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BackgroundMyrciaria dubia is an Amazonian fruit shrub that produces numerous bioactive phytochemicals, but is best known by its high L-ascorbic acid (AsA) content in fruits. Pronounced variation in AsA content has been observed both within and among individuals, but the genetic factors responsible for this variation are largely unknown. The goals of this research, therefore, were to assemble, characterize, and annotate the fruit transcriptome of M. dubia in order to reconstruct metabolic pathways and determine if multiple pathways contribute to AsA biosynthesis.ResultsIn total 24,551,882 high-quality sequence reads were de novo assembled into 70,048 unigenes (mean length = 1150 bp, N50 = 1775 bp). Assembled sequences were annotated using BLASTX against public databases such as TAIR, GR-protein, FB, MGI, RGD, ZFIN, SGN, WB, TIGR_CMR, and JCVI-CMR with 75.2 % of unigenes having annotations. Of the three core GO annotation categories, biological processes comprised 53.6 % of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1 %, respectively. Based on the KEGG pathway assignment of the functionally annotated transcripts, five metabolic pathways for AsA biosynthesis were identified: animal-like pathway, myo-inositol pathway, L-gulose pathway, D-mannose/L-galactose pathway, and uronic acid pathway. All transcripts coding enzymes involved in the ascorbate-glutathione cycle were also identified. Finally, we used the assembly to identified 6314 genic microsatellites and 23,481 high quality SNPs.ConclusionsThis study describes the first next-generation sequencing effort and transcriptome annotation of a non-model Amazonian plant that is relevant for AsA production and other bioactive phytochemicals. Genes encoding key enzymes were successfully identified and metabolic pathways involved in biosynthesis of AsA, anthocyanins, and other metabolic pathways have been reconstructed. The identification of these genes and pathways is in agreement with the empirically observed capability of M. dubia to synthesize and accumulate AsA and other important molecules, and adds to our current knowledge of the molecular biology and biochemistry of their production in plants. By providing insights into the mechanisms underpinning these metabolic processes, these results can be used to direct efforts to genetically manipulate this organism in order to enhance the production of these bioactive phytochemicals.The accumulation of AsA precursor and discovery of genes associated with their biosynthesis and metabolism in M. dubia is intriguing and worthy of further investigation. The sequences and pathways produced here present the genetic framework required for further studies. Quantitative transcriptomics in concert with studies of the genome, proteome, and metabolome under conditions that stimulate production and accumulation of AsA and their precursors are needed to provide a more comprehensive view of how these pathways for AsA metabolism are regulated and linked in this species.Electron...

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Biodiesel production from microalgae triacylglycerols is growing, because this feedstock is a more sustainable and advantageous alternative. In this study, we isolated and identified fourteen strains of native microalgae from the Peruvian Amazon. These strains showed great heterogeneity in biomass productivity, lipid productivity and lipid content, and thus, three of them (Acutodesmus obliquus, Ankistrodesmus sp. and Chlorella lewinii) were selected for further evaluation under culture of nitrogen-sufficient (+N) and nitrogen-deficient (−N) Chu medium No. 10. These microalgae species showed modifications in biomolecule content (protein, lipid and carbohydrate) with a pronounced increase of lipids and carbohydrate and a decrease of protein content under stress culture. Furthermore, the fatty acid profile was peculiar for each species, and these patterns showed evident changes, particularly in the proportion of saturated and monounsaturated fatty acids. The results of this research suggest that the isolated native microalgae, from the Peruvian Amazon, could be suitable candidates for biodiesel production.

The aim of this work was to elucidate the molecular and biochemical mechanisms that control L-ascorbic acid (AsA) content variation in Myrciaria dubia. The AsA was quantified by high-performance liquid chromatography, gene expression by real-time quantitative PCR, and enzyme activities by spectrophotometric methods from leaves and immature fruits of two genotypes (Md-60,06 and Md-02,04) with pronounced (about 2 times) differences in the AsA content. In either genotype, the fruit peel had ~ 1.5 times more AsA than the fruit pulp and ~ 15.0 times more than the leaf. All tissues examined demonstrated the capability for AsA biosynthesis through the D-mannose/L-galactose pathway because mRNAs of the six key genes [GDP-D-mannose pyrophosphorylase (GMP), GDP-D-mannose-3ꞌ,5ꞌ-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1-4-lactone dehydrogenase (GLDH)] and catalytic activities of the corresponding enzymes (GMP, GDH, and GLDH) were detected. The differential expressions of genes and enzyme activities mostly correlated with the respective AsA content. Thus, the expression of several genes of the D-mannose/ L-galactose pathway determined the AsA content variation in tissues of M. dubia.

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Camu-camu is a shrub, native to the Amazon that thrives in areas where flooding is frequent. Genetically, the plant is characterized by a diploid genome and moderate genetic diversity. Several parts of the plant are used in traditional folk medicine to treat a variety of acute and chronic diseases. For over 50 years, the exceptionally high vitamin C content of camu-camu has attracted worldwide attention that continues today because of the recent discovery of several healthpromoting phytochemicals with corroborated biological activities (e.g., antioxidant, anti-obesity, antidiabetic). All of these beneficial attributes are well supported by in vitro and in vivo studies as well as human clinical trials. The metabolic precursors of these phytochemicals are synthesized in key metabolic pathways (i.e., the shikimate pathway, the mevalonate pathway). Of these metabolic pathways, we show details for the biosynthesis of betulinic acid, transresveratrol, and syringic acid. In conclusion, camu-camu is an exceptional plant for its ability to produce and accumulate significant amounts of a variety of health-promoting phytochemicals. Although several metabolic pathways responsible for the biosynthesis of these phytochemicals have been reconstructed based on fruit and seedling transcriptomes, detailed knowledge of the vast majority of metabolic pathways and their molecular regulatory mechanisms is lacking. Consequently, we must increase our knowledge of the metabolic processes using multi-omic approaches so that we can acquire the skills necessary to develop genetically improved varieties of camu-camu and implement biotechnological applications for the production of these bioactive phytochemicals.

Camu camu is a typical Amazon native fruit shrub that possesses a diploid genome, moderate genetic diversity, and population structure. The fruits accumulate several essential nutrients and synthesize L-ascorbic acid (vitamin C) in great quantities and an array of diverse secondary metabolites with corroborated in vitro and in vivo health-promoting activities. These beneficial effects include antioxidative and antiinflammatory activities, antiobesity, hypolipidemic, antihypertensive and antidiabetic effects, DNA damage and cancer protection effects, and other bioactivities. Many health-promoting phytochemicals are biosynthesized in several metabolic pathways of camu camu. Their reconstruction from the fruit transcriptome database was accomplished by our research group. These include basic metabolic pathways such as glycolysis and pentose phosphate pathway, vitamin C biosynthesis pathways, and pathways involved in secondary metabolites production. Due to their agronomic potential and fruits growing demand, recently, based on an ideotype, programs were initiated for their domestication and genetic improvement, but so far with very negligible achievements. Consequently, we propose new strategies to accelerate the processes of domestication and genetic improvement based on state of the art technologies for multiomic data analysis and innovative molecular tools.