Enhancement of vitamin B6 levels in rice expressing Arabidopsis vitamin B6 biosynthesis de novo genes

Vitamin B₆ (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B₆ content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B₆ in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B₆ biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B₆ levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B₆ biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B₆ in leaves (up to 28.3‐fold) and roots (up to 12‐fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B₆ did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B₆ content could also be achieved in rice seeds (up to 3.1‐fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B₆‐enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.     

Role of vitamin B 6 biosynthetic rate in the study of vitamin B 6 synthesis in Escherichia coli

Nutritional auxotrophs of Escherichia coli synthesize vitamin B(6) compounds at a rate of 1 x 10(-10) to 2 x 10(-10) moles per hr per mg (dry weight) of cells when they are suspended in minimal medium lacking their required nutrients. A few auxotrophs have been found to stop or reduce vitamin B(6) synthesis during such an experiment. These include thiamineless, citrate synthaseless, and pyridoxineless mutants as well as mutants which require four carbon compounds for growth. Glycolaldehyde was found to restore vitamin B(6) synthesis in the last named of these mutants without restoring normal growth. A class of pyridoxineless mutants which responded with normal growth to 0.4 mm glycolaldehyde or 0.15 x 10(-3) mm pyridoxol was also found. The results suggest that a thiamine pyrophosphate-requiring step as well as glycolaldehyde may be involved in pyridoxal phosphate biosynthesis.     

Photo-induced processes in vitamin B6 compounds

In this work, we applied multi-wavelength stopped-flow spectroscopy (MSFS) to study the chemical equilibria between tautomeric or hydrated forms of various vitamin B6 compounds and the Schiff base formed by epsilon-aminocaproic acid (= 6-aminohexanoic acid) with pyridoxal 5'-phosphate at 25 degrees and variable pH. Since some of these compounds are photosensitive, we analyzed the possible occurrence of any secondary photo-induced processes under the conditions of irradiation in the MSFS equipment (continuous irradiation with light from a 75-W Xe lamp spanning the wavelength range of 200-700 nm). To determine the tautomeric composition of these compounds, the electronic absorption spectra were analyzed by means of log-normal curves. Continuous irradiation of pyridoxamine and pyridoxal 5'-phosphate over the wavelength range of 200-700 nm displaces the chemical equilibrium between the tautomeric or hydrated forms of these compounds. However, the Schiff base of epsilon-aminocaproic acid with pyridoxal 5'-phosphate is insensitive to the radiation used. The photo-induced processes detected in pyridoxamine and pyridoxal 5'-phosphate should be taken into account in examining vitamers by MSFS. In fact, these additional processes should be considered in studying the mechanism of action of vitamin B6-dependent enzymes by the MSFS technique, whenever some free vitamer may be present in solution.     

Rationalising vitamin B6 biofortification in crop plants

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Transport and metabolism of vitamin B6 in lactic acid bacteria.

Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.     

Effects of vitamin B6 supplementation in rats during lactation on vitamin B6 concentration and transaminase activities in the offspring

The aim of the present investigation was to study the effect of a varying maternal vitamin B₆ supplementation during lactation period on vitamin B₆ levels in blood, liver and total body, and on the activity of two transaminase enzymes in the offspring. Therefore, eighty female Sprague‐Dawley rats were fed a semi‐synthetic diet (0.2 mg vitamin B₆ per kg) which was supplemented during gravidity with 5 mg vitamin B₆ per kg diet. During the following lactation period the rats were assigned to one of 10 vitamin B₆ treatment groups (supplementation of 0, 3, 6, 9, 12, 15, 18, 36, 360, 3600 mg vitamin B₆ per kg diet). At day 14 of lactation the pubs of all dams were decapitated and blood, liver, and carcass were used for analysis of vitamin B₆ concentration, activities of two transaminases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in plasma, erythrocytes, and liver, and of haematological parameters.

While the liver and total body wet weights as well as the haematological parameters (red blood cells, haemoglobin concentration, hematocrit, middle corpuscular cell volume, middle corpuscular haemoglobin, middle corpuscular haemoglobin concentration) did not differ within the experimental groups, the present data clearly show that in blood, liver and total body of the offspring exists a slight dose‐response relationship between the maternal dietary vitamin B₆ supplementation and the vitamin B₆ concentration. Concerning the activities of the transaminases a dietary supplementation above 3mg vitamin B₆ per kg diet had no influence on the AST and ALT activities in offspring plasma. In the erythrocytes no statistical significant influence of the vitamin B₆ supplementation during lactation on the activities of AST and ALT was found. The activities of ALT and AST in liver were not consistently altered by the vitamin B₆ supplementation of the dams during lactation. In conclusion these results indicate that a minimal maternal dietary vitamin B₆ supply of 3.1 mg per kg diet is necessary with regard to health and development of their offspring. But not all of the analysed parameters as the liver and total body weights, the activities of AST and ALT in the erythrocytes, and the haematological parameters were influenced by a deficient maternal dietary vitamin B₆ supply.     

Carpal tunnel syndrome and vitamin B6

We reviewed 1075 patients presenting over a 12-year period with symptoms of carpal tunnel syndrome. A total of 994 had a final diagnosis of carpal tunnel syndrome. There were 444 male and 550 female patients with a mean age of 42 years. Three-hundred and ninety-five related symptoms to their job. Surgery was performed in 27 percent of the total diagnosed cases with approximately 97 percent relief of symptoms. Satisfactory alleviation of symptoms was obtained in 14.3 percent of patients treated conservatively prior to 1980, with one or a combination of splinting anti-inflammatory agents, job or activity change, and steroid injections. In 1980, vitamin B6 (pyridoxine) was added as a method of conservative treatment. Satisfactory improvement was obtained in 68 percent of 494 patients treated with a controlled dosage (100 mg b.i.d.). While our findings were not the result of a controlled scientific study, we feel they suggest that regulated use of vitamin B6 may be helpful in treating many cases of carpal tunnel syndrome.