversion into para-aminobenzoic acid followed by high performance liquid chromatography combined with on-line postcolumn derivatization and fluorescence detection.

A procedure involving chemical conversion of all forms of folate present in plant material into para-aminobenzoic acid (PABA) and a liquid chromatographic-fluorimetric determination with on-line postcolumn derivatization is reported. All folates are cleaved with liberation of PABA by hydrogen peroxide followed by acid hydrolysis using concentrated hydrochloric acid (37%) at 110 degrees C for 6 h. The reaction yield for individual folates conversion to PABA ranged from 44.

Gamma-glutamyl transpeptidase in transgenic tobacco plants. Cellular localization, processing, and biochemical properties.

gamma-Glutamyl transpeptidase (gamma-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the gamma-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for gamma-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound gamma-GT activity was localized outside the cell in transgenic plants.