Carotenoid metabolism: New insights and synthetic approaches

Carotenoids are well-known isoprenoid pigments naturally produced by plants, algae, photosynthetic bacteria as well as by several heterotrophic microorganisms. In plants, they are synthesized in plastids where they play essential roles in light-harvesting and in protecting the photosynthetic apparatus from reactive oxygen species (ROS). Carotenoids are also precursors of bioactive metabolites called apocarotenoids, including vitamin A and the phytohormones abscisic acid (ABA) and strigolactones (SLs). Genetic engineering of carotenogenesis made possible the enhancement of the nutritional value of many crops. New metabolic engineering approaches have recently been developed to modulate carotenoid content, including the employment of CRISPR technologies for single-base editing and the integration of exogenous genes into specific “safe harbors” in the genome. In addition, recent studies revealed the option of synthetic conversion of leaf chloroplasts into chromoplasts, thus increasing carotenoid storage capacity and boosting the nutritional value of green plant tissues. Moreover, transient gene expression through viral vectors allowed the accumulation of carotenoids outside the plastid. Furthermore, the utilization of engineered microorganisms allowed efficient mass production of carotenoids, making it convenient for industrial practices. Interestingly, manipulation of carotenoid biosynthesis can also influence plant architecture, and positively impact growth and yield, making it an important target for crop improvements beyond biofortification. Here, we briefly describe carotenoid biosynthesis and highlight the latest advances and discoveries related to synthetic carotenoid metabolism in plants and microorganisms

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

Carotenoids are a core plastid component and yet their regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids and their derivatives under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a z-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid retrograde signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the repressor of photomorphogenesis, DEETIOLATED1 (DET1), to transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5). The unknown apocarotenoid signal restored POR protein levels and PLB formation in det1, thereby controlling plastid development

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

Carotenoids are a core plastid component and yet their regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids and their derivatives under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a ζ-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid retrograde signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the repressor of photomorphogenesis, DEETIOLATED1 (DET1), to transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5). The unknown apocarotenoid signal restored POR protein levels and PLB formation in det1, thereby controlling plastid development.This work was supported by Grant CE140100008 (BJP) and DP130102593 (CIC)

Light-limited photosynthesis under energy-saving film decreases eggplant yield

Glasshouse films with adjustable light transmittance and energy-efficient designs have the potential to reduce (up to 80%) the high energy cost for greenhouse horticulture operations. Whether these films compromise the quantity and quality of light transmission for photosynthesis and crop yield remains unclear. A “Smart Glass” film ULR-80 (SG) was applied to a high-tech greenhouse horticulture facility, and two experimental trials were conducted by growing eggplant (Solanum melongena) using commercial vertical cultivation and management practices. SG blocked 85% of ultraviolet (UV), 58% of far-red, and 26% of red light, leading to an overall reduction of 19% in photosynthetically active radiation (PAR, 380–699 nm) and a 25% reduction in total season fruit yield. There was a 53% (season mean) reduction in net short-wave radiation (radiometer range, 385–2,105 nm upward; 295–2,685 nm downward) that generated a net reduction of 8% in heat load and reduced water and nutrient consumption by 18%, leading to improved energy and resource use efficiency. Eggplant adjusted to the altered SG light environment via decreased maximum light-saturated photosynthetic rates (Amax) and lower xanthophyll de-epoxidation state. The shift in light characteristics under SG led to reduced photosynthesis, which may have reduced source (leaf) to sink (fruit) carbon distribution, increased fruit abortion and decreased fruit yield, but did not affect nutritional quality. We conclude that SG increases energy and resource use efficiency, without affecting fruit quality, but the reduction in photosynthesis and eggplant yield is high. The solution is to re-engineer the SG to increase penetration of UV and PAR, while maintaining blockage of glasshouse heat gain

Feedback regulation of carotenoid biosynthesis by an apocarotenoid sensing RNA structural switch

Carotenoid biosynthesis and accumulation in plants are tightly coordinated to maintain cellular homeostasis by regulating several processes, including plastid development, production of phytohormones and signalling molecules associated with metabolic regulation and environmental stress response. The EPSILON LYCOPENE CYCLASE (εLCY) and BETA LYCOPENE CYCLASE (βLCY) enzymes control metabolic flux through the α- and β-branches of the carotenoid pathway and regulate the production and accumulation of downstream carotenoids. Previous research showed that the changes in εLCY gene expression might be associated with an altered abundance of downstream carotenoids such as β-carotene or controlled with an apocarotenoid retrograde signal originated from cis-carotenes. Here we characterise the substrate of the apocarotenoid signal (ACS) and describe a molecular mechanism regulating εLCY expression. Analysis using cis-carotene mutants, ccr2, ziso and ccr2 ziso showed that the εLCY regulatory ACS could not be derived from a cis-carotene intermediate downstream of ZISO metabolic step. The exogenic expression of a bacterial carotenoid isomerase (CrtI) bypassed the metabolic steps between PDS and CRTISO and decreased the cis-carotene content and enhanced the accumulation of β-branch carotenoids as well as εLCY expression in dark-grown ccr2 seedlings. The de-etiolation of seedlings under constant light caused higher accumulation of β-branch carotenoids and enhanced the εLCY expression. Metabolic and gene expression analysis with green leaves at different stages of development demonstrated that carotenoid accumulation, as well as εLCY expression, was higher in leaves at an early stage of development. These findings suggest that feedback control of εLCY expression was required throughout the plant development. Subsequent analysis with norflurazon (NFZ) treated dark-grown wild-type, ccr2 and CrtI expressing transgenic seedlings revealed that cis-carotenes, including phytoene and phytofluene, could not be involved in the regulation of εLCY expression. The εLCY expression was upregulated in β-carotene-rich cotyledon tissues of dark-grown Arabidopsis ccd4 mutants with wild-type and ccr2 background. These pieces of evidence showed that an unknown β-apocarotenoid retrograde signal might control the εLCY expression in order to maintain the balance between α- and β-carotene accumulation. Treatment with D15, an inhibitor of CCD catalytic activity, revealed that CCDs were not involved in generating an εLCY regulatory ACS; however, they might have enhanced the β-carotene substrate accumulation in embryonic tissues to generate β-apocarotenoids. Therefore, we conclude that the β-apocarotenoid signal which regulates εLCY was generated by the oxidative cleavage of β-carotene rather than by enzymatic catalysis. Previous research shows that the disruption of εLCY 5’end by mutations or transposon insertion enhances the β-carotene abundance in crop species. We hypothesized that the εLCY 5’end may harbour a regulatory unit that might interact with the β- apocarotenoid signal to control εLCY expression. In order to test this, we generated Arabidopsis transgenic lines harbouring an εLCY promoter-reporter gene fusion and confirmed that luciferase transcript levels were reduced in etiolated tissues of ccr2 and NFZ treated seedlings, and up-regulated by 30-fold following light exposure. The regulation of reporter gene expression was parallel to the changes in β-carotene abundance in plant tissues. In-silico and in-vitro analysis of the εLCY promoter region revealed three alternative transcription start sites (TSS) separated by a highly conserved 60-70bp AT-rich segments and a putative RNA regulatory switch in the 5’ untranslated leader region (UTR) having two alternative secondary structures resembling an RNA riboswitch. A mutated version of the εLCY 5’UTR having an altered secondary structure reduced CaMV35S promoter-driven luciferase activity in light-grown tissues. In-silico analysis of RNA secondary structures in εLCY 5’UTR showed that a conserved hairpin motif, which is eliminated in the mutated version of the 5’UTR, might be involved in shifting the RNA structure between transcriptionally active and inhibitor form of RNA structures. We propose that a β- apocarotenoid signal (ligand) can move out of the chloroplast and regulate εLCY gene expression through an RNA structural switch (ligand-binding domain) in the 5’UTR in order to fine-tune carotenoid flux through the pathway branch during development and in response to environmental changes

Identification and sequence analysis of alkaloid biosynthesis genes in Papaver section Oxytona

The benzylisoquinoline alkaloids (BIA) are secondary metabolites that are produced by many of the Papaver species including Papaver bracteatum, Papaver pseudo-orientale, and Papaver orientale, three representative members of the section Oxytona Bernh. The sequences and expression levels of the genes positioned on the BIA biosynthesis pathway were previously defined in opium poppy (Papaver somniferum), one of the mostly studied species of the same genus. Nevertheless, the majority of predicted BIA-related gene sequences in the section Oxytona have not been specified. Here we are presenting new cDNA sequences that belong to the Oxytona species. In this study, partial sequences of norcoclaurine-6-O methyltransferase (6OMT), NADPH-dependent codeinone reductase (COR), salutaridinol 7-O-acetyltransferase (SalAT), and (S)-tetrahydroprotoberberine cis-N methyltransferase (TNMT) in P. pseudo-orientale; 3-hydroxy-N-methylcoclaurine 4´-O-methyltransferase (4OMT) in P. bracteatum; and TNMT and 6OMT in P. orientale were identified for the first time, and some of the previously sequenced genes were resequenced in the section Oxytona. Furthermore, expressions of those genes were also detected by using qRT-PCR in leaves. The findings were used to construct phylogenetic trees demonstrating the evolutionary relationships of BIA-related genes among Oxytona species

Plant apocarotenoids : from retrograde signaling to interspecific communication

Carotenoids are isoprenoid compounds synthesized by all photosynthetic and some nonphotosynthetic 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 (ROS) or is catalyzed by enzymes generally belonging to the carotenoid cleavage dioxygenase (CCD) family. Apocarotenoids include the phytohormones abscisic acid (ABA) and strigolactones (SLs), signaling molecules, and growth regulators. ABA 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, βcyclogeranic acid, β-ionone, and loliolide, are involved in plant growth and development, and/or contribute to different processes, including arbuscular mycorrhiza (AM) symbiosis, abiotic stress response, plant-plant and plant-herbivore interactions, and plastid retrograde signaling. There are also indications for the presence of structurally unidentifiedlinearcis-carotene-derived apocarotenoids (LCDAs), which are presumed to modulateplastid 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 plants’ communication

Epigenetic control of carotenogenesis during plant development

Carotenoids are secondary metabolites synthesized in plastids that function in photosynthesis, photoprotection, growth and development of plants. Carotenoids contribute to the yellowish, orange and pinkish-red hues of leaves, flowers and fruits, as well as various aromas. They provide substrates for the biosynthesis of phytohormones and are cleavable into smaller apocarotenoids that function as retrograde signals and/or mediate intracellular communication as well as regulate gene transcription and/or protein translation. Carotenoid biosynthesis and gene regulation are tightly coordinated with tissue-specific plastid differentiation, seedling morphogenesis, fruit development, and prevailing environmental growth conditions such as light, temperature and mycorrhizal interactions. In the last decade, epigenetic processes have been linked to the regulation of carotenoid biosynthesis, accumulation and degradation during plant development. Next-generation sequencing approaches have shed new light on key rate-limiting steps in carotenoid pathways targeted by epigenetic modifications that synchronize carotenoid accumulation with plastid development and morphogenesis. We discuss how histone modifications (methylation and acetylation), DNA methylation and demethylation, as well as small RNA gene silencing processes can modulate carotenoid biosynthesis, accumulation and apocarotenoid generation throughout the plants’ life cycle: from seed germination to fruit morphogenesis. This review highlights how apocarotenoid signals regulate plastid biogenesis and gene expression in sync with chromatin alterations during skotomorphogenesis and photo-morphogenesis. We provide a new perspective based upon emerging evidence that supports a likely role for carotenoids in contributing to the programming and/or maintenance of the plants' epigenetic landscape

cis-carotene biosynthesis, evolution and regulation in plants : the emergence of novel signaling metabolites

Carotenoids are isoprenoid pigments synthesised by plants, algae, photosynthetic bacteria as well as some non-photosynthetic bacteria, fungi and insects. Abundant carotenoids found in nature are synthesised via a linear route from phytoene to lycopene after which the pathway bifurcates into cyclised α- and β-carotenes. Plants evolved additional steps to generate a diversity of cis-carotene intermediates, which can accumulate in fruits or tissues exposed to an extended period of darkness. Enzymatic or oxidative cleavage, light-mediated photoisomerization and histone modifications can affect cis-carotene accumulation. cis-carotene accumulation has been linked to the production of signaling metabolites that feedback and forward to regulate nuclear gene expression. When cis-carotenes accumulate, plastid biogenesis and operational control can become impaired. Carotenoid derived metabolites and phytohormones such as abscisic acid and strigolactones can fine-tune cellular homeostasis. There is a hunt to identify a novel cis-carotene derived apocarotenoid signal and to elucidate the molecular mechanism by which it facilitates communication between the plastid and nucleus. In this review, we describe the biosynthesis and evolution of cis-carotenes and their links to regulatory switches, as well as highlight how cis-carotene derived apocarotenoid signals might control organelle communication, physiological and developmental processes in response to environmental change

cis/trans carotenoid extraction, purification, detection, quantification and profiling in plant tissues

Reverse phase high-performance liquid chromatography (HPLC) is the method of choice used in biological, health, and food research to identify, quantify, and profile carotenoid species. The identification and quantification of cis- and/or trans-carotene and xanthophyll isomers in plant tissues can be affected by the method of sample preparation and extraction, as well as the HPLC column chemistry and the solvent gradient. There is a high degree of heterogeneity in existing methods in terms of their ease, efficiency, and accuracy. We describe a simple carotenoid extraction method and two different optimised HPLC methods utilizing C18 or C30 reverse-phase columns. We outline applications, advantages, and disadvantages for using these reverse phase columns to detect xanthophylls and cis-carotenes in wild-type photosynthetic leaves and mutant dark-grown etiolated seedlings, respectively. Resources are provided to profile individual species based upon their spectral properties and retention time, as well as quantify carotenoids by their composition and absolute levels in different plant tissues