Supplementary Far-Red and Blue Lights Influence the Biomass and Phytochemical Profiles of Two Lettuce Cultivars in Plant Factory.

Three different LED spectra (W: White light; WFR: W + far-red light; WB: W + blue light) with similar photosynthetic photon flux density (PPFD) were designed to explore the effects of supplementary far-red and blue lights on leaf color, biomass and phytochemicals of two cultivars of red-leaf lettuce ("Yanzhi" and "Red Butter") in an artificial lighting plant factory. Lettuce plants under WB had redder leaf color and significantly higher contents of pigments, such as chlorophyll a, chlorophyll b, chlorophyll (a + b) and anthocyanins. The accumulation of health-promoting compounds, such as vitamin C, vitamin A, total phenolic compounds, total flavonoids and anthocyanins in the two lettuce cultivars were obviously enhanced by WB. Lettuce under WFR showed remarkable increase in fresh weight and dry weight; meanwhile, significant decreases of pigments, total phenolic compounds, total flavonoids and vitamin C were found. Thus, in the plant factory system, the application of WB can improve the coloration and quality of red leaf lettuce while WFR was encouraged for the purpose of elevating the yield of lettuce.

Enrichment of provitamin A content in durum wheat grain by suppressing ß-carotene hydroxylase 1 genes with a TILLING approach.

KEY MESSAGE: The suppression of the HYD-1 gene by a TILLING approach increases the amount of ß-carotene in durum wheat kernel. Vitamin A deficiency is a major public health problem that affects numerous countries in the world. As humans are not able to synthesize vitamin A, it must be daily assimilated along with other micro- and macronutrients through the diet. Durum wheat is an important crop for Mediterranean countries and provides a discrete amount of nutrients, such as carbohydrates and proteins, but it is deficient in some essential micronutrients, including provitamin A. In the present work, a targeting induced local lesions in genomes strategy has been undertaken to obtain durum wheat genotypes biofortified in provitamin A. In detail, we focused on the suppression of the ß-carotene hydroxylase 1 (HYD1) genes, encoding enzymes involved in the redirection of ß-carotene toward the synthesis of the downstream xanthophylls (neoxanthin, violaxanthin and zeaxanthin). Expression analysis of genes involved in carotenoid biosynthesis revealed a reduction of the abundance of HYD1 transcripts greater than 50% in mutant grain compared to the control. The biochemical profiling of carotenoid in the wheat mutant genotypes highlighted a significant increase of more than 70% of ß-carotene compared to the wild-type sibling lines, with no change in lutein, α-carotene and zeaxanthin content. This study sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat and provides new genotypes that represent a good genetic resource for future breeding programs focused on the provitamin A biofortification through non-transgenic approaches.

Carotenoids and fatty liver disease: Current knowledge and research gaps.

Carotenoids form an important part of the human diet, consumption of which has been associated with many health benefits. With the growing global burden of liver disease, increasing attention has been paid on the possible beneficial role that carotenoids may play in the liver. This review focuses on carotenoid actions in non-alcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD). Indeed, many human studies have suggested an association between decreased circulating levels of carotenoids and increased incidence of NAFLD and ALD. The literature describing supplementation of individual carotenoids in rodent models of NAFLD and ALD is reviewed, with particular attention paid to ß-carotene and lycopene, but also including ß-cryptoxanthin, lutein, zeaxanthin, and astaxanthin. The effect of beta-carotene oxygenase 1 and 2 knock-out mice on hepatic lipid metabolism is also discussed. In general, there is evidence to suggest that carotenoids have beneficial effects in animal models of both NAFLD and ALD. Mechanistically, these benefits may occur via three possible modes of action: 1) improved hepatic antioxidative status broadly attributed to carotenoids in general, 2) the generation of vitamin A from ß-carotene and ß-cryptoxanthin, leading to improved hepatic retinoid signaling, and 3) the generation of apocarotenoid metabolites from ß-carotene and lycopene, that may regulate hepatic signaling pathways. Gaps in our knowledge regarding carotenoid mechanisms of action in the liver are highlighted throughout, and the review ends by emphasizing the importance of dose effects, mode of delivery, and mechanism of action as important areas for further study. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

Metabolic engineering for the production of fat-soluble vitamins: advances and perspectives.

Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and ß-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and ß-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes.

Impacts of deletion and ichthyosis prematurity syndrome-associated mutations in fatty acid transport protein 4 on the function of RPE65.

The retinal pigment epithelium-specific 65 kDa (RPE65) isomerase plays a pivotal role in photoreceptor survival and function. RPE65-catalyzed synthesis of 11-cis-retinol from all-trans-retinyl esters in the visual cycle is negatively regulated, through a heretofore unknown mechanism, by the fatty acid transport protein FATP4, mutations in which are associated with ichthyosis prematurity syndrome (IPS). Here, we analyzed the interaction between deletion mutants of FATP4 and RPE65 and the impacts of IPS-associated FATP4 mutations on RPE65 expression, 11-cis-retinol synthesis, and all-trans-retinyl ester synthesis. Our results suggest that the interaction between FATP4 and RPE65 contributes to the inhibition of RPE65 function and that IPS-associated nonsense and missense mutations in FATP4 have different effects on the visual cycle.

Insights into the role of bacteria in vitamin A biosynthesis: Future research opportunities.

Significant efforts have been made to address the hidden hunger challenges due to iron, zinc, iodine, and vitamin A since the beginning of the 21st century. Prioritizing the vitamin A deficiency (VAD) disorders, many countries are looking for viable alternative strategies such as biofortification. One of the leading causes of VAD is the poor bioconversion of ß-carotene into retinoids. This review is focused on the opportunities of bacterial biosynthesis of retinoids, in particular, through the gut microbiota. The proposed hypothesis starts with the premise that an animal can able to store and timely convert carotenoids into retinoids in the liver and intestinal tissues. This theory is experimental with many scientific insights. The syntrophic metabolism, potential crosstalk of bile acids, lipocalins and lipopolysaccharides of gut microbiota are reported to contribute significantly to the retinoid biosynthesis. The gut bacteria respond to these kinds of factors by genetic restructuring driven mainly by events like horizontal gene transfer. A phylogenetic analysis of ß-carotene 15, 15'-mono (di) oxygenase enzymes among a selected group of prokaryotes and eukaryotes was carried out to validate the hypotheses. Shedding light on the probiotic strategies through non-genetically modified organism such as gut bacteria capable of synthesizing vitamin A would address the VAD disorders.

Peanut butter increases the bioavailability and bioconversion of kale ß-carotene to vitamin A.

BACKGROUND AND OBJECTIVES: Kale is a rich source of provitamin A- ß-carotene. This study used intrinsically labeled kale [2H9] ß-carotene to determine the effect of peanut butter on the bioconversion of kale ß-carotene to vitamin A in preschool children. METHODS AND STUDY DESIGN: Preschool children (n=37; age 12-36 mo) were randomly assigned to 50 g cooked kale (1.5 mg ß-carotene content) with either 33 g peanut butter (PBG) or with 16 g lard (LG) and a reference dose of 1 mg [13C10] retinyl acetate capsule. Blood samples were processed to serum and analyzed by Negative Chemical Ionization-Gas Chromatography Mass Spectrometry (NCI-GCMS) for the enrichments of [2H] retinol from kale [2H9] ß-carotene and [13C10] retinol from reference dose. RESULTS: The area under curves (AUCs) of molar enrichment at days 1, 2, 3, 6, 15, and 21 after the labeled doses was 56.3±10.5 and 84.8±16.2 (nmole) for [2H] retinol from LG and PBG kale [2H9] ß-carotene, respectively. The AUC of [13C10] retinol from reference dose was 432.6±54.9 (LG) and 560.3±156.7 (nmole) (PBG), respectively. The calculated ß-carotene conversion factors were 13.4±3.1 and 11.0±3.9 to 1 (p>0.05) by weight for LG and PBG, respectively. CONCLUSIONS: This study showed that peanut butter enhances the vitamin A value of kale.

A Simple Plasma Retinol Isotope Ratio Method for Estimating ß-Carotene Relative Bioefficacy in Humans: Validation with the Use of Model-Based Compartmental Analysis.

Background: Provitamin A carotenoids are an important source of dietary vitamin A for many populations. Thus, accurate and simple methods for estimating carotenoid bioefficacy are needed to evaluate the vitamin A value of test solutions and plant sources. ß-Carotene bioefficacy is often estimated from the ratio of the areas under plasma isotope response curves after subjects ingest labeled ß-carotene and a labeled retinyl acetate reference dose [isotope reference method (IRM)], but to our knowledge, the method has not yet been evaluated for accuracy.Objectives: Our objectives were to develop and test a physiologically based compartmental model that includes both absorptive and postabsorptive ß-carotene bioconversion and to use the model to evaluate the accuracy of the IRM and a simple plasma retinol isotope ratio [(RIR), labeled ß-carotene-derived retinol/labeled reference-dose-derived retinol in one plasma sample] for estimating relative bioefficacy.Methods: We used model-based compartmental analysis (Simulation, Analysis and Modeling software) to develop and apply a model that provided known values for ß-carotene bioefficacy. Theoretical data for 10 subjects were generated by the model and used to determine bioefficacy by RIR and IRM; predictions were compared with known values. We also applied RIR and IRM to previously published data.Results: Plasma RIR accurately predicted ß-carotene relative bioefficacy at 14 d or later. IRM also accurately predicted bioefficacy by 14 d, except that, when there was substantial postabsorptive bioconversion, IRM underestimated bioefficacy. Based on our model, 1-d predictions of relative bioefficacy include absorptive plus a portion of early postabsorptive conversion.Conclusion: The plasma RIR is a simple tracer method that accurately predicts ß-carotene relative bioefficacy based on analysis of one blood sample obtained at ≥14 d after co-ingestion of labeled ß-carotene and retinyl acetate. The method also provides information about the contributions of absorptive and postabsorptive conversion to total bioefficacy if an additional sample is taken at 1 d.

Myrciaria dubia, an Amazonian fruit: population structure and its implications for germplasm conservation and genetic improvement.

Myrciaria dubia (camu-camu) is an Amazon tree that produces a tart fruit with high vitamin C content. It is probably the fruit with the highest vitamin C content among all Brazilian fruit crops and it can be used to supplement daily vitamin C dose. This property has attracted the attention of consumers and, consequently, encouraged fruit farmers to produce it. In order to identify and select potential accessions for commercial exploitation and breeding programs, M. dubia has received considerable research attention. The identification and characterization of genetic diversity, as well as identification of the population structure of accessions preserved in germplasm banks are fundamental for the success of any breeding program. The objective of this study was to evaluate the genetic variability of 10 M. dubia populations obtained from the shores of Reis Lake, located in the municipality of Caracaraí, Roraima, Brazil. Fourteen polymorphic inter simple sequence repeat (ISSR) markers were used to study the population genetic diversity, which resulted in 108 identified alleles. Among the 14 primers, GCV, UBC810, and UBC827 produced the highest number of alleles. The study illustrated the suitability and efficiency of ISSR markers to study the genetic diversity of M. dubia accessions. We also revealed the existence of high genetic variability among both accessions and populations that can be exploited in future breeding programs and conservation activities of this species.

Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2.

The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-cis-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11-cis-retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11-cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis-retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols and thus enables preferential synthesis of 11-cis-retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity.

Formation and Clearance of All-Trans-Retinol in Rods Investigated in the Living Primate Eye With Two-Photon Ophthalmoscopy.

Purpose: Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown that all-trans-retinol is likely the chief source of time-varying TPEF from photoreceptors. Endogenous TPEF from retinol could provide the specificity desired for tracking the visual cycle. However, in vivo characterization of native retinol kinetics is complicated by visual stimulation from the imaging beam. We have developed an imaging scheme for overcoming these challenges and monitored the formation and clearance of retinol. Methods: Three macaques were imaged by using an in vivo two-photon ophthalmoscope. Endogenous TPEF was excited at 730 nm and recorded through the eye's pupil for more than 90 seconds. Two-photon excited fluorescence increased with onset of light and plateaued within 40 seconds, at which point, brief incremental stimuli were delivered at 561 nm. The responses of rods to stimulation were analyzed by using first-order kinetics. Results: Two-photon excited fluorescence resulting from retinol production corresponded to the fraction of rhodopsin bleached. The photosensitivity of rhodopsin was estimated to be 6.88 ± 5.50 log scotopic troland. The rate of retinol clearance depended on intensity of incremental stimulation. Clearance was faster for stronger stimuli and time constants ranged from 50 to 300 seconds. Conclusions: This study demonstrates a method for rapidly measuring the rate of clearance of retinol in vivo. Moreover, TPEF generated due to retinol can be used as a measure of rhodopsin depletion, similar to densitometry. This enhances the utility of two-photon ophthalmoscopy as a technique for evaluating the visual cycle in the living eye.

Biosynthesis of Carotenoids in Plants: Enzymes and Color.

Carotenoids are the most important biocolor isoprenoids responsible for yellow, orange and red colors found in nature. In plants, they are synthesized in plastids of photosynthetic and sink organs and are essential molecules for photosynthesis, photo-oxidative damage protection and phytohormone synthesis. Carotenoids also play important roles in human health and nutrition acting as vitamin A precursors and antioxidants. Biochemical and biophysical approaches in different plants models have provided significant advances in understanding the structural and functional roles of carotenoids in plants as well as the key points of regulation in their biosynthesis. To date, different plant models have been used to characterize the key genes and their regulation, which has increased the knowledge of the carotenoid metabolic pathway in plants. In this chapter a description of each step in the carotenoid synthesis pathway is presented and discussed.

Carotenoid Biosynthesis in Daucus carota.

Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota.

Manipulation of Carotenoid Content in Plants to Improve Human Health.

Carotenoids are essential components for human nutrition and health, mainly due to their antioxidant and pro-vitamin A activity. Foods with enhanced carotenoid content and composition are essential to ensure carotenoid feasibility in malnourished population of many countries around the world, which is critical to alleviate vitamin A deficiency and other health-related disorders. The pathway of carotenoid biosynthesis is currently well understood, key steps of the pathways in different plant species have been characterized and the corresponding genes identified, as well as other regulatory elements. This enables the manipulation and improvement of carotenoid content and composition in order to control the nutritional value of a number of agronomical important staple crops. Biotechnological and genetic engineering-based strategies to manipulate carotenoid metabolism have been successfully implemented in many crops, with Golden rice as the most relevant example of ß-carotene improvement in one of the more widely consumed foods. Conventional breeding strategies have been also adopted in the bio-fortification of carotenoid in staple foods that are highly consumed in developing countries, including maize, cassava and sweet potatoes, to alleviate nutrition-related problems. The objective of the chapter is to summarize major breakthroughs and advances in the enhancement of carotenoid content and composition in agronomical and nutritional important crops, with special emphasis to their potential impact and benefits in human nutrition and health.

The Biochemical Basis of Vitamin A3 Production in Arthropod Vision.

Metazoan photochemistry involves cis-trans isomerization of a retinylidene chromophore bound to G protein coupled receptors. Successful production of chromophores is critical for photoreceptor function and survival. For chromophore production, animals have to choose from more than 600 naturally occurring carotenoids and process them by oxidative cleavage and geometric isomerization of double bonds. Vertebrates employ three carotenoid cleavage oxygenases to tailor the carotenoid precursor in the synthesis of 11-cis-retinal (vitamin A1). Lepidoptera (butterfly and moth) possess only one such enzyme, NinaB, which faces the challenge to catalyze these reactions in unison to produce 11-cis-3-hydroxy-retinal (vitamin A3). We here showed that key to this multitasking is a bipartite substrate recognition site that conveys regio- and stereoselectivity for double bond processing. One side performed the specific C11, C12 cis-isomerization and preferentially binds 3-OH-ß-ionone rings sites. The other side maintained a trans configuration in the resulting product and preferentially binds noncanonical ionone ring sites. Concurrent binding of carotenoids containing two cyclohexyl rings to both domains is required for specific oxidative cleavage at position C15, C15' of the substrate. The unique reaction sequence follows a dioxygenase mechanism with a carbocation/radical intermediate. This ingenious quality control system guarantees 11-cis-3-hydroxy-retinal production, the essential retinoid for insect (vitamin A3) vision.

Metabolic Engineering of Wheat Provitamin A by Simultaneously Overexpressing CrtB and Silencing Carotenoid Hydroxylase (TaHYD).

Increasing the provitamin A content in staple crops via carotenoid metabolic engineering is one way to address vitamin A deficiency. In this work a combination of methods was applied to specifically increase ß-carotene content in wheat by metabolic engineering. Endosperm-specific silencing of the carotenoid hydroxylase gene (TaHYD) increased ß-carotene content 10.5-fold to 1.76 µg g(-1) in wheat endosperm. Overexpression of CrtB introduced an additional flux into wheat, accompanied by a ß-carotene increase of 14.6-fold to 2.45 µg g(-1). When the "push strategy" (overexpressing CrtB) and "block strategy" (silencing TaHYD) were combined in wheat metabolic engineering, significant levels of ß-carotene accumulation were obtained, corresponding to an increase of up to 31-fold to 5.06 µg g(-1). This is the first example of successful metabolic engineering to specifically improve ß-carotene content in wheat endosperm through a combination of methods and demonstrates the potential of genetic engineering for specific nutritional enhancement of wheat.

The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry.

Euglena gracilis is a highly complex alga belonging to the green plant line that shows characteristics of both plants and animals, while in evolutionary terms it is most closely related to the protozoan parasites Trypanosoma and Leishmania. This well-studied organism has long been known as a rich source of vitamins A, C and E, as well as amino acids that are essential for the human diet. Here we present de novo transcriptome sequencing and preliminary analysis, providing a basis for the molecular and functional genomics studies that will be required to direct metabolic engineering efforts aimed at enhancing the quality and quantity of high value products from E. gracilis. The transcriptome contains over 30,000 protein-encoding genes, supporting metabolic pathways for lipids, amino acids, carbohydrates and vitamins, along with capabilities for polyketide and non-ribosomal peptide biosynthesis. The metabolic and environmental robustness of Euglena is supported by a substantial capacity for responding to biotic and abiotic stress: it has the capacity to deploy three separate pathways for vitamin C (ascorbate) production, as well as producing vitamin E (α-tocopherol) and, in addition to glutathione, the redox-active thiols nor-trypanothione and ovothiol.

Efficient two-step chemo-enzymatic synthesis of all-trans-retinyl palmitate with high substrate concentration and product yield.

A new two-step chemo-enzymatic approach for highly efficient synthesis of all-trans-retinyl palmitate is constructed in this study. In the first step, retinyl acetate as starting material was fully hydrolyzed to retinol by potassium hydroxide. In the hydrolysis system, anhydrous ethanol was the best co-solvent to increase the solubility of retinyl acetate. The addition amounts of 5 M potassium hydroxide and anhydrous ethanol were 8 and 10 mL against 10 g retinyl acetate, respectively, and 100 % hydrolysis rate was obtained. In the second step, esterification was catalyzed by immobilized lipase on macroporous acrylic resin AB-8 using the extracted retinol and palmitic acid as substrates in non-aqueous system. After optimization, the parameters of esterification reaction were confirmed as follows: non-aqueous solvent was selected as n-hexane, washing times of extraction solution was four times, retinol concentration was 300 g/L, substrate molar ratio of retinol to palmitic acid was 1:1.1, the amount of immobilized enzyme was 10 g/L, and the esterification temperature was 30 °C. Under the optimal conditions, this protocol resulted in a 97.5 % yield of all-trans-retinyl palmitate in 700-L reactor. After purification, all-trans-retinyl palmitate was obtained with above 99 % of purity and 88 % of total recovery rate. This methodology provides a promising strategy for the large-scale production of all-trans-retinyl palmitate.

The lycopene ß-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm.

BACKGROUND: Lycopene ß-cyclase (LCYB) is a key enzyme catalyzing the biosynthesis of ß-carotene, the main source of provitamin A. However, there is no documented research about this key cyclase gene's function and relationship with ß-carotene content in wheat. Therefore, the objectives of this study were to clone TaLCYB and characterize its function and relationship with ß-carotene biosynthesis in wheat grains. We also aimed to obtain more information about the endogenous carotenoid biosynthetic pathway and thus provide experimental support for carotenoid metabolic engineering in wheat. RESULTS: In the present study, a lycopene ß-cyclase gene, designated TaLCYB, was cloned from the hexaploid wheat cultivar Chinese Spring. The cyclization activity of the encoded protein was demonstrated by heterologous complementation analysis. The TaLCYB gene was expressed differentially in different tissues of wheat. Although TaLCYB had a higher expression level in the later stages of grain development, the ß-carotene content still showed a decreasing tendency. The expression of TaLCYB in leaves was dramatically induced by strong light and the ß-carotene content variation corresponded with changes of TaLCYB expression. A post-transcriptional gene silencing strategy was used to down-regulate the expression of TaLCYB in transgenic wheat, resulting in a decrease in the content of ß-carotene and lutein, accompanied by the accumulation of lycopene to partly compensate for the total carotenoid content. In addition, changes in TaLCYB expression also affected the expression of several endogenous carotenogenic genes to varying degrees. CONCLUSION: Our results suggest that TaLCYB is a genuine lycopene cyclase gene and plays a crucial role in ß-carotene biosynthesis in wheat. Our attempt to silence it not only contributes to elucidating the mechanism of carotenoid accumulation in wheat but may also help in breeding wheat varieties with high provitamin A content through RNA interference (RNAi) to block specific carotenogenic genes in the wheat endosperm.

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI.

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 µg g(-1) of seed dry weight, a ß-carotene increase of 65-fold to 3.21 µg g(-1) of seed dry weight, and a provitamin A content (sum of α-carotene, ß-carotene, and ß-cryptoxanthin) increase of 76-fold to 3.82 µg g(-1) of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm.