Plant carotenoid cleavage oxygenases and their apocarotenoid products

The oxidative cleavage of carotenoids leads to the production of apocarotenoids and is catalyzed by a family of carotenoid cleavage dioxygenases (CCDs). CCDs often exhibit substrate promiscuity, which probably contributes to the diversity of apocarotenoids found in nature. Biologically and commercially important apocarotenoids include the phytohormone abscisic acid, the visual and signaling molecules retinal and retinoic acid, and the aromatic volatile beta-ionone. Unexpected properties associated with the CCD catalytic products emphasize their role in many aspects of plant growth and development. For instance, CCD7 and CCD8 produce a novel, graft-transmissible hormone that controls axillary shoot growth in plants. Here, CCDs are discussed according to their roles in the biosynthesis of these products. Recent studies regarding their mechanism of action are also addressed.     

Plant apocarotenoids: from retrograde signaling to interspecific communication

Carotenoids are isoprenoid compounds synthesized by all photosynthetic and some non-photosynthetic organisms. They are essential for photosynthesis and contribute to many other aspects of a plant's life. The oxidative breakdown of carotenoids gives rise to the formation of a diverse family of essential metabolites called apocarotenoids. This metabolic process either takes place spontaneously through reactive oxygen species or is catalyzed by enzymes generally belonging to the CAROTENOID CLEAVAGE DIOXYGENASE family. Apocarotenoids include the phytohormones abscisic acid and strigolactones (SLs), signaling molecules and growth regulators. Abscisic acid and SLs are vital in regulating plant growth, development and stress response. SLs are also an essential component in plants’ rhizospheric communication with symbionts and parasites. Other apocarotenoid small molecules, such as blumenols, mycorradicins, zaxinone, anchorene, β-cyclocitral, β-cyclogeranic acid, β-ionone and loliolide, are involved in plant growth and development, and/or contribute to different processes, including arbuscular mycorrhiza symbiosis, abiotic stress response, plant–plant and plant–herbivore interactions and plastid retrograde signaling. There are also indications for the presence of structurally unidentified linear cis-carotene-derived apocarotenoids, which are presumed to modulate plastid biogenesis and leaf morphology, among other developmental processes. Here, we provide an overview on the biology of old, recently discovered and supposed plant apocarotenoid signaling molecules, describing their biosynthesis, developmental and physiological functions, and role as a messenger in plant communication.     

Die facettenreiche Welt der Apocarotinoide. Farben,Düfte,Aromen und Hormone

Apocarotenoids are tailored from carotenoids by highly specific oxidative enzymes cleaving different double bonds. New chromophores can be generated, which make additional nuances of the yellow‐red color spectrum available. Colorless C13 apocarotenoids can constitute potent scent and aroma compounds. Many apocarotenoid hormone functions are well‐known (abscisic acid in plants, trisporic acid in fungi, retinoic acid in mammals). A new class of apocarotenoid plant hormones, which take part in determining shoot branching has recently been identified as strigolactones. In the biosyntheses of strigolactones and mycorrhiza‐induced C13/C14 apocarotenoids several sequential cleavage reactions occur. The knowledge about biosynthetic pathways and functions of apocarotenoids opens up new perspectives for its application in horticulture and in the control of parasitic weeds as well as in the manipulation of flower scents and fruit aromas.     

Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin.     

Occurrence and localization of apocarotenoids in arbuscular mycorrhizal plant roots

The core structure of the yellow pigment from arbuscular mycorrhizal (AM) maize roots contains the apocarotenoids mycorradicin (an acyclic C14 polyene) and blumenol C cellobioside (a C13 cyclohexenone diglucoside). The pigment seems to be a mixture of different esterification products of these apocarotenoids. It is insoluble in water and accumulates as hydrophobic droplets in the vacuoles of root cortical cells. Screening 58 species from 36 different plant families, we detected mycorradicin in mycorrhizal roots of all Liliopsida analyzed and of a considerable number of Rosopsida, but also species were found in which mycorradicin was undetectable in mycorrhizal roots. Kinetic experiments and microscopic analyses indicate that accumulation of the yellow pigment is correlated with the concomitant degradation of arbuscules and the extensive plastid network covering these haustorium-like fungal structures. The role of the apocarotenoids in mycorrhizal roots is still unknown. The potential C40 carotenoid precursors, however, are more likely to be of functional importance in the development and functioning of arbuscules.     

Carotenoid oxygenases: cleave it or leave it

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.     

Identification of carotenoid cleavage dioxygenases from Nostoc sp. PCC 7120 with different cleavage activities

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes that oxidatively cleave carotenoids into apocarotenoids. Dioxygenases have been identified in plants and animals and produce a wide variety of cleavage products. Despite what is known about apocarotenoids in higher organisms, very little is known about apocarotenoids and CCDs in microorganisms. This study surveyed cleavage activities of ten putative carotenoid cleavage dioxygenases from five different cyanobacteria in recombinant Escherichia coli cells producing different carotenoid substrates. Three CCD homologs identified in Nostoc sp. PCC 7120 were purified, and their cleavage activities were investigated. Two of the three enzymes showed cleavage of beta,beta-carotene at the 9,10 and 15,15' positions, respectively. The third enzyme did not cleave full-length carotenoids but cleaved the apocarotenoid beta-apo-8'-carotenal at the 9,10 position. 9,10-Apocarotenoid cleavage specificity has previously not been described. The diversity of carotenoid cleavage activities identified in one cyanobacteria suggests that CCDs not only facilitate the degradation of photosynthetic pigments but generate apocarotenals with yet to be determined biological roles in microorganisms.     

Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta

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.     

Synergistic Interactions between Carotene Ring Hydroxylases Drive Lutein Formation in Plant Carotenoid Biosynthesis

Plant carotenoids play essential roles in photosynthesis, photoprotection, and as precursors to apocarotenoids. The plastid-localized carotenoid biosynthetic pathway is mediated by well-defined nucleus-encoded enzymes. However, there is a major gap in understanding the nature of protein interactions and pathway complexes needed to mediate carotenogenesis. In this study, we focused on carotene ring hydroxylation, which is performed by two structurally distinct classes of enzymes, the P450 CYP97A and CYP97C hydroxylases and the nonheme diiron HYD enzymes. The CYP97A and HYD enzymes both function in the hydroxylation of β-rings in carotenes, but we show that they are not functionally interchangeable. The formation of lutein, which involves hydroxylation of both β- and ε-rings, was shown to require the coexpression of CYP97A and CYP97C enzymes. These enzymes were also demonstrated to interact in vivo and in vitro, as determined using bimolecular fluorescence complementation and a pull-down assay, respectively. We discuss the role of specific hydroxylase enzyme interactions in promoting pathway flux and preventing the formation of pathway dead ends. These findings will facilitate efforts to manipulate carotenoid content and composition for improving plant adaptation to climate change and/or for enhancing nutritionally important carotenoids in food crops.     

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system

Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron β-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites.     

In vitro characterization of a carotenoid cleavage dioxygenase from Nostoc sp. PCC 7120 reveals a novel cleavage pattern, cytosolic localization and induction by highlight

Carotenoid oxygenases catalyse the cleavage of C-C double bonds forming apocarotenoids, a diverse group of compounds, including retinoids and the precursors of some phytohormones. Some apocarotenoids, like β-ionone (C₁₃), are ecologically important volatiles released by plants and cyanobacteria. In this work, we elucidated the activity of the N ostoc c arotenoid c leavage d ioxygenase (NosCCD, previously named NSC1) using synthetic and cyanobacterial substrates. NosCCD converted bicyclic and monocyclic xanthophylls, including myxoxanthophylls, glycosylated carotenoids that are essential for thylakoid and cell wall structure. The products identified revealed two different cleavage patterns. The first is observed with bicyclic xanthophylls and is identical with that of plant orthologues, while the second is novel and occurs upon cleavage of monocyclic substrates at the C9-C10 and C7'-C8' double bonds. These properties enable the enzyme to produce a plenitude of different C₁₀ and C₁₃ apocarotenoids. Expression analyses indicated a role of NosCCD in response to highlight stress. Western blot analyses of Nostoc cells revealed NosCCD as a soluble enzyme in the cytosol, which also accomodates NosCCD substrates. Incubation of the corresponding fraction with synthetic substrates revealed the activity of the native enzyme and confirmed its induction by highlight.     

Lipid signaling

Various lipids are involved in mediating plant growth, development and responses to biotic and abiotic cues, and their production is regulated by lipid-signaling enzymes. Lipid-hydrolyzing enzymes play a pivotal role both in the production of lipid messengers and in other processes, such as cytoskeletal rearrangement, membrane trafficking, and degradation. Studies on the downstream targets and modes of action of lipid signals in plants are still in their early stages but distinguishing features of plant lipid-based signaling are being recognized. Phospholipase D enzymes and phosphatidic acid may play a broader role in lipid signaling in plants than in other systems.     

Casein kinase-2-type protein kinases in plants: possible targets of polyamine action during growth regulation?

The biochemical mechanisms by which polyamines influence plant growth and development are not known. One of mechanisms frequently proposed is that polyamines can bind to key cellular enzymes and modulate their activity. Polyamines have been reported to alter the activity of a number of enzymes in vitro. Among these the casein kinase-2 protein kinases are of particular interest, not only because of increasing recognition of the major role of protein phosphorylation in regulating plant cell metabolism, but also because these kinases have been specifically implicated in the phosphorylation of trans-acting factors and thus could regulate gene expression. Casein kinase-2-type protein kinases have been purified and characterized from both plants and animals. Their structural and biochemical properties appear to have been remarkably conserved throughout evolution. Most are stimulated by mM levels of polyamines. Although this concentration is within the range estimated to occur in plant cells, not enough is known about [polyamine] in subcellular compartments and about how rapidly this concentration can be altered by hormonal and environmental signals to predict whether polyamines play a major role in the regulation of casein kinase-2 protein kinase activity in vivo.     

Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation

During colonization by arbuscular mycorrhizal (AM) fungi plant roots frequently accumulate two types of apocarotenoids (carotenoid cleavage products). Both compounds, C(14) mycorradicin and C(13) cyclohexenone derivatives, are predicted to originate from a common C(40) carotenoid precursor. Mycorradicin is the chromophore of the "yellow pigment" responsible for the long-known yellow discoloration of colonized roots. The biosynthesis of apocarotenoids has been investigated with a focus on the two first steps of the methylerythritol phosphate (MEP) pathway catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). In Medicago truncatula and other plants the DXS2 isogene appears to be specifically involved in the AM-mediated accumulation of apocarotenoids, whereas in the case of DXR a single gene contributes to both housekeeping and mycorrhizal (apo)carotenoid biosynthesis. Immunolocalization of DXR in mycorrhizal maize roots indicated an arbuscule-associated protein deposition, which occurs late in arbuscule development and accompanies arbuscule degeneration and breakdown. The DXS2 isogene is being developed as a tool to knock-down apocarotenoid biosynthesis in mycorrhizal roots by an RNAi strategy. Preliminary results from this approach provide starting points to suggest a new kind of function for apocarotenoids in mycorrhizal roots.