Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers

Background  

Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold.     

SSR markers in transcripts of genes linked to post-transcriptional and transcriptional regulatory functions during vegetative and reproductive development of Elaeis guineensis

Background  

The oil palm (Elaeis guineensis Jacq.) is a perennial monocotyledonous tropical crop species that is now the world's number one source of edible vegetable oil, and the richest dietary source of provitamin A. While new elite genotypes from traditional breeding programs provide steady yield increases, the long selection cycle (10-12 years) and the large areas required to cultivate oil palm make genetic improvement slow and labor intensive. Molecular breeding programs have the potential to make significant impacts on the rate of genetic improvement but the limited molecular resources, in particular the lack of molecular markers for agronomic traits of interest, restrict the application of molecular breeding schemes for oil palm.     

Simultaneous extraction of carotenoids and transfructosylating enzyme from <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> by a bead beater

Purpose of work  

Conditions for the cell disruption of Xanthophyllomyces dendrorhous by using a bead beater have been optimized with respect to maximizing the recovery of carotenoids and transfructosylating enzymes in a one-step process.     

A Foundation for Provitamin A Biofortification of Maize: Genome-Wide Association and Genomic Prediction Models of Carotenoid Levels

Efforts are underway for development of crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency. As a global staple crop with considerable variation in kernel carotenoid composition, maize (Zea mays L.) could have a widespread impact. We performed a genome-wide association study (GWAS) of quantified seed carotenoids across a panel of maize inbreds ranging from light yellow to dark orange in grain color to identify some of the key genes controlling maize grain carotenoid composition. Significant associations at the genome-wide level were detected within the coding regions of zep1 and lut1, carotenoid biosynthetic genes not previously shown to impact grain carotenoid composition in association studies, as well as within previously associated lcyE and crtRB1 genes. We leveraged existing biochemical and genomic information to identify 58 a priori candidate genes relevant to the biosynthesis and retention of carotenoids in maize to test in a pathway-level analysis. This revealed dxs2 and lut5, genes not previously associated with kernel carotenoids. In genomic prediction models, use of markers that targeted a small set of quantitative trait loci associated with carotenoid levels in prior linkage studies were as effective as genome-wide markers for predicting carotenoid traits. Based on GWAS, pathway-level analysis, and genomic prediction studies, we outline a flexible strategy involving use of a small number of genes that can be selected for rapid conversion of elite white grain germplasm, with minimal amounts of carotenoids, to orange grain versions containing high levels of provitamin A.     

Lipid-dissolved γ-carotene, β-carotene,and lycopene in globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits

Main conclusion

High levels of β-carotene, lycopene, and the rare γ-carotene occur predominantly lipid-dissolved in the chromoplasts of peach palm fruits. First proof of their absorption from these fruits is reported. The structural diversity, the physical deposition state in planta, and the human bioavailability of carotenoids from the edible fruits of diverse orange and yellow-colored peach palm (Bactris gasipaes Kunth) varieties were investigated. HPLC–PDA–MSⁿ revealed a broad range of carotenes, reaching total carotenoid levels from 0.7 to 13.9 mg/100 g FW. Besides the predominant (all-E)-β-carotene (0.4–5.4 mg/100 g FW), two (Z)-isomers of γ-carotene (0.1–3.9 mg/100 g FW), and one (Z)-lycopene isomer (0.04–0.83 mg/100 g FW) prevailed. Approximately 89–94 % of total carotenoid content pertained to provitamin A carotenoids with retinol activity equivalents ranging from 37 to 609 µg/100 g FW. The physical deposition state of these carotenoids in planta was investigated using light, transmission electron, and scanning electron microscopy. The plastids found in both orange and yellow-colored fruit mesocarps were amylo-chromoplasts of the globular type, containing carotenoids predominantly in a lipid-dissolved form. The hypothesis of lipid-dissolved carotenoids was supported by simple solubility estimations based on carotenoid and lipid contents of the fruit mesocarp. In our study, we report first results on the human bioavailability of γ-carotene, β-carotene, and lycopene from peach palm fruit, particularly proving the post-prandial absorption of the rarely occurring γ-carotene. Since the physical state of carotenoid deposition has been shown to be decisive for carotenoid bioavailability, lipid-dissolved carotenoids in peach palm fruits are expected to be highly bioavailable, however, further studies are required.     

The effect of enhanced carotenoid content of transgenic maize grain on fungal colonization and mycotoxin content

Novel strategies that address vitamin A deficiency have been developed, such as high-carotenoid maize, a biofortified transgenic maize line rich in carotenoids generated by genetic transformation. The South African white maize inbred (M37W), which is devoid of carotenoids, was engineered to accumulate high levels of β-carotene (provitamin A), lutein, and zeaxanthin. Maize seeds contaminated with fumonisins and other mycotoxins pose a serious threat to both humans and livestock. During three consecutive harvests, the fungal incidence and the fumonisin and aflatoxin content of maize seeds grown in an experimental field in Catalonia (Northeastern Spain) were evaluated. Fungal infection was similar in high-carotenoid maize and its isogenic line, with Fusarium verticillioides being the most prevalent fungus in all the harvests. Neither Aspergillus spp. nor aflatoxin contamination was found. Fumonisin levels were lower in high carotenoid than in its isogenic line, but this reduction was statistically significant in only 2 of the 3 years of study. Our results suggest that high carotenoid content reduces fumonisin levels in maize grains.     

Carotenoid dynamics in Atlantic salmon

Background  

Carotenoids are pigment molecules produced mainly in plants and heavily exploited by a wide range of organisms higher up in the food-chain. The fundamental processes regulating how carotenoids are absorbed and metabolized in vertebrates are still not fully understood. We try to further this understanding here by presenting a dynamic ODE (ordinary differential equation) model to describe and analyse the uptake, deposition, and utilization of a carotenoid at the whole-organism level. The model focuses on the pigment astaxanthin in Atlantic salmon because of the commercial importance of understanding carotenoid dynamics in this species, and because deposition of carotenoids in the flesh is likely to play an important life history role in anadromous salmonids.     

Carotenoid metabolism in mammals,including man: formation,occurrence, and function of apocarotenoids: Thematic Review Series: Fat-Soluble Vitamins: Vitamin A

Vitamin A was recognized as an essential nutrient 100 years ago. In the 1930s, it became clear that dietary β-carotene was cleaved at its central double to yield vitamin A (retinal or β-apo-15′-carotenal). Thus a great deal of research has focused on the central cleavage of provitamin A carotenoids to form vitamin A (retinoids). The mechanisms of formation and the physiological role(s) of noncentral (eccentric) cleavage of both provitamin A carotenoids and nonprovitamin A carotenoids has been less clear. It is becoming apparent that the apocarotenoids exert unique biological activities themselves. These compounds are found in the diet and thus may be absorbed in the intestine, or they may form from enzymatic or nonenzymatic cleavage of the parent carotenoids. The mechanism of action of apocarotenoids in mammals is not fully worked out. However, as detailed in this review, they have profound effects on gene expression and work, at least in part, through the modulation of ligand-activated nuclear receptors. Understanding the interactions of apocarotenoids with other lipid-binding proteins, chaperones, and metabolizing enzymes will undoubtedly increase our understanding of the biological roles of these carotenoid metabolites.     

ASTER-B regulates mitochondrial carotenoid transport and homeostasis

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by β-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon β-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by β-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-β-cyclodextrin treatment enhanced zeaxanthin but not β-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.     

Antioxidant potentials of vitamin A and carotenoids and their relevance to heart disease

Despite being one of the first vitamins to be discovered, the full range of biological activities for vitamin A remains to be defined. Structurally similar to vitamin A, carotenoids are a group of nearly 600 compounds. Only about 50 of these have provitamin A activity. Recent evidence has shown vitamin A, carotenoids and provitamin A carotenoids can be effective antioxidants for inhibiting the development of heart disease. Vitamin A must be obtained from the diet: green and yellow vegetables, dairy products, fruits and organ meats are some of the richest sources. Within the body, vitamin A can be found as retinol, retinal and retinoic acid. Because all of these forms are toxic at high concentrations, they are bound to proteins in the extracellular fluids and inside cells. Vitamin A is stored primarily as long chain fatty esters and as provitamin carotenoids in the liver, kidney and adipose tissue. The antioxidant activity of vitamin A and carotenoids is conferred by the hydrophobic chain of polyene units that can quench singlet oxygen , neutralize thiyl radicals and combine with and stabilize peroxyl radicals. In general, the longer the polyene chain, the greater the peroxyl radical stabilizing ability. Because of their structures, vitamin A and carotenoids can autoxidize when O2 tension increases, and thus are most effective antioxidants at low oxygen tensions that are typical of physiological levels found in tissues. Overall, the epidemiological evidence suggests that vitamin A and carotenoids are important dietary factors for reducing the incidence of heart disease. Although there is considerable discrepancy in the results from studies in humans regarding this relationship, carefully controlled experimental studies continue to indicate that these compounds are effective for mitigating and defending against many forms of cardiovascular disease. More work, especially concerning the relevance of how tissue concentrations, rather than plasma levels, relate to the progression of tissue damage in heart disease is required. This review assembles information regarding the basic structure and metabolism of vitamin A and carotenoids as related to their antioxidant activities. Epidemiological, intervention trials and experimental evidence about the effectiveness of vitamin A and carotenoids for reducing cardiovascular disease is also reviewed.     

Biological actions of carotenoids

Of 600 carotenoids from natural sources that have been characterized, fewer than 10% serve as precursors of vitamin A. Many dietary carotenoids, both with and without provitamin A activity, are found in the blood and tissues of humans. beta-Carotene, the most nutritionally active carotenoid, comprises 15-30% of total serum carotenoids. Vitamin A is formed primarily by the oxygen-dependent central cleavage of beta-carotene and other provitamin A carotenoids. Several carotenoids show enhancement of the immune response, inhibition of mutagenesis, reduction of induced nuclear damage, and protection from various neoplastic events in cells, tissues, and whole animals. Carotenoids also protect against photo-induced tissue damage. Some carotenoids, including beta-carotene, quench highly reactive singlet oxygen under certain conditions and can block free radical-mediated reactions. In epidemiological studies, the intake of carotenoid-rich fruits and vegetables has been correlated with protection from some forms of cancer, particularly lung cancer. Similarly, serum beta-carotene levels have been associated with a decreased chance of developing lung cancer. It must be stressed, however, that these epidemiological associations do not show cause and effect. In this regard, long-term intervention trials with beta-carotene supplements are in progress. Whatever the results of these trials, carotenoids clearly show biological actions in animals distinct from their function as precursors of vitamin A.     

Generation of transgenic maize with enhanced provitamin A content

Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as beta-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of 'Golden Rice 2' which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and zeta-carotene desaturase in plants), under the control of a 'super gamma-zein promoter' for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of beta-carotene in the maize endosperm. The levels attained approach those estimated to have a significant impact on the nutritional status of target populations in developing countries. The high beta-carotene trait was found to be reproducible over at least four generations. Gene expression analyses suggest that increased accumulation of beta-carotene is due to an up-regulation of the endogenous lycopene beta-cylase. These experiments set the stage for the design of transgenic approaches to generate provitamin A-rich maize that will help alleviate VAD.     

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology,for the production of human-therapeutic glycoproteins

Background  

Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.     

Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.     

Provitamin A conversion to retinal via the beta,beta-carotene-15,15'-oxygenase (bcox) is essential for pattern formation and differentiation during zebrafish embryogenesis

The egg yolk of vertebrates contains carotenoids, which account for its characteristic yellow color in some species. Such plant-derived compounds, e.g. beta-carotene, serve as the natural precursors (provitamins) of vitamin A, which is indispensable for chordate development. As egg yolk also contains stored vitamin A, carotenoids have so far been solely discussed as pigments for the coloration of the offspring. Based on our recent molecular identification of the enzyme catalyzing provitamin A conversion to vitamin A, we address a possible role of provitamin A during zebrafish (Danio rerio) development. We cloned the zebrafish gene encoding the vitamin A-forming enzyme, a beta,beta-carotene-15,15'-oxygenase. Analysis of its mRNA expression revealed that it is under complex spatial and temporal control during development. Targeted gene knockdown using the morpholino antisense oligonucleotide technique indicated a vital role of the provitamin A-converting enzyme. Morpholino-injected embryos developed a morphological phenotype that included severe malformation of the eyes, the craniofacial skeleton and pectoral fins, as well as reduced pigmentation. Analyses of gene expression changes in the morphants revealed that distinct retinoic acid-dependent developmental processes are impaired, such as patterning of the hindbrain and differentiation of hindbrain neurons, differentiation of neural crest derivatives (including the craniofacial skeleton), and the establishment of the ventral retina. Our data provide strong evidence that, for several developmental processes, retinoic acid generation depends on local de novo formation of retinal from provitamin A via the carotene oxygenase, revealing an unexpected, essential role for carotenoids in embryonic development.     

Combined mutagenesis of <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> for improved production of carotenoids and lipids

Objectives

To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains.

Results

Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW.

Conclusions

A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.