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Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Gold Rice endosperm suggests apocarotenoids as substrates in planta
Abstract
Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.
Supplementary resource (1)
Data
August 2010
... Additionally, the rice CCD1 enzyme showed an in vitro enzymatic activity that targets the C7-C8 bond of lycopene (Figure 2), generating a C 10 flavor compound, geranial (Ilg et al., 2009). However, CCD1 enzymes are localized to cytoplasm, therefore they seem to cleave already destructed carotenoids transported to cytoplasm (i.e., apocarotenoids) rather than carotenoid substrates in plastid (Ilg et al., 2009(Ilg et al., , 2010. Consistent with this speculation, overexpression of rice CCD1 in Golden Rice did not lead to significant decrease of the carotenoid content in endosperm (Ilg et al., 2010). ...
... However, CCD1 enzymes are localized to cytoplasm, therefore they seem to cleave already destructed carotenoids transported to cytoplasm (i.e., apocarotenoids) rather than carotenoid substrates in plastid (Ilg et al., 2009(Ilg et al., , 2010. Consistent with this speculation, overexpression of rice CCD1 in Golden Rice did not lead to significant decrease of the carotenoid content in endosperm (Ilg et al., 2010). CCD2, a specific CCD type that is restricted to the Crocus and Freesia genus of the Iridaceae family, is closely related to the cytoplasmic CCD1 subfamily, although it is localized in plastids (Frusciante et al., 2014;Ahrazem et al., 2016b;Fang et al., 2020). ...
In plants, carotenoids are subjected to enzyme-catalyzed oxidative cleavage reactions as well as to non-enzymatic degradation processes, which produce various carbonyl products called apocarotenoids. These conversions control carotenoid content in different tissues and give rise to apocarotenoid hormones and signaling molecules, which play important roles in plant growth and development, response to environmental stimuli, and in interactions with surrounding organisms. In addition, carotenoid cleavage gives rise to apocarotenoid pigments and volatiles that contribute to the color and flavor of many flowers and several fruits. Some apocarotenoid pigments, such as crocins and bixin, are widely utilized as colorants and additives in food and cosmetic industry and also have health-promoting properties. Considering the importance of this class of metabolites, investigation of apocarotenoid diversity and regulation has increasingly attracted the attention of plant biologists. Here, we provide an update on the plant apocarotenoid biosynthetic pathway, especially highlighting the diversity of the enzyme carotenoid cleavage dioxygenase 4 (CCD4) from different plant species with respect to substrate specificity and regioselectivity, which contribute to the formation of diverse apocarotenoid volatiles and pigments. In addition, we summarize the regulation of apocarotenoid metabolic pathway at transcriptional, post-translational, and epigenetic levels. Finally, we describe inter- and intraspecies variation in apocarotenoid production observed in many important horticulture crops and depict recent progress in elucidating the genetic basis of the natural variation in the composition and amount of apocarotenoids. We propose that the illustration of biochemical, genetic, and evolutionary background of apocarotenoid diversity would not only accelerate the discovery of unknown biosynthetic and regulatory genes of bioactive apocarotenoids but also enable the identification of genetic variation of causal genes for marker-assisted improvement of aroma and color of fruits and vegetables and CRISPR-based next-generation metabolic engineering of high-value apocarotenoids.
... Furthermore, the results from recent experiments indicated that, in planta, the substrate for CCD1 was an apocarotenoid rather than the carotenoid, and it was suggested that, in fact, CCD1s could perform more of a scavenging role for cytosolic apocarotenoids [49], whereas CCD4s (leaves and berries) or CCD7s (roots and stems) should be regarded as the primary cleavage enzymes for carotenoids, as they are co-located with their substrates in plastids [35,49], and for delivering the C 27 intermediate substrate for the action by CCD1 in the cytosol [36]. ...
... Furthermore, the results from recent experiments indicated that, in planta, the substrate for CCD1 was an apocarotenoid rather than the carotenoid, and it was suggested that, in fact, CCD1s could perform more of a scavenging role for cytosolic apocarotenoids [49], whereas CCD4s (leaves and berries) or CCD7s (roots and stems) should be regarded as the primary cleavage enzymes for carotenoids, as they are co-located with their substrates in plastids [35,49], and for delivering the C 27 intermediate substrate for the action by CCD1 in the cytosol [36]. ...
Olfactory cues are key drivers of our multisensory experiences of food and drink. For example, our perception and enjoyment of the flavour and taste of a wine is primarily steered by its aroma. Making sense of the underlying smells that drive consumer preferences is integral to product innovation as a vital source of competitive advantage in the marketplace, which explains the intense interest in the olfactory component of flavour and the sensory significance of individual compounds, such as one of the most important apocarotenoids for the bouquet of wine, β-ionone (violet and woody notes). β-Ionone is formed directly from β-carotene as a by-product of the actions of carotenoid cleavage dioxygenases (CCDs). The biological production of CCDs in microbial cell factories is one way that important aroma compounds can be generated on a large scale and with reduced costs, while retaining the ‘natural’ moniker. The CCD family includes the CCD1, CCD2, CCD4, CCD7 and CCD8; however, the functions, co-dependency and interactions of these CCDs remain to be fully elucidated. Here, we review the classification, actions and biotechnology of CCDs, particularly CCD1 and its action on β-carotene to produce the aromatic apocarotenoid β-ionone.
... However, it remains to be answered whether PGM48 is a positive regulator of carotenoid content. CCD1 enzymes seem to have less impact on carotenoid content [154] and are thought to cleave already destructed carotenoids, i.e. apocarotenoids, rather than carotenoids in planta [125,161]. Indeed, overexpression of rice CCD1 in Golden Rice endosperm did not significantly affect carotenoid content [161]. Considering that the products of NCEDs and CCD7/CCD8 are hormone precursors occurring at low concentrations, it can be assumed that the activity of these enzymes does not play a major role in determining total carotenoid content. ...
... CCD1 enzymes seem to have less impact on carotenoid content [154] and are thought to cleave already destructed carotenoids, i.e. apocarotenoids, rather than carotenoids in planta [125,161]. Indeed, overexpression of rice CCD1 in Golden Rice endosperm did not significantly affect carotenoid content [161]. Considering that the products of NCEDs and CCD7/CCD8 are hormone precursors occurring at low concentrations, it can be assumed that the activity of these enzymes does not play a major role in determining total carotenoid content. ...
Carotenoids are indispensable for human health, required as precursors of vitamin A and efficient antioxidants. However, these plant pigments that play a vital role in photosynthesis are represented at insufficient levels in edible parts of several crops, which creates a need for increasing their content or optimizing their composition through biofortification. In particular, vitamin A deficiency, a severe health problem affecting the lives of millions in developing countries, has triggered the development of a series of high-provitamin A crops, including Golden Rice as the best-known example. Further carotenoid-biofortified crops have been generated by using genetic engineering approaches or through classical breeding. In this review, we depict carotenoid metabolism in plants and provide an update on the development of carotenoid-biofortified plants and their potential to meet needs and expectations. Furthermore, we discuss the possibility of using natural variation for carotenoid biofortification and the potential of gene editing tools.
... CCD1 from maize has been reported to cleave specifically at 9 and 10 positions of lycopene, β-carotene and zeaxanthin, representing both cyclic and acyclic carotenoids [43]. Additionally, CCD1 from rice can cleave lycopene at 7,8 double bond [44]. SlCCD1A and SlCCD1B show flexibility in the substrate and cleavage site specificity through oxidative cleavage of cis, all-trans-carotenoids as well as of different apocarotenoids at many other double-bond positions [45]. ...
... The VvCCD1 has been shown to cleave zeaxanthin symmetrically yielding 3-hydroxy-β-ionone, a C(13)-norisoprenoidic compound and a C(14)-dialdehyde in V. vinifera [74]. Another interesting case was observed in Golden Rice 2 for its color, where OsCCD1 was reported to act upon apocarotenoid as substrate instead of carotenoid, as silencing of OsCCD1 had no impact on the carotenoid content [44]. In O. fragrans, OfCCD1 cleaved carotenes to α-ionone and β-ionone responsible for its fragrance [75]. ...
A plant communicates within itself and with the outside world by deploying an array of agents that include several attractants by virtue of their color and smell. In this category, the contribution of 'carotenoids and apocarotenoids' is very significant. Apocarotenoids, the carotenoid-derived compounds, show wide representation among organisms. Their biosynthesis occurs by oxidative cleavage of carotenoids, a high-value reaction, mediated by carotenoid cleavage oxygenases or carotenoid cleavage dioxygenases (CCDs)-a family of non-heme iron enzymes. Structurally, this protein family displays wide diversity but is limited in its distribution among plants. Functionally, this protein family has been recognized to offer a role in phytohormones, volatiles and signal production. Further, their wide presence and clade-specific functional disparity demands a comprehensive account. This review focuses on the critical assessment of CCDs of higher plants, describing recent progress in their functional aspects and regulatory mechanisms, domain architecture, classification and localization. The work also highlights the relevant discussion for further exploration of this multi-prospective protein family for the betterment of its functional understanding and improvement of crops.
... Additional cleavage sites in apocarotenoids have also been documented (Ilg et al., 2014). Moreover, it was assumed that CCD1-type enzymes convert apocarotenoids rather than carotenoids in planta and that they may act as a scavenger for oxidatively damaged carotenoids (Scherzinger and Al-Babili, 2008;Ilg et al., 2010). In contrast, CCD7 and CCD8 are devoted to the biosynthesis of SLs (Gomez-Roldan et al., 2008;Umehara et al., 2008;Al-Babili and Bouwmeester, 2015). ...
... The resulting rate constants demonstrate that (i) unhydroxylated bicyclic C 40 carotenes are preferred over C 27 apocarotenoids; (ii) the presence of one or more OH groups in the C 40 substrate reduces cleavage activity; and (iii) the latter does not apply to C 27 apocarotenoids which, hydroxylated or not, were converted with very similar albeit low rate constants. It is worth noting that AtCCD1, an enzyme also targeting the C9-C10 double bond, prefers apocarotenoids over bicyclic carotenoids (Schmidt et al., 2006;Ilg et al., 2010). This difference is probably due to different biological functions and might be reflected by the cytoplasmatic localization of CCD1 that may contribute to carotenoid degradation in planta (Gonzalez-Jorge et al., 2013; see the Introduction). ...
The Arabidopsis carotenoid cleavage dioxygenase 4 (AtCCD4) is a negative regulator of the carotenoid content of seeds and has recently been suggested as a candidate for the generation of retrograde signals that are thought to derive from the cleavage of poly-cis-configured carotene desaturation intermediates. In this work, we investigated the activity of AtCCD4 in vitro and used dynamic modeling to determine its substrate preference. Our results document strict regional specificity for cleavage at the C9–C10 double bond in carotenoids and apocarotenoids, with preference for carotenoid substrates and an obstructing effect on hydroxyl functions, and demonstrate the specificity for all-trans-configured carotenes and xanthophylls. AtCCD4 cleaved substrates with at least one ionone ring and did not convert acyclic carotene desaturation intermediates, independent of their isomeric states. These results do not support a direct involvement of AtCCD4 in generating the supposed regulatory metabolites. In contrast, the strigolactone biosynthetic enzyme AtCCD7 converted 9-cis-configured acyclic carotenes, such as 9-cis-ζ-carotene, 9'-cis-neurosporene, and 9-cis-lycopene, yielding 9-cis-configured products and indicating that AtCCD7, rather than AtCCD4, is the candidate for forming acyclic retrograde signals.
... Therefore, it is speculated that plant CCD1s convert the plastid-released apocarotenoids that have arisen through either non-enzymatic oxidative cleavage processes or enzymatic cleavage by other CCDs (CCD4 and/or CCD7). This scenario might explain the multiple cleavage sites and the wide substrate specificity displayed by CCD1 enzymes (Ilg et al. 2010). CCD1 enzymes are involved in the cleavage of the 5,6 (5 0 ,6 0 ) (Vogel et al. 2008), 7,8 (7 0 ,8 0 ) (Ilg et al. 2009) and 9,10 (9 0 ,10 0 ) (Schwartz et al. 2001) double bonds to produce a variety of volatiles. ...
... However, during senescence, when the chloroplast membranes disintegrate, CCD1s will have access to its substrates. C 27 apocarotenoids have rarely been found in nature, perhaps due to the activity of CCD1s in plant tissues and it is speculated that plant CCD1s also convert the plastid-released C 27 apocarotenoids that have arisen through the non-enzymatic oxidative cleavage processes (Ilg et al. 2010). Recently, a novel plant CCD enzyme from saffron, CsCCD2, which catalyzes the cleavage step leading to crocetin biosynthesis starting from the precursor, zeaxanthin, was reported (Frusciante et al. 2014). ...
Carotenoids are precursors of carotenoid derived molecules termed apocarotenoids, which include isoprenoids with important functions in plant-environment interactions such as the attraction of pollinators and the defense against pathogens and herbivores. Apocarotenoids also include volatile aromatic compounds that act as repellents, chemoattractants, growth simulators and inhibitors, as well as the phytohormones abscisic acid and strigolactones. In plants, apocarotenoids can be found in several types of plastids (etioplast, leucoplast and chromoplast) and among different plant tissues such as flowers and roots. The structural similarity of some flower and spice isoprenoid volatile organic compounds (β-ionone and safranal) to carotenoids has led to the recent discovery of carotenoid-specific cleavage oxygenases, including carotenoid cleavage dioxygenases and 9-cis-epoxydioxygenases, which tailor and transform carotenoids into apocarotenoids. The great diversity of apocarotenoids is a consequence of the huge amount of carotenoid precursors, the variations in specific cleavage sites and the modifications after cleavage. Lycopene, β-carotene and zeaxanthin are the precursors of the main apocarotenoids described to date, which include bixin, crocin, picrocrocin, abscisic acid, strigolactone and mycorradicin.
The current chapter will give rise to an overview of the biosynthesis and function of the most important apocarotenoids in plants, as well as the current knowledge about the carotenoid cleavage oxygenase enzymes involved in these biosynthetic pathways.
... Therefore, it is speculated that plant CCD1s convert the plastid-released apocarotenoids that have arisen through either non-enzymatic oxidative cleavage processes or enzymatic cleavage by other CCDs (CCD4 and/or CCD7). This scenario might explain the multiple cleavage sites and the wide substrate specificity displayed by CCD1 enzymes (Ilg et al. 2010). CCD1 enzymes are involved in the cleavage of the 5,6 (5 0 ,6 0 ) (Vogel et al. 2008), 7,8 (7 0 ,8 0 ) (Ilg et al. 2009) and 9,10 (9 0 ,10 0 ) (Schwartz et al. 2001) double bonds to produce a variety of volatiles. ...
... However, during senescence, when the chloroplast membranes disintegrate, CCD1s will have access to its substrates. C 27 apocarotenoids have rarely been found in nature, perhaps due to the activity of CCD1s in plant tissues and it is speculated that plant CCD1s also convert the plastid-released C 27 apocarotenoids that have arisen through the non-enzymatic oxidative cleavage processes (Ilg et al. 2010). Recently, a novel plant CCD enzyme from saffron, CsCCD2, which catalyzes the cleavage step leading to crocetin biosynthesis starting from the precursor, zeaxanthin, was reported (Frusciante et al. 2014). ...
This comprehensive, edited book explores carotenoids and their important functional roles in yeast, bacteria and plants and a profound exposition on the structures of carotenoid molecules, focusing in the first of three parts on the biosynthesis of carotenoids. The regulation of carotenoid biosynthesis in photosynthesis as well as in plant, fruits, storage roots and algae is central to the second part, and discoveries about the function of carotenoids in human health feature in the third and final part. Many helpful illustrations, explanations, overviews and examples help to bring readers up to date on relevant themes including carotenogenic genes, carotenoids in fruits and metabolic engineering.
The book explores where carotenoids are synthesized in nature, including in carrots and algae. Contributing expert authors examine enzyme functions and plant models, and analyze the structure of carotenoid molecules. The function of carotenoids in photosynthesis and in photosynthetic organs as well as during fruit ripening are then explored. A whole chapter is dedicated to the latest research on apocarotenoids and further chapters cover interesting and novel themes on plastid development and the epigenetic regulation that affects carotenoid synthesis in plants. The metabolic engineering of carotenoids that has been done in fruits, plants, and seeds is another area that readers can explore, along with evidences on the function of carotenoids in human nutrition, as antioxidants, as in the control of lipid metabolism and in the absorption of carotenoids.
This is a highly informative and wide-ranging work which will update researchers in the field, as well as supporting students of plant physiology and biotechnology, as supplementary reading.
... Lycopene and β-carotene were found commonly to occur in chloroplasts and chloroplast, respectively, and to affect the color of some fruits and flowers (Jabeen et al. 2013;Jarquín-Enríquez et al. 2013;Nagal et al. 2012). In recent years, carotenoids were also found to play important roles in various types of cleavage products in plants, bacteria, fungi, and animals (Heo et al. 2013;Huang et al. 2009;Ilg et al. 2010;Liang et al. 2011;Sui et al. 2013). Carotenoid cleavage products, also known as apocarotenoids, were obtained by carotenoid cleavage oxygenases (CCOs) of specific cleavage of carotenoids. ...
... To date, all CCD1 genes identified from other plant species are single copy (Ibdah et al. 2006;Ilg et al. 2009Ilg et al. , 2010Simkin et al. 2004a;Vogel et al. 2008), with an exception of the SlCCD1 which encoded by two closely related genes (SlCCD1a and SlCCD1b). It suggested that a duplication event had occurred in the course of evolution. ...
Carotenoid cleavage dioxygenases (CCDs) in plant species is one of the most important enzymes in the carotenoid metabolism. In this study, we performed a comprehensive analysis for the CCDs family in Solanum lycopersicum based on the whole tomato genome sequences and explored their expression pattern. At least seven CCD genes were discovered in the tomato genome sequence. Two pairs of them were arranged in tandem. The tandem duplication events could be dating to approximately 14 and 21 Mya, and the tandem duplication genes experienced a purifying selection during the course of evolution after diversification. Additionally, subcellular localization revealed that four members were predicted to be cytoplasm-localized and the three remaining members plastids-localized. Subsequently, a number of cis-regulatory elements, which were involved in light responsiveness, hormone regulation, and abiotic and biotic stresses, were identified in the promoter sequences of SlCCD genes. Phylogenetic tree revealed that the CCDs from Solanaceae crops have a closer genetic relationship. The difference in abundance and distinct expression patterns during the vegetative and reproductive development suggests different functions for these seven SlCCDs. Our findings suggest that SlCCDs family play important roles throughout the whole life course and will lay the foundation for further elaborating the regulatory mechanism of each member in tomato.
... A study by Ren et al. [58] suggested that a member of the β-glucosidase protein family, Os06gGlu24 plays a role in seed germination and root elongation, while interacting with indole-3-acetic acid (IAA) and abscisic acid (ABA) signaling. Likewise, Ilg et al. [59] proposed the CCD1 gene as being involved in the control of endosperm color in rice. ...
This study investigated novel quantitative traits loci (QTLs) associated with the control of grain shape and size as well as grain weight in rice. We employed a joint-strategy multiple GAPIT (Genome Association and Prediction Integrated Tool) models [(Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK)), Fixed and random model Circulating Probability Uniform (FarmCPU), Settlement of MLM Under Progressive Exclusive Relationship (SUPER), and General Linear Model (GLM)]–High-Density SNP Chip DNA Markers (60,461) to conduct a Genome-Wide Association Study (GWAS). GWAS was performed using genotype and grain-related phenotypes of 143 recombinant inbred lines (RILs). Data show that parental lines (Ilpum and Tung Tin Wan Hein 1, TTWH1, Oryza sativa L., ssp. japonica and indica, respectively) exhibited divergent phenotypes for all analyzed grain traits), which was reflected in their derived population. GWAS results revealed the association between seven SNP Chip makers and QTLs for grain length, co-detected by all GAPIT models on chromosomes (Chr) 1–3, 5, 7, and 11, were qGL1-1BFSG (AX-95918134, Chr1: 3,820,526 bp) explains 65.2–72.5% of the phenotypic variance explained (PVE). In addition, qGW1-1BFSG (AX-273945773, Chr1: 5,623,288 bp) for grain width explains 15.5–18.9% of PVE. Furthermore, BLINK or FarmCPU identified three QTLs for grain thickness independently, and explain 74.9% (qGT1Blink, AX-279261704, Chr1: 18,023,142 bp) and 54.9% (qGT2-1Farm, AX-154787777, Chr2: 2,118,477 bp) of the observed PVE. For the grain length-to-width ratio (LWR), the qLWR2BFSG (AX-274833045, Chr2: 10,000,097 bp) explains nearly 15.2–32% of the observed PVE. Likewise, the major QTL for thousand-grain weight (TGW) was detected on Chr6 (qTGW6BFSG, AX-115737727, 28,484,619 bp) and explains 32.8–54% of PVE. The qTGW6BFSG QTL coincides with qGW6-1Blink for grain width and explained 32.8–54% of PVE. Putative candidate genes pooled from major QTLs for each grain trait have interesting annotated functions that require functional studies to elucidate their function in the control of grain size, shape, or weight in rice. Genome selection analysis proposed makers useful for downstream marker-assisted selection based on genetic merit of RILs.
... A study by Ren, et al. [58] suggested that a member of the β-glucosidase protein family, Os06gGlu24 plays a role in seed germination and root elongation, while interacting with indole-3-acetic acid (IAA) and abscisic acid (ABA) signaling. Likewise, Ilg, et al. [59] proposed the CCD1 gene as being involved in the control of endosperm color in rice. ...
This study investigated novel quantitative traits loci (QTLs) associated with the control of grain shape and size as well as grain weight in rice. We employed a joint strategy multiple GAPIT (Genome Association and Prediction Integrated Tool) models [(Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK)), Fixed and random model Circulating Probability Uniform (FarmCPU), Settlement of MLM Under Progressive Exclusive Relationship (SUPER), and General Linear Model (GLM)]–High Density SNP Chip DNA Markers (60,461) to conduct a Genome-Wide Association Study (GWAS). GWAS was performed using genotype and grain-related phenotypes of 143 recombinant inbred lines (RILs). Data show that parental lines (Ilpum and Tung Tin Wan Hein 1, TTWH1, Oryza sativa L., ssp. japonica and indica, respectively) exhibited divergent phenotypes for all analyzed grain traits), which was reflected in their derived population. GWAS results revealed the association between seven SNP Chip makers and quantitative trait loci (QTLs) for grain length, co-detected by all GAPIT models on (Chr) 1–3, 5, 7, and 11), were qGL1-1BFSG (AX-95918134, Chr1: 3820526 bp) explains 65.2%–72.5% of the phenotypic variance explained (PVE). In addition, qGW1-1BFSG (AX-273945773, Chr1: 5623288 bp) for grain width explains 15.5%–18.9% of PVE. Furthermore, BLINK or FarmCPU identified three QTLs for grain thickness independently, and explain 74.9% (qGT1Blink, AX-279261704, Chr1: 18023142 bp) and 54.9% (qGT2-1Farm, AX-154787777, Chr2: AX-154787777 bp) of the observed PVE. For t length-to-width ratio, the qLWR2BFSG (AX-274833045, Chr2: 10000097 bp) explains nearly 15.2%–32% of PVE for LWR. Likewise, the major QTL for thousand-grain weight (TGW) was detected on Chr6 (qTGW6BFSG, AX-115737727, 28484619 bp) and explains 32.8%–54% of PVE. The qTGW6BFSG QTL coincides with qGW6-1Blink for grain width and explained 32.8%–54% of PVE. Putative Candidate genes pooled from major QTLs for each grain traits have interesting annotated functions that require functional studies to elucidate their function in the control of grain size, shape, or weight in rice. Genome selection analysis proposed makers useful for downstream marker-assisted selection based on genetic merit of RILs.
... CCDs generate apocarotenoids through a carotenoid oxidative cleavage process [9]. CCD1 functions in the cleavage of carotene, zeaxanthin, and apocarotenoids for the synthesis of apocarotenoid flavor and aroma volatiles, such as α-ionone [16][17][18][19]. CCD4 acts to produce β-cyclocitral or β-ionone through the cleavage of β-carotene in different ways, making CCD4 a potential determinant of fruit or flower color [20][21][22][23]. ...
Carotenoids are key metabolites in goji (Lycium), a traditional Chinese medicine plant; however, the carotenoid content varies in fruits of different goji species, and the mechanism of this variation is not clear. Carotenoids participate in signal transduction and photosynthesis, and function as colorants and photoprotectors. Members of the carotenoid cleavage oxygenase (CCO) gene family are involved in the regulation of phytohormones, pigments, and aromatic substances, such as abscisic acid (ABA), β-carotenoid, and α-ionone, by degrading carotenoids. Some CCO genes are also related to an abiotic stress response. Here, a total of 12 LbCCO genes were identified and analyzed from the L. barbarum genome. CCO genes were divided into six subfamilies based on the constructed phylogenetic tree, including LbNCEDs, LbCCD1, LbCCD3, LbCCD4, LbCCD7, and LbCCD8. Among them, CCD3 was reported for the first time. The gene structure and motif analysis revealed the conservation of CCO subfamilies. Pseudogene generation and the importance of each subfamily in CCOs were revealed by collinearity analysis. The spatiotemporal transcriptomes of L. barbarum and L. ruthenicum were compared, suggesting that CCD4-1 may dominate carotenoid degradation in goji fruits. Cis-acting elements prediction and environment responsive gene expression analyses indicated that salt-alkali stress and photothermal conditions might influence the expression of CCOs in goji. The results of this study enhance our understanding of the carotenoid degradation pathway, and the functions and responses of CCOs in goji species.
... The apocarotenoid by-products of this CCD4 cleavage are one C 13 molecule called β-ionone and a C 27 β-apo-10 -carotenal, which then becomes the substrate for another of the CCD family, CCD1, in the hydrophilic cytosol, releasing another molecule of β-ionone and leaving a C 14 dialdehyde (Floss et al. 2008, Walter et al. 2010). The C 27 moiety, passing through the plastidial membrane to the cytosol, is one of a number of substrates to be cleaved by the promiscuous CCD1, which acts as an apocarotenoid scavenger in the cytosol (Ilg et al. 2010). In contrast to the above in planta sequential reactions, the in microbiota reaction, when CCD1 is heterologously expressed, is simplified to a symmetrical cleavage of both ends of β-carotene at the 9/10 and 9 /10 positions, releasing two molecules of β-ionone; this is seen when the CCD1 gene is engineered in laboratory strains of both the prokaryote Escherichia coli and the eukaryote S. cerevisiae (Schwartz et al. 2001, Beekwilder et al. 2014, Meng et al. 2019, Lopez et al. 2015. ...
Wine is composed of multitudinous flavour components and volatile organic compounds that provide this beverage with its attractive properties of taste and aroma. The perceived quality of a wine can be attributed to the absolute and relative concentrations of favourable aroma compounds; hence, increasing the detectable levels of an attractive aroma, such as β-ionone with its violet and berry notes, can improve the organoleptic qualities of given wine styles. We here describe the generation of a new grape-must fermenting strain of Saccharomyces cerevisiae that is capable of releasing β-ionone through the heterologous expression of both the enzyme carotenoid cleavage dioxygenase 1 (CCD1) and its substrate, β-carotene. Haploid laboratory strains of S. cerevisiae were constructed with and without integrated carotenogenic genes and transformed with a plasmid containing the genes of CCD1. These strains were then mated with a sporulated diploid wine industry yeast, VIN13, and four resultant crosses—designated MQ01 to MQ04–which were capable of fermenting the must to dryness were compared for their ability to release β-ionone. Analyses of their fermentation products showed that the MQ01 strain produced a high level of β-ionone and offers a fermenting hybrid yeast with the potential to enhance the organoleptic qualities of wine.
... More in detail, CCD1 and CCD4 expression levels were reduced in the transgenic tubers. CCD1 enzymes have been proposed to act as scavengers of carotenoids that have been depleted by non-enzymatic oxidation (90) and their transcript level in potato tubers has been found to be inversely correlated with carotenoid accumulation (91). Thus, their reduced expression in our transgenic tubers might entail an increase in the carotenoid pool for the biosynthesis of saffron apocarotenoids. ...
Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41–360 to 105–800 μg/g DW, respectively, with crocetin, crocin 1 [(crocetin-(β-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(β-D-glucosyl)-(β-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value.
... SiMADS34, encoding an E-class MADS-box transcription factor, was proved to influence grain yield in foxtail millet by regulating the inflorescence architecture, which was similar to the function of its homologous gene OSMADS34 in rice [10,55]. The SiCCD1 and OsCCD1 genes encoding carotenoid cleavage dioxygenase 1 affect grain color and carotenoid content by regulating lutein degradation in millet and rice, respectively [56,57]. Furthermore, the orthologs of some well-known rice genes such as OsSD1, OsPSY1, and OsAUX1 were also found to exhibit similar expression patterns and functions in foxtail millet [5,[58][59][60]. ...
Panicle traits are important factors affecting yield, and their improvement has long been a critical goal in foxtail millet breeding. In order to understand the genetic basis of panicle formation, a large-scale genome-wide association study (GWAS) was performed in this study for six panicle-related traits based on 706,646 high-polymorphism SNP loci in 407 accessions. As a result, 87 quantitative trait loci (QTL) regions with a physical distance of less than 100 kb were detected to be associated with these traits in three environments. Among them, 27 core regions were stably detected in at least two environments. Based on rice–foxtail millet homologous comparison, expression, and haplotype analysis, 27 high-confidence candidate genes in the QTL regions, such as Si3g11200 (OsDER1), Si1g27910 (OsMADS6), Si7g27560 (GS5), etc., affected panicle-related traits by involving multiple plant growth regulator pathways, a photoperiod response, as well as panicle and grain development. Most of these genes showed multiple effects on different panicle-related traits, such as Si3g11200 affecting all six traits. In summary, this study clarified a strategy based on the integration of GWAS, a homologous comparison, and haplotype analysis to discover the genomic regions and candidate genes for important traits in foxtail millet. The detected QTL regions and candidate genes could be further used for gene clone and marker-assisted selection in foxtail millet breeding.
... In plants CCD1 was localized in the cytoplasm and can use various carotenoids and apocarotenoids as substrates (Auldridge et al., 2006b;Ilg et al., 2010), which can have great influences on the flavor, fragrance and quality. Loss of AtCCD1 results in increases in seed carotenoid contents but plants had no other changed phenotype (Auldridge et al., 2006b). ...
The carotenoids are the most widely distributed secondary metabolites in plants and can be degraded by carotenoid cleavage dioxygenase (CCD) to form apocarotenoids including an important C13 compound beta-ionone. Volatile beta-ionone can confer the violet and woody fragrance to plant essential oils, flowers, fruits, and vegetables, which therefore has been used in various industries. Dendrobium officinale is a traditional medicinal plant. However, there was limited information on the key enzymes involved in the biosynthesis of beta-ionone in D. officinale . In the present study, beta-ionone was detected in stems and leaves of D. officinale and genome-wide identification and expression profiles of CCD genes were subsequently carried out. There were nine DoCCD members in D. officinale . According to the phylogenetic relationship, DoCCD proteins were classified into six subfamilies including CCD1, CCD4, CCD7, CCD8, nine-cis-epoxycarotenoid dioxygenase (NCED) and zaxinone synthase (ZAS). DoCCD genes showed distinctive expression profiles and DoCCD1 gene was abundantly expressed in eight tissues. Induced expression of DoCCD1 gene resulted in discoloration of Escerichia coli strains that can accumulate carotenoids. Analysis of Gas Chromatography/Mass Spectrometer showed that DoCCD1 enzyme can cleave lycopene to produce 6-methyl-5-hepten-2-one and pseudoionone and also catalyze beta-carotene to form beta-ionone. Expression of DoCCD1 gene in Nicotiana benthamiana leaf resulted in production of abundant beta-ionone. Overall, the present study first provides valuable information on the CCD gene family in D. officinale , function of DoCCD1 gene as well as production of beta-ionone through genetic modification.
... Previous in vitro enzyme assays showed that wheat CCD1, not CCD4, could use b-carotene as substrate (violaxanthin was not tested) with low efficiency and CCD-A1, but not CCD-B1 and CCD4 homoeologs, was expressed in endosperms (Qin et al., 2016), further pinpointing CCD-A1 as the potential activity for cleaving b-carotene and b-carotene-derived xanthophylls into apocarotenoids. However, overexpression of OsCCD1 in Golden Rice endosperm did not affect carotenoid content in one study (Ilg et al., 2010), and a similar experiment in a different Golden Rice background led to up to 1.4-fold higher grain carotenoid accumulation with very little change in bcarotene content in another study (Ko et al., 2018). It remains to be determined whether eliminating CCD1 activity could further increase b-carotene in endosperms of mutant combinations with blocked LCYe and HYD2 activities. ...
Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non-provitamin A carotenoid lutein—with low natural variation in provitamin A β-carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene ε-cyclase (LCYe) and β-carotene hydroxylase (HYD) function in diverting carbons from β-carotene to lutein biosynthesis and catalyzing the turnover of β-carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase β-carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss-of-function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye-A, lcye-B, hyd-A2, and hyd-B2. Hyd-A2 hyd-B2, lcye-A hyd-A2 hyd-B2, lcye-B hyd-A2 hyd-B2, and lcye-A lcye-B hyd-A2 hyd-B2 achieved significantly increased β-carotene in endosperm, with lcye-A hyd-A2 hyd-B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent β-carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that β-carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe-B expression in endosperm is adequate for lutein production in the absence of LCYe-A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing-based approach in hexaploid wheat.
... Arabidopsis CCDs are divided into nine-cis-epoxycarotenoid cleavage dioxygenases (NCED2, NCED3, NCED5, NCED6, and NCED9) that form the ABA precursor xanthoxin from the cleavage of 9-cisepoxycarotenoids, and CCDs with different substrate and regiospecificities (10,11). The latter group includes CCD1, which forms a plentitude of C 13 , C 10 , and C 8 volatiles from different apocarotenoids and C 40 -carotenoids (12); CCD4, which cleaves all-trans--carotene into -ionone (C 13 ) and -apo-10′-carotenal (C 27 ) (13); the SL biosynthesis enzyme CCD7 (MAX3), which breaks 9-cis--carotene into -ionone (C 13 ); and CCD8 (MAX4), which converts 9-cis--apo-10′-carotenal into the SL biosynthesis intermediate carlactone (8). CCD8 can also cleave all-trans--apo-10′-carotenal into the ketone -apo-13-carotenone (d'orenone) but with low activity (14). ...
Anchor roots (ANRs) arise at the root-shoot junction and are the least investigated type of Arabidopsis root. Here, we show that ANRs originate from pericycle cells in an auxin-dependent manner and a carotenogenic signal to emerge. By screening known and assumed carotenoid derivatives, we identified anchorene, a presumed carotenoid-derived dialdehyde (diapocarotenoid), as the specific signal needed for ANR formation. We demonstrate that anchorene is an Arabidopsis metabolite and that its exogenous application rescues the ANR phenotype in carotenoid-deficient plants and promotes the growth of normal seedlings. Nitrogen deficiency resulted in enhanced anchorene content and an increased number of ANRs, suggesting a role of this nutrient in determining anchorene content and ANR formation. Transcriptome analysis and treatment of auxin reporter lines indicate that anchorene triggers ANR formation by modulating auxin homeostasis. Together, our work reveals a growth regulator with potential application to agriculture and a new carotenoid-derived signaling molecule.
... In Arabidopsis, there are five different types of CCDs. The nine-cis-epoxycarotenoid cleavage dioxygenases (NCED represented by 5 enzymes; NCED2, 3, 5, 6, and 9) are involved in the biosynthesis of abscisic acid, while the four other CCD types, designated as CCD1, CCD4, CCD7, and CCD8 exert different biological functions and have, accordingly, different substrates and regiospecificities [23,25,44,47]. For example, CCD1 enzymes catalyze the conversion of carotenoids into a plenitude of volatiles, such as geranial, pseudoionone, and β-ionone, and are likely involved in scavenging of destructed carotenoids/apocarotenoids; the Arabidopsis CCD4 converts all-trans-β-carotene into β-ionone and all-trans-β-apo-10 0 -carotenal and determines carotenoid content; The strigolactone biosynthesis enzyme CCD7 (MAX3) cleaves 9-cis-β-carotene to form β-ionone and 9-cis-β-apo-10 0 -carotenal, the substrate of CCD8 (MAX4) that forms the central strigolactone biosynthesis intermediate carlactone. ...
We developed a chemical derivatization based ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap MS) analytical method to identify low-abundant and instable carotenoid-derived dialdehydes (DIALs, diapocarotenoids) from plants. Application of this method enhances the MS response signal of DIALs, enabling the detection of diapocarotenoids, which is crucial for understanding the function of these compounds and for elucidating the carotenoid oxidative metabolic pathway in plants.
... They often display a high carotene cleavage can generate β-ionone and β-cyclocitral from 9,10 (9′,10′) and 7,8 (7′,8′) bonds respectively [15]. In addition, CCD1 enzymes target different sites in monocyclic and acyclic carotenoids as well as in apocarotenoids of different chain-length and are reported to produce a plenitude of mono-apocarotenoids and dialdehyde products with different chain lengths, including cis-pseudoionone, neral, geranial, rosafluene and farnesylacetone [16][17][18]. CCD4 enzymes from several plant species have also been characterized, and showed 9,10 (9',10') [19,20], 7,8 (7′,8′) [21,22] and 5,6 (5',6') [23] double bond cleavage activity. ...
In plants the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including β-ionone, 6-methyl-5-hepten-2-ol, and α-ionone; these compounds are important in herbivore-plant communication. Combined chemical, biochemical, and molecular studies were conducted to evaluate the differential accumulation of carotenoids and volatile apocarotenoids during the development of pollinated and parthenocarpic fig fruits. Pollinated fig fruits showed less emission of apocarotenoid volatiles than the parthenocarpic figs, while in the case of carotenoid pigments, pollinated figs manifested higher accumulation. The apocarotenoids, 6-methyl-5-hepten-2-ol and β-cyclogeraniol, showed a marked increase after the two weeks of hand-pollination in pollinated and parthenocarpic figs; but afterwards these volatile levels decreased during further fruit development. In addition, we report a transcriptome-based identification and functional characterization of the carotenoid cleavage dioxygenase (FcCCD) genes. These genes were overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant FcCCD1A enzyme showed specificity for the 9,10 (9',10') double bond position of cyclic carotenoids to generate α-ionone and β-ionone, while FcCCD1B cleaved lycopene and an acyclic moiety of δ-carotene, producing 6-methyl-5-hepten-2-one. The qRT-PCR analysis of FcCCD genes revealed differential gene expression during fig fruit development. Our results suggest a role for the FcCCD1genes in apocarotenoid biosynthesis in fig fruits.
... When incubated in vitro with various carotenoids and apocarotenoids, OsCCD1-overexpressing rice (Oryza sativa) endosperms metabolized apocarotenoids more effectively than carotenoids 111 . Ilg et al. proposed that the apocarotenoid cleavage activity observed with OsCCD1 is due to a function of CCD1s as scavengers of non-enzymatic cleavage products of carotenoids resulting from high light stress 111 . Similarly, the role of VviCCD1 may be to catalyze the cleavage of apocarotenoids that have been exported into the cytoplasm after partial metabolism by other CCDs such as CCD4s, whose expression is upregulated during berry development 16 . ...
The grape is one of the oldest and most important horticultural crops. Grape and wine aroma has long been of cultural and scientific interest. The diverse compound classes comprising aroma result from multiple biosynthetic pathways. Only fairly recently have researchers begun to elucidate the genetic mechanisms behind the biosynthesis and metabolism of grape volatile compounds. This review summarizes current findings regarding the genetic bases of grape and wine aroma with an aim towards highlighting areas in need of further study. From the literature, we compiled a list of functionally characterized genes involved in berry aroma biosynthesis and present them with their corresponding annotation in the grape reference genome.
... CCD1 enzymes cleave different cyclic and acyclic all-trans-carotenoids as well as apocarotenoids at several positions, leading to a wide range of products. It has been assumed that the primary function of CCD1 is the scavenging of damaged carotenoids, allowing their replacement by intact ones (Ilg et al. 2010;Scherzinger and Al-Babili 2008). However, CCD1 has also been implicated in the synthesis of special pigments formed upon colonization of plant roots by symbiotic mycorrhizal fungi (Floss et al. 2008;Walter 2013). ...
Strigolactones (SLs) are a group of carotenoid derivatives that act as a hormone regulating plant development and response to environmental stimuli. SLs are also released into soil as a signal indicating the presence of a host for symbiotic arbuscular mycorrhizal fungi and root parasitic weeds. In this chapter, we provide an overview on the enormous progress that has been recently made in elucidating SL biosynthesis and signal transduction. We describe the tailoring pathway from the carotenoid precursor to the central intermediate carlactone, highlighting the stereospecificity of the involved enzymes, the all-trans/9-cis-β-carotene isomerase (D27), the 9-cis-specific CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), as well as CCD8 and its unusual catalytic activity. We then outline the oxidation of carlactone by cytochrome P450 enzymes, such as the Arabidopsis MORE AXILLARY GROWTH 1 (MAX1), into different SLs and the role of other enzymes in generating this diversity, and discuss why plants produce many different SLs. This is followed by depicting hormonal and nutritional factors that regulate SL biosynthesis and release, and by a description of transport mechanisms. In the second part of our chapter, we focus on SL perception and signal transduction, describing the SL receptor DECREASED APICAL DOMINANCE 2 (DAD2)/DWARF14 (D14) and its unique features, the central function of protein degradation mediated by the F-box protein MAX2 and its homologs. We also discuss the latest advances in understanding how SLs regulate the transcription of target genes and the role of SMXL/D53 transcription inhibitors.
... CCD1 enzymes cleave different cyclic and acyclic all-trans-carotenoids as well as apocarotenoids at several positions, leading to a wide range of products. It has been assumed that the primary function of CCD1 is the scavenging of damaged carotenoids, allowing their replacement by intact ones (Ilg et al. 2010;Scherzinger and Al-Babili 2008). However, CCD1 has also been implicated in the synthesis of special pigments formed upon colonization of plant roots by symbiotic mycorrhizal fungi (Floss et al. 2008;Walter 2013). ...
In the last decade strigolactones have been recognized as a novel type of plant hormones. They are involved in the control of key developmental processes such as lateral shoot outgrowth and leaf and root development, among others. In addition, strigolactones modulate plant responses to abiotic stresses like phosphate starvation and drought. Here we summarize the current knowledge of the widely conserved functions of strigolactones in the control of plant development and stress responses as well as some of their reported species-specific roles. In addition, we will review their known genetic and functional interactions with other phytohormones. The newly discovered activities of strigolactones as plant hormones raise the possibility of using these compounds and their signalling pathways as tools to optimise species of agronomical importance.
... CCD1 cleaves carotenoids at 9,10 (9′,10′) double bonds and contributes to the emission of β-and α-ionones, important fragrance components, in flowers of petunia and Osmanthus fragrans (Baldermann et al. 2010, Simkin et al. 2004b. However, lack of correlation between CCD1 expression and carotenoid content has been demonstrated in petals of Japanese morning glory (Yamamizo et al. 2010), rice endosperm (Ilg et al. 2010), tomato fruit (Simkin et al. 2004a), and citrus fruit (Kato et al. 2006), possibly because CCD1 is located in the cytoplasm and has limited access to its substrates in chromoplasts (Bouvier et al. 2003, McCarty andKlee 2006). We previously reported that CCD1 is constitutively expressed in corollas of both white-flowered and pale-yellow-flowered cultivars (Kishimoto et al. 2018). ...
Petunia (Petunia hybrida) is an important ornamental plant with a wide range of corolla colors. Although pale-yellow-flowered cultivars, with a low amount of carotenoids in their corollas, are now available, no deep-yellow-flowered cultivars exist. To find why petunia cannot accumulate enough carotenoids to have deep-yellow flowers, we compared carotenoid profiles and expression of carotenoid metabolic genes between pale-yellow-flowered petunia and deep-yellow-flowered calibrachoa (Calibrachoa hybrida), a close relative. The carotenoid contents and the ratios of esterified xanthophylls to total xanthophylls in petunia corollas were significantly lower than those in calibrachoa, despite similar carotenoid components. A lower esterification rate of trans-xanthophylls than of cis-xanthophylls in petunia suggests that petunia xanthophyll esterase (XES) has low substrate specificity for trans-xanthophylls, which are more abundant than cis-xanthophylls in petunia corolla. The expression of genes encoding key enzymes of carotenoid biosynthesis was lower and that of a carotenoid catabolic gene was higher in petunia. XES expression was significantly lower in petunia. The results suggest that low biosynthetic activity, high cleavage activity, and low esterification activity cause low carotenoid accumulation in petunia corollas.
... The further enzymes represent the four other plant CCD subfamilies, designated as CCD1, CCD4, CCD7, and CCD8 19 . CCD1 enzymes are likely scavengers of destructed carotenoids 20 , generating a plentitude of different products from a wide range of carotenoids and apocarotenoids [21][22][23] . CCD4 enzymes cleave all-trans-cyclic carotenoids 24 , determining carotenoid content in different tissues 25 , and forming apocarotenoid pigments in some fruits 12 . ...
Carotenoid cleavage dioxygenases (CCDs) form hormones and signaling molecules. Here we show that a member of an overlooked plant CCD subfamily from rice, that we name Zaxinone Synthase (ZAS), can produce zaxinone, a novel apocarotenoid metabolite in vitro. Loss-of-function mutants (zas) contain less zaxinone, exhibit retarded growth and showed elevated levels of strigolactones (SLs), a hormone that determines plant architecture, mediates mycorrhization and facilitates infestation by root parasitic weeds, such as Striga spp. Application of zaxinone can rescue zas phenotypes, decrease SL content and release and promote root growth in wild-type seedlings. In conclusion, we show that zaxinone is a key regulator of rice development and biotic interactions and has potential for increasing crop growth and combating Striga, a severe threat to global food security.
... Arabidopsis CCDs are divided into nine-cis-epoxycarotenoid cleavage dioxygenases (NCED2, 3, 5, 6, and 9) that form the ABA precursor xanthoxin from 9-cis-epoxycarotenoids and CCDs with different substrate and regiospecificity (9,10). The latter group includes CCD1, which forms a plentitude of C13, C10 and C8 volatiles from different apocarotenoids and C40-carotenoids (11), CCD4, which cleaves all-trans--carotene into -ionone (C13) and -apo-10'-carotenal (C27) (12), the SL biosynthesis enzyme CCD7 (MAX3), which breaks 9-cis--carotene into -ionone (C13) and 9-cis--apo-10'-carotenal that is converted by CCD8 (MAX4) into the SL biosynthesis intermediate carlactone (8). CCD8 ...
Arabidopsis root development is predicted to be regulated by yet unidentified carotenoid-derived metabolite(s). In this work, we screened known and putative carotenoid cleavage products and identified anchorene, a predicted carotenoid-derived dialdehyde (diapocarotenoid) that triggers anchor root development. Anchor roots are the least characterized type of root in Arabidopsis. They form at the root-shoot junction, particularly upon damage to the root apical meristem. Using Arabidopsis reporter lines, mutants and chemical inhibitors, we show that anchor roots originate from pericycle cells and that the development of this root type is auxin-dependent and requires carotenoid biosynthesis. Transcriptome analysis and treatment of auxin-reporter lines indicate that anchorene triggers anchor root development by modulating auxin homeostasis. Exogenous application of anchorene restored anchor root development in carotenoid-deficient plants, indicating that this compound is the carotenoid-derived signal required for anchor root development. Chemical modifications of anchorene led to a loss of anchor root promoting activity, suggesting that this compound is highly specific. Furthermore, we demonstrate by LC-MS analysis that anchorene is a natural, endogenous Arabidopsis metabolite. Taken together, our work reveals a new member of the family of carotenoid-derived regulatory metabolites and hormones.
Significance
Unknown carotenoid-derived compounds are predicted to regulate different aspects of plant development. Here, we characterize the development of anchor roots, the least characterized root type in Arabidopsis, and show that this process depends on auxin and requires a carotenoid-derived metabolite. We identified a presumed carotenoid-derivative, anchorene, as the likely, specific signal involved in anchor root formation. We further show that anchorene is a natural metabolite that occurs in Arabidopsis. Based on the analysis of auxin reporter lines and transcriptome data, we provide evidence that anchorene triggers the growth of anchor roots by modulating auxin homeostasis. Taken together, our work identifies a novel carotenoid-derived growth regulator with a specific developmental function.
... Simkin et al. (2004a, b) reported that CCD1 expression paralleled emission of β-ionone, an important fragrance component in petunia flowers and tomato fruits, although the correlation between CCD1 activity and carotenoid accumulation in chromoplasts is unclear. Ilg et al. (2010) reported that the introduction of sense or antisense constructs of rice CCD1 into golden rice did not affect carotenoid accumulation in the endosperm. Here, CCD1 was constitutively expressed in corollas of both pale yellow and white cultivars. ...
Petunia (Petunia hybrida) is an important ornamental plant, with corolla colors ranging widely from pink to red to purple, owing mainly to anthocyanins. Although there are no bright-yellow-flowered cultivars, some pale-yellow-flowered cultivars accumulate a small amount of carotenoids. To find a key regulatory step that controls carotenoid content in petunia corollas, we compared the expression of carotenoid metabolism genes and carotenoid composition in corollas of white-flowered and pale-yellow-flowered cultivars. Pale yellow corollas tended to have higher expression of biosynthesis genes. The most prominent result was the complete lack of carotenoid cleavage dioxygenase 4a (CCD4a) transcripts in pale yellow corollas. We found two insertions, one in the putative promoter region and the other in the coding region, of the genomic CCD4a sequence of a pale-yellow-flowered cultivar relative to that of a white-flowered cultivar. We consider this the main reason for the lack of CCD4a transcripts. The results suggest that pale yellow corollas have higher carotenoid biosynthesis activity and lower catabolism activity than white corollas. We propose that carotenoid content in petunia corollas is determined by the balance of the degradation and biosynthesis of carotenoids.
... CCD7 and CCD8 are related to the production of strigolactones, which act as a branching signal (Gomez-Roldan et al. 2008;Umehara et al. 2008). CCD1 plays roles in the formation of volatile compounds responsible for flavour and aroma and in carotenoid turnover (Schwartz et al. 2001;Auldridge et al. 2006;Ilg et al. 2010). The functions of CCD4 are similar to those of CCD1, but some CCD4s are involved in the degradation of carotenoids and prevent carotenoid accumulation in the petals. ...
Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.
... CCD7 and CCD8 are also key enzymes for the biosynthesis of a newly discovered plant hormone strigolactone, which regulates branching in plants and tiller formation in rice (Umehara et al. 2010;Cardoso et al. 2014). However, the overexpression of rice CCD1 in Golden Rice in both sense and antisense forms does not affect the accumulation of carotenoids in rice endosperm, because rice CCD1 specifically degrades apocarotenoids rather than carotenoids (Ilg et al. 2010). Thus, it is interesting to know if the knockout of other carotene catabolic genes can be a possible strategy for the improvement of carotenoid content in rice. ...
Key message:
Targeted mutations in five carotenoid catabolism genes failed to boost carotenoid accumulation in rice seeds, but produced dwarf and high tillering mutants when OsCCD7 gene was knocked out. Carotenoids play an important role in human diet as a source of vitamin A. Rice is a major staple food in Asia, but does not accumulate carotenoids in the endosperm because of the low carotenoid biosynthesis or the degradation in metabolism. In this study, the CRISPR/Cas9 system was investigated in the targeted knockout of five rice carotenoid catabolic genes (OsCYP97A4, OsDSM2, OsCCD4a, OsCCD4b and OsCCD7) and in an effort to increase β-carotene accumulation in rice endosperm. Transgenic plants that expressed OsNLSCas9 and sgRNAs were generated by Agrobacterium-mediated transformation. Various knockout mutations were identified at the T0 generation of the transgenic rice by TILLING and direct sequencing of the PCR products amplified from the target sites. Carotenoids were not accumulated in both mono-allelic and bi-allelic knockout mutations of the five genes. However, transgenic plants with homozygous or bi-allelic mutations to the OsCCD7 gene were extremely dwarfish with more tillers and lower seed setting than other transgenic or nontransgenic plants. This phenotype was similar to the previously reported ccd7 mutants, which are defective in the biosynthesis of strigolactone, a plant hormone that regulates branching in plants and tiller formation in rice.
... CCDs are the key enzymes that catalyze the oxidative cleavage of carotenoids with strict regional and substrate specificity. In Arabidopsis, it has been reported that AtCCD1 preferred to cleave the apocarotenoids rather than bicyclic carotenoids, while AtCCD4 was specific to all-trans-configured bicyclic and monocyclic carotenoids (Bruno et al., 2016;Ilg, Yu, Schaub, Beyer, & Al-Babili, 2010;Schmidt, Kurtzer, Eisenreich, & Schwab, 2006). To date, however, the information on the substrate specificity of CCDs towards free and esterified xanthophylls was still limited. ...
In this study, to investigate the xanthophyll accumulation in citrus fruits, the major fatty acid esters of β-cryptoxanthin and β-citraurin were identified, and changes in their contents were investigated in two Satsuma mandarin varieties, 'Miyagawa-wase' and 'Yamashitabeni-wase', during the ripening process. The results showed that β-cryptoxanthin and β-citraurin were mainly esterified with lauric acid, myristic acid, and palmitic acid in citrus fruits. During the ripening process, β-cryptoxanthin laurate, myristate, and palmitate were accumulated gradually in the flavedos and juice sacs of the two varieties. In the flavedo of 'Yamashitabeni-wase', β-citraurin laurate, myristate, and palmitate were specifically accumulated, and their contents increased rapidly with a peak in November. In addition, functional analyses showed that CitCCD1 and CitCCD4 efficiently cleaved the free β-cryptoxanthin, but not the β-cryptoxanthin esters in vitro. The substrate specificity of CitCCDs towards free β-cryptoxanthin indicated that β-cryptoxanthin esters might be more stable than free β-cryptoxanthin in citrus fruits.
... Some of the apocarotenoids derived from carotenoid cleavage are important determinants of flavor in agricultural products. In addition, some products of carotenoid-derived zeaxanthin aldehyde, which can be transformed into the plant hormone abscisic acid, can regulate stress, seed development, and other important functions (Winterhal and Schreier, 1995;Huang et al., 2009;Ilg et al., 2010;Liang et al., 2011;Heo et al., 2013;Sui et al., 2013). These cleavage products are mainly catalyzed by the carotenoid cleavage oxygenases (CCOs) (Bouvier et al., 2005;Heo et al., 2013). ...
Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five subgroups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.
... Therefore, an asymmetric cleavage is observed in such cases. The C5-C6 or C5 -C6 activity of CCD1 enzymes on lycopene (or both), leading to the formation of the C 8 ketone 6-methyl-5-hepten-2-one (MHO), was reported for the first time in tomato, maize, and A. thaliana [92] and was later detected in Cucumis melo [84], Rosa damascena [96], Oryza sativa [97], and Vitis vinifera [98]. The cleavage of C7-C8 and C7 -C8 double bonds of linear and monocyclic carotenoids constitutes a novel recognition site for the CCD1 plant subfamily. ...
Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs), a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon-carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.
... More detailed results of the carotenoid composition are given in Supplementary Table S1 other words, CCPs), including hormones, flavors, and/or defense compounds and pigments. The other focus involves altering the carotenoid content to enhance the nutritional value of food crops and to change flower colors in horticultural crops (Bouvier et al. 2005;Auldridge et al. 2006a;Ilg et al. 2010;Walter et al. 2010;Gonzalez-Jorge et al. 2013). In the former group, the types of apocarotenoid products are highly dependent on the enzymatic action of the CCD family and the specific position of the double bonds cleaved on the carotenoids. ...
A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C40) cleavage products, a two-fold increase of β-ionone (C13) and de novo generation of β-cyclocitral (C10) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).
... β-carotene and zeaxanthin) carotenoids symmetrically at C9-C10/C9′-C10′ or C7-C8/ C7′-C8′ positions [15,16,30]. However, overexpression of OsCCD1 in Golden Rice endosperm (engineered for high β-carotene accumulation) did not change its βcarotene content significantly, arguing against a role of OsCCD1 in carotenoid cleavage in rice endosperm [34]. It remains to be determined whether CCD1 enzymes can transform carotenoids into apocarotenoid products in other cereal grains, such as wheat. ...
Background
β-carotene, the most active provitamin A molecule produced by plants, plays important roles in human nutrition and health. β-carotene does not usually accumulate in the endosperm (i.e. flour) of mature wheat grains, which is a major food source of calories for humans. Therefore, enriching β-carotene accumulation in wheat grain endosperm will enable a sustainable dietary supplementation of provitamin A. Several metabolic genes affecting β-carotene accumulation have already been isolated from wheat, including phytoene synthase 1 (PSY1), lycopene ε-cyclase (LCYe) and carotenoid β-ring hydroxylase1/2 (HYD1/2).
Results
In this work, we cloned and biochemically characterized two carotenoid cleavage dioxygenases (CCDs), CCD1 and CCD4, from wheat. While CCD1 homoeologs cleaved β-apo-8′-carotenal, β-carotene, lutein and zeaxanthin into apocarotenoid products, CCD4 homoeologs were inactive towards these substrates in in vitro assays. When analyzed by real-time qPCR, PSY1, LCYe, HYD1/2 and CCD1/4 homoeologs showed distinct expression patterns in vegetative tissues and sections of developing tetraploid and hexaploid wheat grains, suggesting that carotenoid metabolic genes and homoeologs are differentially regulated at the transcriptional level in wheat.
Conclusions
The CCD1/4 enzyme activity and the spatial-temporal gene expression data provide critical insights into the specific carotenoid metabolic gene homoeologs that control β-carotene accumulation in wheat grain endosperm, thus establishing the knowledge base for generation of wheat varieties with enhanced β-carotene in the endosperm through breeding and genome editing approaches.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-016-0848-7) contains supplementary material, which is available to authorized users.
... In fact, a similar carotenoid content was observed in both GR2 and antisense lines. Surprisingly, in vitro analyses suggested that apocarotenoids were the primary substrates of OsCCD1 (Ilg et al., 2010). ...
Carotenoid pigments provide fruits and flowers with distinctive red, orange and yellow colours as well as a number of aromas, which make them commercially important in agriculture, food, health and the cosmetic industries. Carotenoids comprise a large family of C 40 polyenes that are critical for the survival of plants and animals alike. β-carotene and its derivatives contain unmodified β-ionone groups, which serve as precursors for vitamin A and are therefore essential dietary components for mammals. Significant progress has been made towards producing staple food crops with elevated provitamin A carotenoids, an important first step in alleviating worldwide vitamin A deficiency. Recent insights into the regulatory processes that control carotenoid composition and content may further advance biofortification projects.
... CCD7 and CCD8 are involved in strigolactone biosynthesis and are localized in the plastid (Gomez-Roldan et al., 2008;Umehara et al., 2008;Rubio-Moraga et al., 2014a). The constitutively expressed CCD1 enzymes are characterized by broad substrate and cleavage-site specificity (Walter & Strack, 2011), cleaving cyclic and acyclic all-trans-carotenoids as well as apocarotenoids, such as b-apo-8 0 -carotenal, b-apo-10 0 -carotenal and apolycopenals (Vogel et al., 2008;Huang et al., 2009a;Ilg et al., 2009) suggesting a function in scavenging degraded carotenoids (Ilg et al., 2010). CCD4 enzymes from different plants produce b-ionone by cleaving b-carotene or b-apo-8 0carotenal at the C9-C10 double bond (Rubio et al., 2008;Huang et al., 2009b), and also at the 7 0 ,8 0 double bond in b-carotene, b-cryptoxanthin and zeaxanthin, leading to the pigments b-apo-8 0 -carotenal and 3-OH-b-apo-8 0 -carotenal (b-citraurin) (Ma et al., 2013;Rodrigo et al., 2013). ...
The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD 2 ( AIG 94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom.
Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCD s, namely Ca CCD 1, Ca CCD 2 and Ca CCD 4a/b and Ca CCD 4c were first cloned and characterized.
Ca CCD 2 was localized in plastids, and a longer CCD 2 version, Cs CCD 2L, was also localized in this compartment. The activity of Ca CCD 2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCD s was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. Ca CCD 2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the Ca CCD 1 activity in Crocus ancyrensis flowers.
The specific CCD 2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast.
Main conclusion
A new carotenoid cleavage dioxygenase NtCCD10 from tobacco was characterized. There is some difference between NtCCD10 and CCD1 in structure. NtCCD10 can cleave the C5–C6 (C5'–C6') and C9–C10 (C9'–C10') double bonds of carotenoids and has high catalytic activity.
Abstract
Carotenoid cleavage dioxygenases (CCDs) cleave carotenoids to produce a variety of apocarotenoids, which have important biological functions for organisms in nature. There are eleven CCDs subfamilies in the plant kingdom, many of which have been extensively characterized in their functions. However, as a newly classified subfamily, the function of CCD10 has rarely been studied. In this work, the function of an NtCCD10 gene from dicotyledonous Nicotiana tabacum was cloned and characterized, and its phylogeny, molecular structural modeling and protein structure were also systematically analyzed. Like other CCDs, NtCCD10 also possesses a seven bladed β-propeller with Fe²⁺ cofactor in its center constituting the active site of the enzyme. The Fe²⁺ is also coordinated bonding with four conserved histidine residues. Meanwhile, NtCCD10 also has many unique features, such as its α1 and α3 helixes are not anti-parallel, a special β-sheet and a longer access tunnel for substrates. When expressed in engineered Escherichia coli (producing phytoene, lycopene, β-carotene, and zeaxanthin) and Saccharomyces cerevisiae (producing β-carotene), NtCCD10 could symmetrically cleave phytoene and β-carotene at the C9–C10 and C9'–C10' positions to produce geranylacetone and β-ionone, respectively. In addition, NtCCD10 could also cleave the C5-C6 and C5'–C6' double bonds of lycopene to generate 6-methyl-5-heptene-2-one (MHO). NtCCD10 has higher catalytic activity than PhCCD1 in yeast, which provides a good candidate CCD for biosynthesis of β-ionone and has potential applications in biotechnological industry. This study identified the taxonomic position and catalytic activity of the first NtCCD10 in dicotyledonous plants. This will provide a reference for the discovery and functional identification of CCD10 enzymes in dicotyledons.
Foxtail millet is a minor but economically important crop in certain regions of the world. Millet color is often used to judge grain quality, yet the molecular determinants of millet coloration remain unclear. Here, we explored the relationship between SiCCD1 and millet coloration in yellow and white millet varieties. Carotenoid levels declined with grain maturation and were negatively correlated with SiCCD1 expression, which was significantly higher in white millet as compared to yellow millet during the color development stage. Cloning of the SiCCD1 promoter and CDS sequences from these different millet varieties revealed the presence of two additional cis-regulatory elements within the SiCCD1 promoter in white millet varieties, including an enhancer-like GC motif element associated with anoxic specific inducibility and a GCN4-motif element associated with endosperm expression. Dual-luciferase reporter assays confirmed that SiCCD1 promoter fragments containing these additional cis-acting elements derived from white millet varieties were significantly more active than those from yellow millet varieties, consistent with the observed SiCCD1 expression patterns. Further in vitro enzyme detection assays confirmed that SiCCD1 primarily targets and degrades lutein. Together, these data suggest that SiCCD1 promoter variation was a key factor associated with the observed differences in SiCCD1 expression, which in turn led to the difference in millet coloration.
Carotenoid cleavage oxygenases (CCOs) play crucial roles in plant growth and development, as well as in the response to phytohormonal, biotic and abiotic stresses. However, comprehensive and systematic research on the CCO gene family has not yet been conducted in Saccharum. In this study, 47 SsCCO and 14 ShCCO genes were identified and characterized in Saccharum spontaneum and Saccharum spp. R570 cultivar, respectively. The SsCCOs consisted of 38 SsCCDs and 9 SsNCEDs, while ShCCOs contained 11 ShCCDs and 3 ShNCEDs. The SsCCO family could be divided into 7 groups, while ShCCO family into 5 groups. The genes/proteins contained similar compositions within the same group, and the evolutionary mechanisms differed between S. spontaneum and R570. Gene Ontology annotation implied that CCOs were involved in many physiological and biochemical processes. Additionally, 41 SsCCOs were regulated by 19 miRNA families, and 8 ShCCOs by 9 miRNA families. Cis-regulatory elements analysis suggested that CCO genes functioned in the process of growth and development or under the phytohormonal, biotic and abiotic stresses. qRT-PCR analysis indicated that nine CCO genes from different groups exhibited similar expression patterns under abscisic acid treatment, while more divergent profiles were observed in response to Sporisorium scitamineum and cold stresses. Herein, comparative genomics analysis of the CCO gene family between S. spontaneum and R570 was conducted to investigate its evolution and functions. This is the first report on the CCO gene family in S. spontaneum and R570, thus providing valuable information and facilitating further investigation into its function in the future.
In mammals, carotenoids are converted by two carotenoid cleavage oxygenases into apocarotenoids, including vitamin A. Although knowledge about β-carotene oxygenase-1 (BCO1) and vitamin A metabolism has tremendously increased, the function of β-carotene oxygenase-2 (BCO2) remains less well-defined. We here studied the role of BCO2 in the metabolism of long chain β-apocarotenoids, which recently emerged as putative regulatory molecules in mammalian biology. We showed that recombinant murine BCO2 converted the alcohol, aldehyde, and carboxylic acid of a β-apocarotenoid substrate by oxidative cleavage at position C9,C10 into a β-ionone and a diapocarotenoid product. Chain length variation (C20 to C40) and ionone ring site modifications of the apocarotenoid substrate did not impede catalytic activity or alter the regioselectivity of the double bond cleavage by BCO2. Isotope labeling experiments revealed that the double bond cleavage of an apocarotenoid followed a dioxygenase reaction mechanism. Structural modeling and site directed mutagenesis identified amino acid residues in the substrate tunnel of BCO2 that are critical for apocarotenoid binding and catalytic processing. Mice deficient for BCO2 accumulated apocarotenoids in their livers, indicating that the enzyme engages in apocarotenoid metabolism. Together, our study provides novel structural and functional insights into BCO2 catalysis and establishes the enzyme as a key component of apocarotenoid homeostasis in mice.
Carotenoid cleavage dioxygenase (CCD), a key enzyme in carotenoid metabolism, cleaves carotenoids to form apo-carotenoids, which play a major role in plant growth and stress responses. CCD genes had not previously been systematically characterized in Brassica napus (rapeseed), an important oil crop worldwide. In this study, we identified 30 BnCCD genes and classified them into nine subgroups based on a phylogenetic analysis. We identified the chromosomal locations, gene structures, and cis-promoter elements of each of these genes and performed a selection pressure analysis to identify residues under selection. Furthermore, we determined the subcellular localization, physicochemical properties, and conserved protein motifs of the encoded proteins. All the CCD proteins contained a retinal pigment epithelial membrane protein (RPE65) domain. qRT-PCR analysis of expression of 20 representative BnCCD genes in 16 tissues of the B. napus cultivar Zhong Shuang 11 ('ZS11') revealed that members of the BnCCD gene family possess a broad range of expression patterns. This work lays the foundation for functional studies of the BnCCD gene family.
Due to the ongoing prevalence of vitamin A deficiency (VAD) in developing countries there has been a large effort towards increasing the carotenoid content of staple foods via biofortification. Common strategies used for carotenoid biofortification include altering flux through the biosynthesis pathway to direct synthesis to a specific product, generally β-carotene, or via increasing the expression of genes early in the carotenoid biosynthesis pathway. Recently, carotenoid biofortification strategies are turning towards increasing the retention of carotenoids in plant tissues either via altering sequestration within the cell or via downregulating enzymes known to cause degradation of carotenoids. To date, little attention has focused on increasing the stability of carotenoids, which may be a promising method of increasing carotenoid content in staple foods.
Carotenoids are isoprenoid compounds synthesized de novo in all photosynthetic organisms as well as in some nonphotosynthetic bacteria and fungi. In plants, carotenoids are essential for light harvesting and photoprotection. They contribute to the vivid color found in many plant organs. The cleavage of carotenoids produces small molecules (apocarotenoids) that serve as aroma compounds, as well as phytohormones and signals to affect plant growth and development. Since carotenoids provide valuable nutrition and health benefits for humans, understanding of carotenoid biosynthesis, catabolism and storage is important for biofortification of crops with improved nutritional quality. This chapter primarily introduces our current knowledge about carotenoid biosynthesis and degradation pathways as well as carotenoid storage in plants.
Carotenoids of staple food crops have high nutritional value as provitamin A components in the daily diet. To increase the levels of carotenoids, inhibition of carotenoid cleavage dioxygenases (CCDs), which degrade carotenoids, has been considered as a promising target in crop biotechnology. Suppression of the OsCCD1, OsCCD4a, and OsCCD4b genes using RNA interference (Ri) was verified in transgenic rice plants by quantitative RT-PCR and small RNA detection. Leaf carotenoids were statistically increased overall in OsCCD4a_Ri lines of the T1 generation, and the highest accumulation of 1.3-fold was found in the OsCCD4a_Ri 7 line of the T2 generation relative to non-transgenic plants. The effects on seed carotenoids were elucidated via cross-fertilization between β-carotene-producing transgenic rice and one of two independent homozygous lines of OsCCD1_Ri, OsCCD4a_Ri or OsCCD4b_Ri. As a result, seed carotenoids were increased to a maximum of 1.4- and 1.6-fold in OsCCD1_Ri and OsCCD4a_Ri, respectively, with a different preference toward α-ring carotenoids and -ring carotenoids; levels were not established in OsCCD4b_Ri. In addition, the levels of four carotenoids decreased when OsCCD1, OsCCD4a, and OsCCD4b were overexpressed in E. coli strains accumulating phytoene, lycopene, -carotene, and zeaxanthin. OsCCD1 and OsCCD4a had a similar high carotenoid degrading activity, followed by OsCCD4b without substrate specificity. Collectively, the blocking of OsCCD4a activity might have potential as a practical tool for enhancing the carotenoid level of the carotenoid-accumulating seed endosperms as well as leaves of rice plants.
Fluridone (FLU) is widely used as a herbicide and interferes with photosynthesis via induction of leaf bleaching. However, the mechanism of FLU-induced leaf bleaching remains elusive. In this study, the effects of FLU on the metabolism of leaf pigments, including chlorophyll and carotenoids, were investigated. Our results demonstrate that FLU induced rice leaf bleaching in a dose-dependent manner. Treatment with 5 µM FLU strongly induced leaf bleaching by decreasing pigment content, with carotenoids and chlorophyll decreased by 98 and 95%, respectively, in the second leaves of rice seedlings. Our results indicate that the transcription levels of leaf pigment biosynthetic and catabolic genes were significantly downregulated by 5 µM FLU treatment. These results suggest that FLU induces leaf bleaching by decreasing leaf pigment content via downregulation of the transcription levels of leaf pigment biosynthetic genes, and that the downregulation of transcription levels of leaf pigment catabolic genes is a result of feedback inhibition mediated by FLU-decreased leaf pigment content. In addition, to test whether FLU-induced leaf bleaching is due to FLU-induced abscisic acid (ABA) deficiency, the effect of FLU treatment on endogenous ABA content and the recovery effect of ABA on FLU-induced leaf bleaching were investigated. Application of FLU significantly decreased endogenous ABA content, but FLU-induced leaf bleaching was not rescued by ABA application, thus FLU-induced leaf bleaching is not due to FLU-induced ABA deficiency. © 2018 Springer Science+Business Media, LLC, part of Springer Nature
Provitamin A biofortification, the provision of provitamin A carotenoids through agriculture, is regarded as an effective and sustainable intervention to defeat vitamin A deficiency representing a global health problem. This food-based intervention has been questioned in conjunction with negative outcomes for smokers and asbestos-exposed populations of the CARET and ATBC trials in which very high doses of β-carotene were supplemented. The current notion that β-carotene cleavage products (apocarotenoids) represented the harmful agents is the basis of the here-presented research. We have quantitatively analyzed numerous plant food items and can conclude that neither the amounts of apocarotenoids nor of β-carotene provided by plant tissues, be they conventional or provitamin A-biofortified, pose an increased risk. We have also investigated β-carotene degradation pathways over time. This reveals a substantial non-enzymatic proportion of carotene decay and corroborates the quantitative relevance of highly oxidized β-carotene polymers that form in all plant tissues investigated.
Vitamin A deficiency affects an estimated 190 million preschool-aged children and 10 million pregnant women in low-income countries. Prevalent cases of xerophthalmia in young children are believed to number ~5 million. Of these, 10% can be considered potentially blinding, such that this ocular condition remains the leading cause of preventable pediatric blindness in the developing world. Although there has been a significant decrease in the prevalence of vitamin A deficiency during the past couple of decades, vitamin A deficiency remains an underlying cause of at least 157,000 early childhood deaths due to diarrhea, measles, malaria, and other infections each year. Vitamin A deficiency is also recognized as a problem that reflects chronic dietary deficiency that may extend from early childhood into adolescence and adulthood, especially for women of child-bearing age. This chapter describes the chemical structure of vitamin A and its precursors, dietary sources, absorption, metabolism and functions, followed by discussions on the epidemiology of vitamin A deficiency, and its role in morbidity and mortality. The chapter concludes with insights on its diagnosis, treatment, and approaches to prevention through dietary improvement, supplementation, fortification, and biofortification.
Carotenoid cleavage dioxygenases (CCDs) form a multienzyme family, the members of which are involved in the production of a diversity of apocarotenoids. The apocarotenoid module vital physiological and developmental processes in plants. This chapter deals with the different aspects of plant CCDs in general and C. sativus in particular such as structure and reaction mechanisms. Further, this chapter also discusses the role of CCDs in plants and their application in plant biotechnology.
Volatile compounds are the major determinants of aroma and flavor in both grapes and wine. In this study, we investigated the emission of volatile and non-volatile compounds during berry maturation in two grape varieties (Airén and Tempranillo) throughout 2010 and 2011. HS-SPME coupled to gas chromatography and mass spectrometry was applied for the identification and relative quantitation of these compounds. Principal component analysis was performed to search for variability between the two cultivars and evolution during 10 developmental stages. Results showed that there are distinct differences in volatile compounds between cultivars throughout fruit development. Early stages were characterized in both cultivars by higher levels of some apocarotenoids such as β-cyclocitral or β-ionone, terpenoids (E)-linalool oxide and (Z)-linalool oxide and several furans, while the final stages were characterized by the highest amounts of ethanol, benzenoid phenylacetaldehyde and 2-phenylethanol, branched-amino acid-derived 3-methylbutanol and 2-methylbutanol, and a large number of lipid derivatives. Additionally, we measured the levels of the different classes of volatile precursors by using liquid chromatography coupled to high resolution mass spectrometry. In both varieties, higher levels of carotenoid compounds were detected in the earlier stages, zeaxanthin and α-carotene were only detected in Airén while neoxanthin was found only in Tempranillo; more variable trends were observed in the case of the other volatile precursors. Furthermore, we monitored the expression of homolog genes of a set of transcripts potentially involved in the biosynthesis of these metabolites, such as some glycosyl hydrolases family 1, lipoxygenases, alcohol dehydrogenases hydroperoxide lyases, O-methyltransferases and carotenoid cleavage dioxygenases during the defined developmental stages. Finally, based on Pearson correlation analyses, we explored the metabolite-metabolite fluctuations within VOCs/precursors during the berry development; as well as tentatively linking the formation of some metabolites detected to the expression of some of these genes. Our data showed that the two varieties displayed a very different pattern of relationships regarding the precursor/volatile metabolite-metabolite fluctuations, being the lipid and the carotenoid metabolism the most distinctive between the two varieties. Correlation analysis showed a higher degree of overall correlation in precursor/volatile metabolite-metabolite levels in Airén, confirming the enriched aroma bouquet characteristic of the white varieties.
In plants, carotenoids are essential for photosynthesis and photoprotection. However, carotenoids are not the end products of the pathway; apocarotenoids are produced by carotenoid cleavage dioxygenases (CCDs) or non-enzymatic processes. Apocarotenoids are more soluble or volatile than carotenoids but they are not simply breakdown products, as there can be modifications post-cleavage and their functions include hormones, volatiles, and signals. Evidence is emerging for a class of apocarotenoids, here referred to as apocarotenoid signals (ACSs), that have regulatory roles throughout plant development beyond those ascribed to abscisic acid (ABA) and strigolactone (SL). In this context we review studies of carotenoid feedback regulation, chloroplast biogenesis, stress signaling, and leaf and root development providing evidence that apocarotenoids may fine-tune plant development and responses to environmental stimuli.
Advances in research of biosynthetic and degradation pathway of carotenoid, and hence its related carotenogenic gene as well as carotenoid dioxygenase gene were reviewed. Metabolic manipulation of carotenoid was summarized. Strategies, problems and achievements of genetic manipulation of carotenoid metabolism were discussed.
Ripe tomato fruits accumulate large amounts of the red linear carotene, lycopene (a dietary antioxidant) and small amounts of its orange cyclisation product, beta-carotene (pro-vitamin A). Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase (β-Lcy). We introduced, via Agrobacterium-mediated transformation, DNA constructs aimed at up-regulating (OE construct) or down-regulating (AS construct) the expression of the β-Lcy gene in a fruit-specific fashion. Three transformants containing the OE construct show a significant increase in fruit beta-carotene content. The fruits from these plants display different colour phenotypes, from orange to orange-red, depending on the lycopene/beta-carotene ratio. Fruits from AS transformants show up to 50% inhibition of β-Lcy expression, accompanied by a slight increase in lycopene content. Leaf carotenoid composition is unaltered in all transformants. In most transformants, an increase in total carotenoid content is observed with respect to the parental line. This increase occurs in the absence of major variations in the expression of endogenous carotenoid genes.
Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds beta-apo-10'-carotenal and its alcohol into beta-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species.
Oxidative tailoring of C(40) carotenoids by double bond-specific cleavage enzymes (carotenoid cleavage dioxygenases, CCDs) gives rise to various apocarotenoids. AtCCD1 generating C(13) and C(14) apocarotenoids and orthologous enzymes in other plants are the only CCDs acting in the cytosol, while the hitherto presumed C(40) substrate is localized in the plastid. A new model for CCD1 action arising from a RNAi-mediated CCD1 gene silencing study in mycorrhizal hairy roots of Medicago truncatula may solve this contradiction. This approach unexpectedly resulted in the accumulation of C(27) apocarotenoids but not C(40) carotenoids suggesting C(27) as the main substrates for CCD1 in planta. It further implies a consecutive two-step cleavage process, in which another CCD performs the primary cleavage of C(40) to C(27) in the plastid followed by C(27) export and further cleavage by CCD1 in the cytosol. We compare the specificities and subcellular locations of the various CCDs and propose the plastidial CCD7 to be the first player in mycorrhizal apocarotenoid biogenesis.
Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant
species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain
insight into their biological function, CCD4 genes were isolated from apple (Malus×domestica, MdCCD4), chrysanthemum (Chrysanthemum×morifolium, CmCCD4a), rose (Rosa×damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved β-carotene well to yield β-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost
inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-ζ-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8′-apo-β-caroten-8′-al by AtCCD4 and RdCCD4 to β-ionone, while this apocarotenal
was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4
enzymes cleave their substrates at the same position (9,10 and 9′,10′), they might have different biochemical functions as
they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized.
Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C(40) carotenoid substrates at 9,10 and 9',10' positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C(13) cyclohexenone and C(14) mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C(27) apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C(27) apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C(27) apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C(27)-->C(14)+C(13)), while the first step (C(40)-->C(27)+C(13)) may be catalyzed by CCD7 and/or CCD4 inside plastids.
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.
Colonisation of maize roots by arbuscular mycorrhizal (AM) fungi leads to the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives). Other root apocarotenoids (strigolactones) are involved in signalling during early steps of the AM symbiosis but also in stimulation of germination of parasitic plant seeds. Both apocarotenoid classes are predicted to originate from cleavage of a carotenoid substrate by a carotenoid cleavage dioxygenase (CCD), but the precursors and cleavage enzymes are unknown. A Zea mays CCD (ZmCCD1) was cloned by RT-PCR and characterised by expression in carotenoid accumulating E. coli strains and analysis of cleavage products using GC-MS. ZmCCD1 efficiently cleaves carotenoids at the 9, 10 position and displays 78% amino acid identity to Arabidopsis thaliana CCD1 having similar properties. ZmCCD1 transcript levels were shown to be elevated upon root colonisation by AM fungi. Mycorrhization led to a decrease in seed germination of the parasitic plant Striga hermonthica as examined in a bioassay. ZmCCD1 is proposed to be involved in cyclohexenone and mycorradicin formation in mycorrhizal maize roots but not in strigolactone formation.
A carotenoid-derived hormonal signal that inhibits shoot branching in plants has long escaped identification. Strigolactones are compounds thought to be derived from carotenoids and are known to trigger the germination of parasitic plant seeds and stimulate symbiotic fungi. Here we present evidence that carotenoid cleavage dioxygenase 8 shoot branching mutants of pea are strigolactone deficient and that strigolactone application restores the wild-type branching phenotype to ccd8 mutants. Moreover, we show that other branching mutants previously characterized as lacking a response to the branching inhibition signal also lack strigolactone response, and are not deficient in strigolactones. These responses are conserved in Arabidopsis. In agreement with the expected properties of the hormonal signal, exogenous strigolactone can be transported in shoots and act at low concentrations. We suggest that endogenous strigolactones or related compounds inhibit shoot branching in plants. Furthermore, ccd8 mutants demonstrate the diverse effects of strigolactones in shoot branching, mycorrhizal symbiosis and parasitic weed interaction.
Shoot branching is a major determinant of plant architecture and is highly regulated by endogenous and environmental cues. Two classes of hormones, auxin and cytokinin, have long been known to have an important involvement in controlling shoot branching. Previous studies using a series of mutants with enhanced shoot branching suggested the existence of a third class of hormone(s) that is derived from carotenoids, but its chemical identity has been unknown. Here we show that levels of strigolactones, a group of terpenoid lactones, are significantly reduced in some of the branching mutants. Furthermore, application of strigolactones inhibits shoot branching in these mutants. Strigolactones were previously found in root exudates acting as communication chemicals with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Thus, we propose that strigolactones act as a new hormone class-or their biosynthetic precursors-in regulating above-ground plant architecture, and also have a function in underground communication with other neighbouring organisms.
Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially
in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin
A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries,
including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional
value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.
Tomato products are the principal dietary sources of lycopene and major source of beta-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme phytoene desaturase, which converts phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the beta-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.
The general scheme of carotenoid biosynthesis has been known for more than three decades. However, molecular description of the pathway in plants began only in the 1990s after the genes for the carotenogenic enzymes were cloned. Recent data on the biochemistry of carotenogenesis and its regulation in vivo present the possibility of genetically manipulating this pathway in crop plants.
The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor. Enzymes that catalyze this carotenoid cleavage reaction, nine-cis epoxy-carotenoid dioxygenases, have been identified in several plant species. Similar proteins, whose functions are not yet known, are present in diverse organisms. A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes. However, the overall homology with known abscisic acid biosynthetic enzymes is low. To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays. The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' positions. In most instances, the enzyme cleaves the substrate symmetrically to produce a C(14) dialdehyde and two C(13) products, which vary depending on the carotenoid substrate. Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom. A similar protein in beans catalyzes the same reaction in vitro. The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.
Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T(0)) were 2-4-fold higher than the controls, whereas phytoene, lycopene, beta-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T(1) and T(2) generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5-10-fold elevations in total phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into phytoene) were not significantly altered by the presence of the bacterial phytoene synthase.
Ripe tomato fruits accumulate significant amounts of the linear carotene lycopene, but only trace amounts of xanthophylls (oxygenated carotenoids). We overexpressed the lycopene beta-cyclase (b-Lcy) and beta-carotene hydroxylase (b-Chy) genes under the control of the fruit-specific Pds promoter. Transgene and protein expression was followed through semi-quantitative reverse transcription-PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of beta-carotene, beta-cryptoxanthin and zeaxanthin. The carotenoid composition of leaves remained unaltered. The transgenes and the phenotype are inherited in a dominant Mendelian fashion. This is the first example of successful metabolic engineering of xanthophyll content in tomato fruits.
Carotenoid cleavage products (apocarotenoids) are widespread in living organisms and exert key biological functions. In animals, retinoids function as vitamins, visual pigments and signalling molecules. In plants, apocarotenoids play roles as hormones, pigments, flavours, aromas and defence compounds. The first step in their biosynthesis is the oxidative cleavage of a carotenoid catalysed by a non-heme iron oxygenase. A novel family of enzymes, which can cleave different carotenoids at different positions, has been characterized.
As an important step toward free access and, thus, impact of GoldenRice, a freedom-to-operate situation has been achieved for developing countries for the technology involved. Specifically, to carry the invention beyond its initial "proof-of-concept" status in a Japonica rice (Oryza sativa) cultivar, we report here on two transformed elite Indica varieties (IR64 and MTL250) plus one Japonica variety Taipei 309. Indica varieties are predominantly consumed in the areas with vitamin A deficiency. To conform with regulatory constraints, we changed the vector backbone, investigated the absence of beyond-border transfer, and relied on Agrobacterium tumefaciens-mediated transformation to obtain defined integration patterns. To avoid an antibiotic selection system, we now rely exclusively on phosphomannose isomerase as the selectable marker. Single integrations were given a preference to minimize potential epigenetic effects in subsequent generations. These novel lines, now in the T(3) generation, are highly valuable because they are expected to more readily receive approval for follow-up studies such as nutritional and risk assessments and for breeding approaches leading to locally adapted variety development.
Carotenoids are integral and essential components of the photosynthetic membranes in all plants. Within the past few years, genes encoding nearly all of the enzymes required for the biosynthesis of these indispensable pigments have been identified. This review focuses on recent findings as to the structure and function of these genes and the enzymes they encode. Three topics of current interest are also discussed: the source of isopentenyl pyrophosphate for carotenoid biosynthesis, the progress and possibilities of metabolic engineering of plants to alter carotenoid content and composition, and the compartmentation and association of the carotenogenic enzymes. A speculative schematic model of carotenogenic enzyme complexes is presented to help frame and provoke insightful questions leading to future experimentation.
Enzymes that are able to oxidatively cleave carotenoids at specific positions have been identified in animals and plants. The first such enzyme to be identified was a nine-cis-epoxy carotenoid dioxygenase from maize, which catalyzes the rate-limiting step of abscisic acid biosynthesis. Similar enzymes are necessary for the synthesis of vitamin A in animals and other carotenoid-derived molecules in plants. In the model plant, Arabidopsis, there are nine hypothetical proteins that share some degree of sequence similarity to the nine-cis-epoxy carotenoid dioxygenases. Five of these proteins appear to be involved in abscisic acid biosynthesis. The remaining four proteins are expected to catalyze other carotenoid cleavage reactions and have been named carotenoid cleavage dioxygenases (CCDs). The hypothetical proteins, AtCCD7 and AtCCD8, are the most disparate members of this protein family in Arabidopsis. The max3 and max4 mutants in Arabidopsis result from lesions in AtCCD7 and AtCCD8. Both mutants display a dramatic increase in lateral branching and are believed to be impaired in the synthesis of an unidentified compound that inhibits axillary meristem development. To determine the biochemical function of AtCCD7, the protein was expressed in carotenoid-accumulating strains of Escherichia coli. The activity of AtCCD7 was also tested in vitro with several of the most common plant carotenoids. It was shown that the recombinant AtCCD7 protein catalyzes a specific 9-10 cleavage of β-carotene to produce the 10 ▾-apo-β-carotenal (C27) and β-ionone (C13). When AtCCD7 and AtCCD8 were co-expressed in a β-carotene-producing strain of E. coli, the 13-apo-β-carotenone (C18) was produced. The C18 product appears to result from a secondary cleavage of the AtCCD7-derived C27 product. The sequential cleavages of β-carotene by AtCCD7 and AtCCD8 are likely the initial steps in the synthesis of a carotenoid-derived signaling molecule that is necessary for the regulation lateral branching.
For the first time in its history, the Annual Maize Genetics Conference was held in Mexico City, near the center of origin for many Zea species, including maize. Maize research has made many contributions to our understanding of plant physiology and development, the regulation of transposable elements and chromosome structure, and the epigenetic control of gene expression. In addition to the reported advances in these research fields, this year's meeting emphasized the tremendous genetic diversity present within maize races. Explorations of this variation in studies of domestication, population genetics, and crop improvement hinted at the tremendous potential that lies within the maize genome. Tapping into this potential will soon be made easier as highlighted in a workshop outlining advances in maize genomics. Remarkable progress has been made toward sequencing the genic regions of the maize genome, and strategies to anchor and finish the entire maize gene space by 2006 were presented and discussed. This report highlights the new developments made in these areas of maize biology.
In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Désirée which normally produces tubers containing c. 5.6 μg carotenoid g−1 DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 μg carotenoid g−1 DW. In developing tubers of transgenic crtB Désirée lines, carotenoid levels reached 35 μg carotenoid g−1 DW and the balance of carotenoids changed radically compared with controls: β-carotene levels in the transgenic tubers reached
c. 11 μg g−1 DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector
transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 μg carotenoid g−1 DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin,
and β-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic
tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes
that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further
modifications of tuber carotenoid content are discussed.
A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically
yielding 3-hydroxy-β-ionone, a C13-norisoprenoidic compound, and a C14-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat
of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching véraison was observed in both
cultivars. In parallel, the C13-norisoprenoid level increased from véraison to maturity in both cultivars.
Apocarotenoids resulting from the oxidative cleavage of carotenoids serve as important signaling and accessory molecules in a variety of biological processes. The enzymes catalyzing these reactions are referred to as carotenases or carotenoid oxygenases. Whether they act according to a monooxygenase mechanism, requiring two oxygens from different sources, or a dioxygenase mechanism is still a topic of controversy. In this study, we utilized the readily available beta-apo-8'-carotenal as a substrate for the heterologously expressed AtCCD1 protein from Arabidopsis thaliana to investigate the oxidative cleavage mechanism of the 9,10 double bond of carotenoids. Beta-ionone and a C(17)-dialdehyde were detected as products by gas and liquid chromatography-mass spectrometry as well as NMR analysis. Labeling experiments using H(2)(18)O or (18) O(2) showed that the oxygen in the keto-group of beta-ionone is derived solely from molecular dioxygen. When experiments were performed in an (18)O(2)-enriched atmosphere, a substantial fraction of the C(17)-dialdehyde contained labeled oxygen. The results unambiguously demonstrate a dioxygenase mechanism for the carotenase AtCCD1 from A. thaliana.
To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.
The accumulation of three major carotenoid derivatives—crocetin glycosides, picrocrocin, and safranal—is in large part responsible for the color, bitter taste, and aroma of saffron, which is obtained from the dried styles of Crocus. We have identified and functionally characterized the Crocus zeaxanthin 7,8(7′,8′)-cleavage dioxygenase gene (CsZCD), which codes for a chromoplast enzyme that initiates the biogenesis of these derivatives. The Crocus carotenoid 9,10(9′,10′)-cleavage dioxygenase gene (CsCCD) also has been cloned, and the comparison of substrate specificities between these two enzymes has shown that the CsCCD enzyme acts on a broader range of precursors. CsZCD expression is restricted to the style branch tissues and is enhanced under dehydration stress, whereas CsCCD is expressed constitutively in flower and leaf tissues irrespective of dehydration stress. Electron microscopy revealed that the accumulation of saffron metabolites is accompanied by the differentiation of amyloplasts and chromoplasts and by interactions between chromoplasts and the vacuole. Our data suggest that a stepwise sequence exists that involves the oxidative cleavage of zeaxanthin in chromoplasts followed by the sequestration of modified water-soluble derivatives into the central vacuole.
Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the 'quality' of the culture used for protoplast isolation.
The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C13 cyclohexenone and C14 mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source–sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids.
Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium ttumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pCNL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable -glucuromdase (GUS) activities. However, plant regeneration following selection on G418 (pCNL56 contained the nptII gene) did not occur. Using the same basic protocol, but inoculating immature embryos of rice with LBA4404 (pTOK233), resulted in efficient (about 27%) production of transgenic plants of the japonica variety, Radon, and an acceptable efficiency (from 1–5%) for the indica varieties IR72 and TCS10. Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), Southern blots for detection of the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Southern blots indicated two to three copies of the gene integrated in most transformants. Transgenic plants of both the japonica and indica varieties were self-fertile and comparable in this respect to seed-grown plants. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the expiant, the use of hygromycin as the selection agent (which does not interfere with rice regeneration), the presence of extra copies of certain vir genes on the binary vector of pTOK233, and maintaining high concentrations of acetosyringone for inducing the vir genes during co-cultivation of embryos with Agrobacterium.
Linseed flax (Linum usitatissimum L.) is an industrially important oil crop, which includes large amounts of alpha-linolenic acid (18:3) and lignan in its seed oil. We report here the metabolic engineering of flax plants to increase carotenoid amount in seeds. Agrobacterium-mediated transformation of flax was performed to express the phytoene synthase gene (crtB) derived from the soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3) under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter or the Arabidopsis thaliana fatty acid elongase 1 gene (FAE1) seed-specific promoter. As a result, eight transgenic flax plants were generated. They formed orange seeds (embryos), in which phytoene, alpha-carotene, and beta-carotene were newly accumulated in addition to increased amounts of lutein, while untransformed flax plants formed light-yellow seeds, in which only lutein was detected. Interestingly, despite the control of the CaMV 35S promoter, the expression of crtB was not observed in the leaves but in the seeds in the transgenic flax plants. Total carotenoid amounts in these seeds were 65.4-156.3 microg/g fresh weight, which corresponded to 7.8- to 18.6-fold increase, compared with those of untransformed controls. These results suggest that the flux of phytoene synthesis from geranylgeranyl diphosphate was first promoted by the expressed crtB gene product (CrtB), and then phytoene was consecutively decomposed to the downstream metabolites alpha-carotene, beta-carotene, and lutein, as catalyzed by endogenous carotenoid biosynthetic enzymes in seeds. The transgenic flaxseeds enriched with the carotenoids could be valuable as nutritional sources for human health.
A bacterial phytoene synthase (crtB) gene was overexpressed in a seed-specific manner and the protein product targeted to the plastid in Brassica napus (canola). The resultant embryos from these transgenic plants were visibly orange and the mature seed contained up to a 50-fold increase in carotenoids. The predominant carotenoids accumulating in the seeds of the transgenic plants were alpha and beta-carotene. Other precursors such as phytoene were also detected. Lutein, the predominant carotenoid in control seeds, was not substantially increased in the transgenics. The total amount of carotenoids in these seeds is now equivalent to or greater than those seen in the mesocarp of oil palm. Other metabolites in the isoprenoid pathway were examined in these seeds. Sterol levels remained essentially the same, while tocopherol levels decreased significantly as compared to non-transgenic controls. Chlorophyll levels were also reduced in developing transgenic seed. Additionally, the fatty acyl composition was altered with the transgenic seeds having a relatively higher percentage of the 18 : 1 (oleic acid) component and a decreased percentage of the 18 : 2 (linoleic acid) and 18 : 3 (linolenic acid) components. This dramatic increase in flux through the carotenoid pathway and the other metabolic effects are discussed.
Carotenoid cleavage products--apocarotenoids--include biologically active compounds, such as hormones, pigments and volatiles. Their biosynthesis is initiated by the oxidative cleavage of C-C double bonds in carotenoid backbones, leading to aldehydes and/or ketones. This step is catalyzed by carotenoid oxygenases, which constitute an ubiquitous enzyme family, including the group of plant carotenoid cleavage dioxygenases 1 (CCD1s), which mediates the formation of volatile C(13) ketones, such as beta-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic and acyclic carotenoids. Recently, it was reported that plant CCD1s also act on the C5-C6/C5'-C6' double bonds of acyclic carotenes, leading to the volatile C(8) ketone 6-methyl-5-hepten-2-one. Using in vitro and in vivo assays, we show here that rice CCD1 converts lycopene into the three different volatiles, pseudoionone, 6-methyl-5-hepten-2-one, and geranial (C(10)), suggesting that the C7-C8/C7'-C8' double bonds of acyclic carotenoid ends constitute a novel cleavage site for the CCD1 plant subfamily. The results were confirmed by HPLC, LC-MS and GC-MS analyses, and further substantiated by in vitro incubations with the monocyclic carotenoid 3-OH-gamma-carotene and with linear synthetic substrates. Bicyclic carotenoids were cleaved, as reported for other plant CCD1s, at the C9-C10 and C9'-C10' double bonds. Our study reveals a novel source for the widely occurring plant volatile geranial, which is the cleavage of noncyclic ends of carotenoids.
A gene encoding a carotenoid cleavage dioxygenase class 1 enzyme (FaCCD1) was identified among a strawberry fruit expressed sequence tag collection. The full-length cDNA was isolated, and the expression profiles along fruit receptacle development and ripening, determined by quantitative real time polymerase chain reaction, showed that FaCCD1 is a ripening-related gene that reaches its maximal level of expression in the red fully ripe stage. FaCCD1 was expressed in Escherichia coli, and the products formed by the recombinant protein through oxidative cleavage of carotenoids were identified by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses. The FaCCD1 protein cleaves zeaxanthin, lutein, and beta-apo-8'-carotenal in vitro. Although beta-carotene is not a good substrate for FaCCD1 in vitro, the expression of FaCCD1 in an engineered carotenoid-producing E. coli strain caused the degradation of beta-carotene in vivo. Additionally, the carotenoid profile in strawberry was analyzed by high-performance liquid chromatography-photodiode detection, and a correlation between the increase of the expression level of FaCCD1 during ripening and the decrease of the lutein content suggests that lutein could constitute the main natural substrate of FaCCD1 activity in vivo.
The Indica rice breeding line IR58 was transformed by particle bombardment with a truncated version of a synthetic cryIA(b) gene from Bacillus thuringiensis. This gene is expressed under control of the CaMV 35S promoter and allows efficient production of the lepidopteran specific delta-endotoxin. R0, R1 and R2 generation plants displayed a significant insecticidal effect on several lepidopterous insect pests. Feeding studies showed mortality rates of up to 100% for two of the most destructive insect pests of rice in Asia, the yellow stem borer (Scirpophaga incertulas) and the striped stem borer (Chilo suppressalis), and feeding inhibition of the two leaffolder species Cnaphalocrocis medinalis and Marasmia patnalis. Introduction of stem borer resistance into the germplasm of an Indica rice breeding line now makes this agronomically important trait available for conventional rice breeding programs.
Plants and certain bacteria use a non-mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids. This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway. We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). This induction is temporarily and spatially correlated with specific and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C13 cyclohexenone derivatives and mycorradicin (C14) conjugates, the latter being a major component of the long-known 'yellow pigment'. A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots. Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identified from mycorrhizal wheat roots after alkaline treatment of an 'apocarotenoid complex' of yellow root constituents. We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor. This is the first report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus.
The accumulation of three major carotenoid derivatives-crocetin glycosides, picrocrocin, and safranal-is in large part responsible for the color, bitter taste, and aroma of saffron, which is obtained from the dried styles of Crocus. We have identified and functionally characterized the Crocus zeaxanthin 7,8(7',8')-cleavage dioxygenase gene (CsZCD), which codes for a chromoplast enzyme that initiates the biogenesis of these derivatives. The Crocus carotenoid 9,10(9',10')-cleavage dioxygenase gene (CsCCD) also has been cloned, and the comparison of substrate specificities between these two enzymes has shown that the CsCCD enzyme acts on a broader range of precursors. CsZCD expression is restricted to the style branch tissues and is enhanced under dehydration stress, whereas CsCCD is expressed constitutively in flower and leaf tissues irrespective of dehydration stress. Electron microscopy revealed that the accumulation of saffron metabolites is accompanied by the differentiation of amyloplasts and chromoplasts and by interactions between chromoplasts and the vacuole. Our data suggest that a stepwise sequence exists that involves the oxidative cleavage of zeaxanthin in chromoplasts followed by the sequestration of modified water-soluble derivatives into the central vacuole.
Bixin, also known as annatto, is a seed-specific pigment widely used in foods and cosmetics since pre-Columbian times. We
show that three genes from Bixa orellana, native to tropical America, govern bixin biosynthesis. These genes code for lycopene cleavage dioxygenase, bixin aldehyde
dehydrogenase, and norbixin carboxyl methyltransferase, which catalyze the sequential conversion of lycopene into bixin. Introduction
of these three genes in Escherichia coli engineered to produce lycopene induced bixin synthesis, thus expanding the supply of this economically important plant product.
Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values.
Plant development is exquisitely environmentally sensitive, with plant hormones acting as long-range signals that integrate developmental, genetic, and environmental inputs to regulate development. A good example of this is in the control of shoot branching, where wide variation in plant form can be generated in a single genotype in response to environmental and developmental cues.
Here we present evidence for a novel plant signaling molecule involved in the regulation of shoot branching. We show that the MAX3 gene of Arabidopsis is required for the production of a graft-transmissible, highly active branch inhibitor that is distinct from any of the previously characterized branch-inhibiting hormones. Consistent with its proposed function in the synthesis of a novel signaling molecule, we show that MAX3 encodes a plastidic dioxygenase that can cleave multiple carotenoids.
We conclude that MAX3 is required for the synthesis of a novel carotenoid-derived long-range signal that regulates shoot branching.
Carotenoids are isoprenoid molecules that are widespread in nature and are typically seen as pigments in fruits, flowers, birds and crustacea. Animals are unable to synthesise carotenoids de novo, and rely upon the diet as a source of these compounds. Over recent years there has been considerable interest in dietary carotenoids with respect to their potential in alleviating age-related diseases in humans. This attention has been mirrored by significant advances in cloning most of the carotenoid genes and in the genetic manipulation of crop plants with the intention of increasing levels in the diet. The aim of this article is to review our current understanding of carotenoid formation, to explain the perceived benefits of carotenoids in the diet and review the efforts that have been made to increase carotenoids in certain crop plants.
Carotenoids are thought to be the precursors of terpenoid volatile compounds that contribute to flavor and aroma. One such volatile, beta-ionone, is important to fragrance in many flowers, including petunia (Petunia hybrida). However, little is known about the factors regulating its synthesis in vivo. The petunia genome contains a gene encoding a 9,10(9',10') carotenoid cleavage dioxygenase, PhCCD1. The PhCCD1 is 94% identical to LeCCD1A, an enzyme responsible for formation of beta-ionone in tomato (Lycopersicon esculentum; Simkin AJ, Schwartz SH, Auldridge M, Taylor MG, Klee HJ [2004] Plant J [in press]). Reduction of PhCCD1 transcript levels in transgenic plants led to a 58% to 76% decrease in beta-ionone synthesis in the corollas of selected petunia lines, indicating a significant role for this enzyme in volatile synthesis. Quantitative reverse transcription-PCR analysis revealed that PhCCD1 is highly expressed in corollas and leaves, where it constitutes approximately 0.04% and 0.02% of total RNA, respectively. PhCCD1 is light-inducible and exhibits a circadian rhythm in both leaves and flowers. beta-Ionone emission by flowers occurred principally during daylight hours, paralleling PhCCD1 expression in corollas. The results indicate that PhCCD1 activity and beta-ionone emission are likely regulated at the level of transcript.
Volatile terpenoid compounds, potentially derived from carotenoids, are important components of flavor and aroma in many fruits, vegetables and ornamentals. Despite their importance, little is known about the enzymes that generate these volatiles. The tomato genome contains two closely related genes potentially encoding carotenoid cleavage dioxygenases, LeCCD1A and LeCCD1B. A quantitative reverse transcriptase-polymerase chain reaction analysis revealed that one of these two genes, LeCCD1B, is highly expressed in ripening fruit (4 days post-breaker), where it constitutes 0.11% of total RNA. Unlike the related neoxanthin cleavage dioxygenases, import assays using pea chloroplasts showed that the LeCCD1 proteins are not plastid-localized. The biochemical functions of the LeCCD1 proteins were determined by bacterial expression and in vitro assays, where it was shown that they symmetrically cleave multiple carotenoid substrates at the 9,10 (9',10') positions to produce a C14 dialdehyde and two C13 cyclohexones that vary depending on the substrate. The potential roles of the LeCCD1 genes in vivo were assessed in transgenic tomato plants constitutively expressing the LeCCD1B gene in reverse orientation. This over-expression of the antisense transcript led to 87-93% reductions in mRNA levels of both LeCCD1A and LeCCD1B in the leaves and fruits of selected lines. Transgenic plants exhibited no obvious morphological alterations. High-performance liquid chromatography analysis showed no significant modification in the carotenoid content of fruit tissue. However, volatile analysis showed a > or =50% decrease in beta-ionone (a beta-carotene-derived C13 cyclohexone) and a > or =60% decrease in geranylacetone (a C13 acyclic product likely derived from a lycopene precursor) in selected lines, implicating the LeCCD1 genes in the formation of these important flavor volatiles in vivo.
Retinal and its derivatives represent essential compounds in many biological systems. In animals, they are synthesized through a symmetrical cleavage of beta-carotene catalysed by a monooxygenase. Here, we demonstrate that the open reading frame sll1541 from the cyanobacterium Synechocystis sp. PCC 6803 encodes the first eubacterial, retinal synthesizing enzyme (Diox1) thus far reported. In contrast to enzymes from animals, Diox1 converts beta-apo-carotenals instead of beta-carotene into retinal in vitro. The identity of the enzymatic product was proven by HPLC, GC-MS and in a biological test. Investigations, of the stereospecifity showed that Diox1 cleaved only the all-trans form of beta-apo-8'-carotenal, yielding all-trans-retinal. However, Diox1 exhibited wide substrate specificity with respect to chain-lengths and functional end-groups. Although with divergent Km and Vmax values, the enzyme converted beta-apo-carotenals, (3R)-3-OH-beta-apo-carotenals as well as apo-lycopenals into retinal, (3R)-3-hydroxy-retinal and acycloretinal respectively. In addition, the alcohols of these substrates were cleaved to yield the corresponding retinal derivatives.
"Golden Rice" is a variety of rice engineered to produce beta-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher beta-carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in beta-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop "Golden Rice 2" introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microg/g) compared to the original Golden Rice and a preferential accumulation of beta-carotene.
A more nutritious version of Golden Rice may offer a practical solution to vitamin A deficiency.
Carotenoids not only play a crucial role in their intact form but also are an important reservoir of lipid-derived bioactive mediators. The process is initiated by tailoring enzymes that cleave carotenoids into apocarotenoids. Apocarotenoids act as visual or volatile signals to attract pollinating and seed dispersal agents, and are also key players in allelopathic interactions and plant defense. Recent studies show that the loss of these cleavage enzymes induces the development of axillary branches, indicating that apocarotenoids convey signals that regulate plant architecture. Here, we describe these molecules and the current understanding of their biosynthesis and functions.
Chemical transformations of isoprenoids in plants and some bacteria and fungi lead to the production of various conjugated products, including carotenoids. Carotenoids can be cleaved to generate apocarotenoid precursors for signaling molecules such as abscisic and retinoic acids, and for the photosensory pigment retinal. The enzymes that catalyze the various transformations of carotenoids and apocarotenoids are closely related. This evolutionarily distant conservation is unexpected and intriguing. Many aspects of the metabolism of retinoids in vertebrates remain controversial and poorly understood. Because few chemical reactions are possible for this group of compounds, furthering our knowledge of isoprenoid transformation in plants could be beneficial to our understanding of how retinoids and carotenoids are transformed in vertebrates.
Tomato near-isogenic lines differing in fruit carotenogenesis genes accumulated different aroma volatiles, in a strikingly similar fashion as compared to watermelon cultivars differing in fruit color. The major volatile norisoprenoids present in lycopene-containing tomatoes and watermelons were noncyclic, such as geranial, neral, 6-methyl-5-hepten-2-one, 2,6-dimethylhept-5-1-al, 2,3-epoxygeranial, (E,E)-pseudoionone, geranyl acetone, and farnesyl acetone, seemingly derived from lycopene and other noncyclic tetraterpenoids. Beta-ionone, dihydroactinodiolide, and beta-cyclocitral were prominent in both tomato and watermelon fruits containing beta-carotene. Alpha-ionone was detected only in an orange-fleshed tomato mutant that accumulates delta-carotene. A yellow flesh (r) mutant tomato bearing a nonfunctional psy1 gene and the yellow-fleshed watermelon Early Moonbeam, almost devoid of carotenoid fruit pigments, also lacked norisoprenoid derivatives and geranial. This study provides evidence, based on comparative genetics, that carotenoid pigmentation patterns have profound effects on the norisoprene and monoterpene aroma volatile compositions of tomato and watermelon and that in these fruits geranial (trans-citral) is apparently derived from lycopene in vivo.
The world is filled with flavors and scents, which are the result of volatile compounds produced and emitted by plants. These specialized metabolites are the products of specific metabolic pathways. The terpenoid, fatty acid, and phenylpropanoid pathways contribute greatly to production of volatile compounds. Mechanisms that lead to evolution of volatile production in plants include gene duplication and divergence, convergent evolution, repeated evolution, and alteration of gene expression, caused by a number of factors, followed by change in enzyme specificity. Many examples of these processes are now available for three important gene families involved in production of volatile metabolites: the small molecule O-methyltransferases, the acyltransferases, and the terpene synthases. Examples of these processes in these gene families are found in roses, Clarkia breweri, and sweet basil, among others. Finally, evolution of volatile emission will be an exciting field of study for the foreseeable future.
Provitamin A accumulates in the grain of Golden Rice as a result of genetic transformation. In developing countries, where vitamin A deficiency prevails, grain from Golden Rice is expected to provide this important micronutrient sustainably through agriculture. Since its original production, the prototype Golden Rice has undergone intense research to increase the provitamin A content, to establish the scientific basis for its carotenoid complement, and to better comply with regulatory requirements. Today, the current focus is on how to get Golden Rice effectively into the hands of farmers, which is a novel avenue for public sector research, carried out with the aid of international research consortia. Additional new research is underway to further increase the nutritional value of Golden Rice.