Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.

MAIN CONCLUSION: Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-L-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-L-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production.

Expression profile analysis of ascorbic acid-related genes in response to temperature stress in the tea plant, Camellia sinensis (L.) O. Kuntze.

Ascorbic acid (AsA), also known as ascorbate or vitamin C, is a natural organic compound in green plants that has antioxidant properties, and is an essential nutrient for humans. The tea plant, Camellia sinensis (L.) O. Kuntze, is an important global economic crop. Here, the expression profiles of genes related to AsA biosynthesis and recycling were analyzed in tea plants in response to temperature stress. Eighteen genes involved in AsA biosynthesis and recycling pathways were identified based on the transcriptome database. The expression levels of CsPGI1 in two varieties of tea plants ('Yingshuang' and 'Huangjinya') increased, peaked at 4 h, and then decreased in response to cold stress. In 'Yingshuang', the genes involved in AsA biosynthesis pathway rapidly responded to heat stress and substantially increased their expression levels at 1 h. The expression levels of CsMDHAR, CsDHAR1, and CsDHAR2 increased sharply at 1 h in response to heat stress in 'Yingshuang'. In contrast, the expression levels of CsMDHAR, CsDHAR1, and CsDHAR2 in 'Huangjinya' gradually increased during heat treatment from 1 to 24 h. The expression trends of two DHAR isoforms differed in 'Huangjinya' during cold stress. The expression patterns of AsA-related genes differed in the different tea plant varieties and depended on temperature. The genes involved in AsA biosynthesis and recycling pathways were induced by heat and cold stress. Our study provides useful data with which to improve the resistance of tea plants to cold and heat stress.

Effect of mitochondrial ascorbic acid synthesis on photosynthesis.

Ascorbic acid (AA) is synthesized in plant mitochondria through the oxidation of l-galactono-1,4-lactone (l-GalL) and then distributed to different cell compartments. AA-deficient Arabidopsis thaliana mutants (vtc2) and exogenous applications of l-GalL were used to generate plants with different AA content in their leaves. This experimental approach allows determining specific AA-dependent effects on carbon metabolism. No differences in O2 uptake, malic and citric acid and NADH content suggest that AA synthesis or accumulation did not affect mitochondrial activity; however, l-GalL treatment increased CO2 assimilation and photosynthetic electron transport rate in vtc2 (but not wt) leaves demonstrating a stimulation of photosynthesis after l-GalL treatment. Increased CO2 assimilation correlated with increased leaf stomatal conductance observed in l-GalL-treated vtc2 plants.

Copper-induced synthesis of ascorbate, glutathione and phytochelatins in the marine alga Ulva compressa (Chlorophyta).

In order to analyze the synthesis of antioxidant and heavy metal-chelating compounds in response to copper stress, the marine alga Ulva compressa (Chlorophyta) was exposed to 10 µM copper for 7 days and treated with inhibitors of ASC synthesis, lycorine, and GSH synthesis, buthionine sulfoximine (BSO). The levels of ascorbate, in its reduced (ASC) and oxidized (DHA) forms, glutathione, in its reduced (GSH) and oxidized (GSSG) forms, and phytochelatins (PCs) were determined as well as activities of enzymes involved in ASC synthesis, L-galactose dehydrogenase (GDH) and L-galactono 1,4 lactone dehydrogenase (GLDH), and in GSH synthesis, γ-glutamylcysteine synthase (γ-GCS) and glutathione synthase (GS). The level of ASC rapidly decreased to reach a minimum at day 1 that remained low until day 7, DHA decreased until day 1 but slowly increased up to day 7 and its accumulation was inhibited by lycorine. In addition, GSH level increased to reach a maximal level at day 5 and GSSG increased up to day 7 and their accumulation was inhibited by BSO. Activities of GDH and GLDH increased until day 7 and GLDH was inhibited by lycorine. Moreover, activities of γ-GCS and GS increased until day 7 and γ-GCS was inhibited by BSO. Furthermore, PC2, PC3 and PC4, increased until day 7 and their accumulation was inhibited by BSO. Thus, copper induced the synthesis of ascorbate, glutathione and PCs in U. compressa suggesting that these compounds are involved in copper tolerance. Interestingly, U. compressa is, until now, the only ulvophyte showing ASC, GSH and PCs synthesis in response to copper excess.

Laranja: em defesa de seu uso como alimento funcional / Orange: in defense of its use as a functional food

Evidences suggest that a diet rich in fruits and vegetables decreases the risk of several degenerative diseases. The world population aging process requires more efforts to delay the onset of such diseases. Therefore, the inclusion of functional foods in the diet is an excellent strategy to improve the quality of life. Brazil is the world`s largest producer of orange, which provides many health benefits. The purpose of this paper is to review the recent literature in order to demonstrate the benefits provided by the inclusion of orange in the diet as a preventive measure for many diseases. Based on the properties demonstrated in this review, a discussion about the inclusion of orange in the category of functional foods is proposed.

Las evidencias sugieren que una dieta rica en frutas y hortalizas reduce el riesgo de varias enfermedades degenerativas. El envejecimiento de la población mundial exige más esfuerzos para retardar la manifestación de esas enfermedades. Por lo tanto, la inclusión de alimentos funcionales en la dieta es una excelente estrategia para mejorar la calidad devida de poblaciones. La naranja, que encuentra en Brasil su mayor productor mundial, tiene varias propiedades benéficas para la salud. El propósito de este trabajo fue revisar la literatura reciente para demostrar los beneficios de la inclusión de naranja en la dieta como medida preventiva de una serie de enfermedades. Con base en las propiedades demostradas e nesta revisión, se propone discutir la inclusión de la naranja en la categoría de alimentos funcionales.

As evidências sugerem que uma dieta rica em frutas e vegetais reduz o risco de várias doenças degenerativas. O envelhecimento da população mundial requer mais esforços no sentido de retardar o aparecimento deste tipo de doença. Portanto, a inclusão de alimentos funcionais na dieta é uma excelente estratégia para melhorar a qualidade de vida da população. A laranja, fruta que tem o Brasil, como o seu maior produtor mundial, possui diversas propriedades benéficas à saúde. O propósito deste artigo é revisar a literatura recente demonstrando os inúmeros benefícios da inclusão da laranja na dieta, como medida preventiva de uma série de doenças. Com base nas propriedades demonstradas nesta revisão, propõe-se a discussão sobre a inclusão da laranja na categoria de alimento funcional.

Control of ascorbic acid synthesis and accumulation and glutathione by the incident light red/far red ratio in Phaseolus vulgaris leaves.

The effects of red/far red (R/FR) ratios on leaf ascorbate (AA) and glutathione (GSH) accumulation were examined in common bean (Phaseolus vulgaris L.). Growth under low R/FR ratios resulted in a "shade" phenotype and much lower leaf AA and GSH contents than high (R/FR) ratios. Photosynthesis rates were unaffected by changes in the R/FR ratio but leaf respiration rates, pyridine nucleotide pools and antioxidant enzyme activities were decreased under the low R/FR regime. The GSH pool changed slowly in response to altered R/FR ratios but leaf ascorbate acclimated over a single photoperiod. We conclude that light quality signals, particularly R/FR ratios, are important regulators of antioxidant synthesis and accumulation. These acclimatory changes are an early response to changing light environment.

Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves.

The effects of growth irradiance and respiration on ascorbic acid (AA) synthesis and accumulation were studied in the leaves of wild-type and transformed Arabidopsis thaliana with modified amounts of the mitochondrial alternative oxidase (AOX) protein. Plants were grown under low (LL; 50 micromol photons m(-2) s(-1)), intermediate (IL; 100 micromol photons m(-2) s(-1)), or high (HL; 250 micromol photons m(-2) s(-1)) light. Increasing growth irradiance progressively elevated leaf AA content and hence the values of dark-induced disappearance of leaf AA, which were 11, 55, and 89 nmol AA lost g(-1) fresh weight h(-1), from LL-, IL-, and HL-grown leaves, respectively. When HL leaves were supplied with L-galactone-1,4-lactone (L-GalL; the precursor of AA), they accumulated twice as much AA and had double the maximal L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activities of LL leaves. Growth under HL enhanced dehydroascorbate reductase and monodehydroascorbate reductase activities. Leaf respiration rates were highest in the HL leaves, which also had higher amounts of cytochrome c and cytochrome c oxidase (CCO) activities, as well as enhanced capacity of the AOX and CCO electron transport pathways. Leaves of the AOX-overexpressing lines accumulated more AA than wild-type or antisense leaves, particularly at HL. Intact mitochondria from AOX-overexpressing lines had higher AA synthesis capacities than those from the wild-type or antisense lines even though they had similar L-GalLDH activities. AOX antisense lines had more cytochrome c protein than wild-type or AOX-overexpressing lines. It is concluded that regardless of limitations on L-GalL synthesis by regulation of early steps in the AA synthesis pathway, the regulation of L-GalLDH activity via the interaction of light and respiratory controls is a crucial determinant of the overall ability of leaves to produce and accumulate AA.

Metabolismo do ácido ascórbico durante o desenvolvimento e amadurecimento da manga (Mangifera indica L. var. Keitt) e amadurecimento da goiaba branca (Psidium guajava L. var. Paloma) / Ascorbic acid metabolism during development and ripening in mango (Mangifera indica L. var. Keitt) and ripening in guava (Psidium guajava L. var. Paloma)

Devido à importância da vitamina C para os organismos humano e vegetal, torna-se útil o estudo dos mecanismos de síntese e degradação do ácido ascórbico (AA) nos vegetais, que fornecem cerca de 90 por cento da recomendação da ingestão diária de vitamina C. Os teores de AA podem variar, especialmente em frutas, durante o amadurecimento, mas pouco se sabe sobre os mecanismos utilizados pelas plantas para a regulação dos níveis deste composto. No presente estudo, procuramos correlacionar as atividades das enzimas de síntese (L-galactono-1,4-lactona desidrogenase - GLDHase), de degradação (ascorbato oxidase - AO e ascorbato peroxidase - APX)...

Biossíntese de ácido L-ascórbico em plantas: estudo com supostos precursores / L-ascorbic acid biosynthesis in plants: study with supposed of precursors

Poucos trabalhos foram publicados envolvendo a biossíntese do AA em plantas desde sua descoberta em 1928. O mecanismo de biossíntese era um mistério até 1998 quando Wheeler, Jones e Smirnoff demonstraram que a L-galactose é precursor chave desta importante vitamina. Utilizando-se acúcares marcados e frios pudemos confirmar o mecanismos Smirnoff-Wheeler de biossíntese do AA. Neste trabalho nós apresentamos os resultados alcançados usando alguns supostos precursores e alguns frutos como o morango, a goiaba e o mamão papaya, e alguns legumes como o brócolis, alguns deles ricos em AA. As técnicas de HPLC e espectrofometria UV/VIS foram utilizadas na determinação do AA. Os vegetais foram mantidos...

Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV.

Ascorbic acid is synthesized from galactono-gamma-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-gamma-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV.

La vitamina C y sus propiedades antimutagénicas / The antimuthagenic properties of vitamin C

Se efectuó el análisis de algunas características de la vitamina C. Para ello se consideraron sus propiedades químicas y biológicas así como su impacto en la salud. Se revisaron las principales evidencias sobre su capacidad antimutagénica (y potencialmente anticarcinogénica) en diversos organismos y sistemas de prueba, y se consideró la posible aplicación en el humano

Como a genética microbiana pode auxiliar a indústria de fermentaçäo / How the microbial genetic can aid the fermentation industry

A fermentaçäo industrial tem recebido, ultimamente, grande impulso, graças às novas técnicas genéticas disponíveis que permitem modificar o patrimônio genético de um microrganismo para que possa ser utilizado mais eficientemente, ou ainda, de modo mais inédito, para fabricar produtos novos que näo podiam ser obtidos por fermentaçäo. Diversos exemplos säo mencionados (ênzimos, antibióticos, proteínas, aminoácidos, entre outros), para ilustrar as enormes potencialidades do melhoramento genético e da engenharia genética como ferramentas valiosíssimas para a indústria de fermentaçöes e säo discutidos os principais problemas oriundos da utilizaçäo desses microrganismos modificados no processo fermentativo