Characterization of cassava ORANGE proteins and their capability to increase provitamin A carotenoids accumulation

Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.

Assessing the Role of Carotenoid Cleavage Dioxygenase 4 Homoeologs in Carotenoid Accumulation and Plant Growth in Tetraploid Wheat

The dietary needs of humans for provitamin A carotenoids arise from their inability to synthesize vitamin A de novo. To improve the status of this essential micronutrient, special attention has been given to biofortification of staple foods, such as wheat grains, which are consumed in large quantities but contain low levels of provitamin A carotenoids. However, there remains an unclear contribution of metabolic genes and homoeologs to the turnover of carotenoids in wheat grains. To better understand carotenoid catabolism in tetraploid wheat, Targeting Induced Local Lesions in Genomes (TILLING) mutants of CCD4, encoding a Carotenoid Cleavage Dioxygenase (CCD) that cleaves carotenoids into smaller apocarotenoid molecules, were isolated and characterized. Our analysis showed that ccd4 mutations co-segregated with Poltergeist-like (pll) mutations in the TILLING mutants of A and B subgenomes, hence the ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B mutants were analyzed in this study. Carotenoid profiles are comparable in mature grains of the mutant and control plants, indicating that CCD4 homoeologs do not have a major impact on carotenoid accumulation in grains. However, the neoxanthin content was increased in leaves of ccd-A4 ccd-B4 pll-A pll-B relative to the control. In addition, four unidentified carotenoids showed a unique presence in leaves of ccd-A4 ccd-B4 pll-A pll-B plants. These results suggested that CCD4 homoeologs may contribute to the turnover of neoxanthin and the unidentified carotenoids in leaves. Interestingly, abnormal spike, grain, and seminal root phenotypes were also observed for ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B plants, suggesting that CCD4 and/or PLL homoeologs could function toward these traits. Overall, this study not only reveals the role of CCD4 in cleavage of carotenoids in leaves and grains, but also uncovers several critical growth traits that are controlled by CCD4, PLL, or the CCD4-PLL interaction.

The Development of Flake Containing Provitamin A Carotenoids From Blend of Banana and Yellow-Fleshed Sweet Potato

As vitamin A deficiency (VAD) remains a health problem in Indonesia, fulfilling the requirement of vitamin A from the daily diet is of importance, especially for children. Consuming food sources rich in provitamin A carotenoids, such as bananas and yellow-fleshed sweet potatoes, is one alternative to reduce the number of VADs. Raw materials have been known for their short shelf life due to high moisture content. Therefore, processing them into dry products such as flakes can be one method to extend the shelf life of a product. The utilisation of Agung Semeru banana and Papua Solossa yellow-fleshed sweet potato into flakes containing provitamin A carotenoids was conducted in this study. Through focus group evaluation, flake F2 that was made from blend of banana puree (BP), banana flour (BF), and sweet potato flour (SPF) with ratio 6:1:3 (w/w/w) was selected as the most preferred flake, and was liked by 77% of panelists in the following organoleptic appraisal. Comprising the total carotenoid by 1926.73 µg/100 g dw, vitamin A activity around 58.45 µg RAE/100 g dw, and high amount of carbohydrate, along with ash that represents minerals, this flake can be considered as a nutritional and energy food resource. However, this product is in short of meeting the recommendation of protein and fat given by Indonesian national standard, with slightly higher moisture content. Hence, it requires some improvement by serving it with milk and/or legumes, as well as selecting a proper packaging material and method.

Vitamins in Cereals: A Critical Review of Content, Health Effects, Processing Losses, Bioaccessibility, Fortification, and Biofortification Strategies for Their Improvement

Around the world, cereals are stapled foods and good sources of vitamins A, B, and E. As cereals are inexpensive and consumed in large quantities, attempts are being made to enrich cereals using fortification and biofortification in order to address vitamin deficiency disorders in a vulnerable population. The processing and cooking of cereals significantly affect vitamin content. Depending on grain structure, milling can substantially reduce vitamin content, while cooking methods can significantly impact vitamin retention and bioaccessibility. Pressure cooking has been reported to result in large vitamin losses, whereas minimal vitamin loss was observed following boiling. The fortification of cereal flour with vitamins B1, B2, B3, and B9, which are commonly deficient, has been recommended; and in addition, region-specific fortification using either synthetic or biological vitamins has been suggested. Biofortification is a relatively new concept and has been explored as a method to generate vitamin-rich crops. Once developed, biofortified crops can be utilized for several years. A recent cereal biofortification success story is the enrichment of maize with provitamin A carotenoids.

Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds

Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification. However, carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains. In this study, we utilized Arabidopsis as a model to establish carotenoid biofortification strategies in seeds. We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase (PSY) increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage, consistent with previous studies of provitamin A biofortified grains. In contrast, stacking with Orange (ORHis), a gene that initiates chromoplast biogenesis, dramatically enhances provitamin A and total carotenoid content and stability. Up to 65- and 10-fold increases of β-carotene and total carotenoids, respectively, with provitamin A carotenoids composing over 63% were observed in the seeds containing ORHis and PSY. Co-expression of Homogentisate geranylgeranyl transferase (HGGT) with ORHis and PSY further increases carotenoid accumulation and stability during seed maturation and storage. Moreover, knocking-out of β-carotene hydroxylase 2 (BCH2) by CRISPR/Cas9 not only potentially facilitates β-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germination. Our findings provide new insights into various processes on carotenoid accumulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis, turnover, and stable storage for carotenoid biofortification in crop seeds.

Ethylene activation of carotenoid biosynthesis by a novel transcription factor CsERF061

Chromoplast-specific lycopene β-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids, in citrus. Its regulatory metabolism is largely unknown. Here, we identified a group I ethylene response factor, CsERF061, in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, and increased the number of chromoplasts needed to sequester the elevated concentrations of carotenoids, which was accompanied by changes in the concentrations of abscisic acid and gibberellin. Electrophoretic mobility shift and dual-luciferase assays verified that CsERF061 activates the promoters of nine other key carotenoid pathway genes, PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which will be useful for manipulating carotenoid accumulation in citrus and other plants.

SPECTRAL COMPOSITION OF LIGHT AS A FACTOR IN THE REGULATION OF PLANT METABOLISM

Plants have many evolutionary developed adaptation mechanisms to ensure functional flexibility under the influence of environmental factors, including the synthesis of secondary metabolites that perform various functions in response to environmental changes, growth, and development. Further, these changes can be caused by various environmental factors, which include local geo-climatic, seasonal changes, temperature conditions, lighting, and humidity. All of them affect the accumulation of biomass and the biosynthesis of primary and secondary plant metabolites. The formation of secondary metabolites by plants is considered their adaptive ability to cope with the stressful conditions of a changing environment. Within the framework of this study, the biochemical status of Brassica oleracea var. Sabellica, Lactuca sativa, Petroselinum crispum plants were determined when grown indoors under various light spectral composition. To determine the biochemical status, the plants were analyzed for the content of ascorbic acid, provitamin A, carotenoids and proteins, and their antioxidant activity was also determined. Results of the study revealed that the blue part of the light spectrum induces the accumulation of ascorbic acid, provitamin A, carotenoids and protein in all studied species. Further, it was also reported that Kale cabbage has the highest accumulation of ascorbic acid, proteins, carotenoids, and also has the highest antioxidant activity as compared to the other studied species.