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Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short-chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type-II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP; C

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity

Vitamin A deficiency is a worldwide public nutrition problem, and β-carotene is the precursor for vitamin A synthesis. Watermelon with golden flesh (gf, due to accumulated abundance of β-carotene) is an important germplasm resource. In this study, a genetic analysis of gf segregating populations indicated that gf was controlled by a single recessive gene. BSA-seq and an initial linkage analysis placed the gf locus in a 290-Kb region on watermelon chromosome 1. Further fine mapping in a large population with over 1,000 F2 plants narrowed this region to 39.08 Kb harboring two genes, Cla97C01G008760 and Cla97C01G008770, which encode phytoene synthase (ClPsy1) and GATA zinc finger domain-containing protein, respectively. Gene sequence alignment and expression analysis between parental lines revealed Cla97C01G008760 as the best possible candidate gene for gf trait. Nonsynonymous SNP mutations in the first exon of ClPsy1 between parental lines cosegregated with the gf trait only among individuals in the genetic population but were not related to flesh color in natural watermelon panels. Promoter sequence analysis of 26 watermelon accessions revealed two SNPs in the cis-acting element sequences corresponding to MYB and MYC2 transcription factors. RNA-seq data and qRT-PCR verification showed that two MYBs and one MYC2 exhibited expression trends similar as ClPsy1 in the parental lines, which may thus play roles in the regulation of ClPsy1 expression. Our research findings indicate that the gf trait is determined not only by ClPsy1 but also by ClLCYB, ClCRTISO and ClNCED7, which play important roles in β-carotene accumulation in watermelon flesh.

Background Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood. Results In this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson’s correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. Conclusion The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

Overexpression of a novel geranylgeranyl pyrophosphate synthase gene (WsGGPPS) in planta resulted in increased levels of gibberellic acid and decrease in withanolide content. Withania somnifera (L.) Dunal, the herb from family Solanaceae is one of the most treasured medicinal plant used in traditional medicinal systems owing to its unique stockpile of pharmaceutically active secondary metabolites. Phytochemical and pharmacological studies in this plant were well established, but the genes affecting the regulation of biosynthesis of major metabolites were not well elucidated. In this study cloning and functional characterization of a key enzyme in terpenoid biosynthetic pathway viz. geranylgeranyl pyrophosphate synthase (EC 2.5.1.29) gene from Withania somnifera was performed. The full length WsGGPPS gene contained 1,104 base pairs that encode a polypeptide of 365 amino acids. The quantitative expression analysis suggested that WsGGPPS transcripts were expressed maximally in flower tissues followed by berry tissues. The expression levels of WsGGPPS were found to be regulated by methyl jasmonate (MeJA) and salicylic acid (SA). Amino acid sequence alignment and phylogenetic studies suggested that WsGGPPS had close similarities with GGPPS of Solanum tuberosum and Solanum pennellii. The structural analysis provided basic information about three dimensional features and physicochemical parameters of WsGGPPS protein. Overexpression of WsGGPPS in planta for its functional characterization suggested that the WsGGPPS was involved in gibberellic acid biosynthesis.

Background Carotenoids play important roles in photosynthesis, hormone signaling, and secondary metabolism. Phytoene synthase (PSY) catalyzes the first step of the carotenoid biosynthetic pathway. In this study, we aimed to characterize the PSY genes in tobacco and analyze their function. Results In this study, we identified three groups of PSY genes, namely PSY1, PSY2, and PSY3, in four Nicotiana species; phylogenetic analysis indicated that these genes shared a high similarity with those in tomato but not with those in monocots such as rice and maize. The expression levels of PSY1 and PSY2 were observed to be highest in leaves compared to other tissues, and they could be elevated by treatment with certain phytohormones and exposure to strong light. No PSY3 expression was detected under these conditions. We constructed virus-induced PSY1 and PSY2 silencing in tobacco and found that the newly emerged leaves in these plants were characterized by severe bleaching and markedly decreased carotenoid and chlorophyll content. Thylakoid membrane protein complex levels in the gene-silenced plants were also less than those in the control plants. The chlorophyll fluorescence parameters such as Fv/Fm, ΦPSII, qP, and NPQ, which reflect photosynthetic system activities, of the gene-silenced plants were also significantly decreased. We further performed RNA-Seq and metabonomics analysis between gene-silenced tobacco and control plants. RNA-Seq results showed that abiotic stress, isoprenoid compounds, and amino acid catabolic processes were upregulated, whereas the biosynthesis of cell wall components was downregulated. Metabolic analysis results were consistent with the RNA-Seq. We also found the downstream genes in carotenoid biosynthesis pathways were upregulated, and putative transcription factors that regulate carotenoid biosynthesis were identified. Conclusions Our results suggest that PSY can regulate carotenoid contents not only by controlling the first biosynthesis step but also by exerting effects on the expression of downstream genes, which would thereby affect photosynthetic activity. Meanwhile, PSY may affect other processes such as amino acid catabolism and cell wall organization. The information we report here may aid further research on PSY genes and carotenoid biosynthesis.

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis as well as photoprotectors for plants to adapt to high light stress. Phytoene synthase (PSY) is the entry enzyme and also the major rate-limiting enzyme in the carotenoid pathway. Here, we report a dehydration-responsive element-binding protein (DREB) transcription factor member in

ilustraciones, gráficas, tablas Se desconoce con claridad la fisiopatología exacta por la cual se forman las cataratas relacionadas con la edad. Se han investigado múltiples relaciones entre la biosíntesis del lanosterol y la formación de cataratas. En nuestro estudio se realizó un estudio de reacción en cadena de polimerasa en tiempo real de las enzimas Farnesyl sintasa, Geranyl sintasa, Lanosterol sintasa y Escualeno sintasa en un cristalino transparente de control y dos cristalinos con catarata senil de diferentes grados de opacidad. En los resultados fue posible evidenciar una diferencia significativa en la expresión relativa de las enzimas Farnesyl sintasa, Geranyl sintasa y Escualeno Sintasa siendo mayor en las muestras 1 y 2 de cristalinos con opacidad nuclear con respecto al cristalino control transparente. Los hallazgos sugieren una modificación patológica en el funcionamiento de la vía metabólica del colesterol bifurcándose hacia un aumento en la expresión de la enzima Geranyl difosfato sintasa. Esta desviación resulta importante al estar este compuesto implicado en el proceso de prenilación de proteínas, lo cual podría explicar la aglomeración de proteínas en cristalinos humanos, conllevando a la opacificación del cristalino con la edad. (Texto tomado de la fuente). The exact pathophysiology by which age-related cataracts develop is not clearly known. Multiple relationships between lanosterol biosynthesis and cataract formation have been investigated. In our study, a real-time polymerase chain reaction assay of the enzymes Farnesyl synthase, Geranyl synthase, Lanosterol synthase and Squalene synthase was performed on a transparent control lens and two senile cataractous lenses with different degrees of opacity. In the results it was possible to evidence a significant difference in the relative expression of the enzymes Farnesyl synthase, Geranyl synthase and Squalene Synthase, being higher in samples 1 and 2 of lenses with nuclear opacity compared to the control lens. These findings suggest a pathological modification in the functioning of the cholesterol metabolic pathway, deviating towards an increase in the expression of the enzyme Geranyl diphosphate synthase. This deviation is important as this compound is involved in the process of protein prenylation, which could explain the agglomeration of proteins in human lenses, leading to lens opacification with age. Especialidades Médicas Especialista en Oftalmología Vía metabólica del Lanosterol

Orange protein (OR) is known to interact with phytoene synthase (PSY) that commits the first step in carotenoid biosynthesis, and functions as a major post-transcriptional regulator on PSY. We here tried to reveal enzymatic characteristics of OR, that is, protein disulfide reductase (PDR) activity of the Arabidopsis thaliana OR protein (AtOR) was analyzed using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The AtOR part containing only the zinc (Zn)-finger motif was found to show PDR activity, with an apparent Km of 12,632 nM, Kcat of 11.85 min−1, and KcatKm−1 of 15.6 × 103 M−1sec−1. To evaluate the significance of the N-terminal region of AtOR, we examined the kinetic parameters of a fusion protein composed of the N-terminal region and the Zn-finger motif from AtOR. Consequently, the fusion protein had lower values for Km (2,074 nM) and Kcat (3.18 min−1) and higher catalytic efficiency (25.9 × 103 M−1sec−1) than that of only the Zn-finger motif part, suggesting that the N-terminal region of AtOR should be important for substrate affinity and catalytic efficiency of PDR activity. Complementation experiments with E. coli further demonstrated that AtOR containing the N-terminal region and the Zn-finger motif increases phytoene synthase activity of AtPSY especially under reduced circumstances retaining a NADPH- and H+-regeneration system.

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

Part of the studies involved in safety assessment of genetically engineered crops includes characterizing the organization, integrity, and stability of the inserted DNA and evaluating the potential allergenicity and toxicity of newly-expressed proteins. Molecular characterization of the introduced DNA in provitamin A biofortified rice event GR2E confirmed insertion of a single copy of the transfer-DNA in the genome and its inheritance as a single locus. Nucleotide sequencing of the inserted DNA confirmed it was introduced without modifications. The phytoene synthase, and carotene desaturase proteins did not display sequence similarity with allergens or toxins. Both proteins were rapidly digested in simulated gastric fluid and their enzymatic activity was inhibited upon heat treatment. Acute oral toxicity testing of the protein in mice demonstrated lack of adverse effects. These evidences substantiated the lack of any identifiable hazards for both proteins and in combination with other existing comparative analyses provided assurance that food derived from this rice is safe. This conclusion is in line with those of the regulatory agencies of US Food and Drug Administration, Health Canada and Food Standard Australia and New Zealand.

Colored apocarotenoids accumulate at high concentrations in few plant species, where display a role in attraction of pollinators and seed dispersers. Among these apocarotenoids, crocins accumulate at high concentrations in the stigma of saffron and are responsible for the organoleptic and medicinal properties of this spice. Phytoene synthase and Orange protein are key for carotenoid biosynthesis and accumulation. We previously isolated four phytoene synthase genes from saffron with differential roles in carotenoid and apocarotenoid biosynthesis. However, the implications of Orange genes in the regulation of apocarotenoid accumulation are unknown. Here, we have identified two Orange genes from saffron, with different expression patterns. CsOr-a was mainly expressed in vegetative tissues and was induced by light and repressed by heat stress. Both CsOr-a and CsOr-b were expressed in stigmas but showed a different profile during the development of this tissue. The interactions of CsOr-a and CsOr-b were tested with all the four phytoene synthase proteins from saffron and with CsCCD2. None interactions were detected with CCD2 neither with the phytoene synthase 2, involved in apocarotenoid biosynthesis in saffron. The obtained results provide evidence of different mechanisms regulating the phytoene synthase enzymes in saffron by Orange for carotenoid and apocarotenoid accumulation in saffron.

Isoprenoids comprise a diverse group of natural products with a broad range of metabolic functions. Isoprenoids are synthesized from prenyl pyrophosphates by prenyltransferases that catalyze the isoprenoid chain-elongation process to different chain lengths. We hereby present the crystal structure of geranylgeranyl pyrophosphate synthase from the marine flavobacterium Nonlabens dokdonensis DSW-6 (NdGGPPS). NdGGPPS forms a hexamer composed of homodimeric trimer, and the monomeric structure is composed of 15 α-helices (α1-α15). In this structure, we observed the binding of one pyrophosphate molecule and two glycerol molecules that mimicked substrate binding to the enzyme. The substrate binding site of NdGGPPS contains large hydrophobic residues such as Phe, His and Tyr, and structural and amino acids sequence analyses thereof suggest that the protein belongs to the short-chain prenyltransferase family.

© 2021 The Authors, Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C30 backbone, rather than the previously predicted canonical C40 phytoene-derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C30 pathway through lateral gene transfer from Planctomycetes. Surprisingly, disruption of carotenoid synthesis did not generate any major growth or membrane biophysical phenotypes, but slightly increased sensitivity to oxidative stress. We further demonstrated that hopanoids but not carotenoids are essential for growth at higher temperatures, membrane permeability and tolerance of low divalent cation concentrations. These observations show that hopanoids and carotenoids serve diverse roles in the outer membrane of M. extorquens PA1., This work was supported by the B CUBE, TU Dresden, a German Federal Ministry of Education and Research BMBF grant (to J.S., project 03Z22EN12), and a VW Foundation “Life” grant (to J.S., project 93090).

BackgroundPlants have remarkable diversity in petal colour through the biosynthesis and accumulation of various pigments. To better understand the mechanisms regulating petal pigmentation inLonicera japonica, we used multiple approaches to investigate the changes in carotenoids, anthocyanins, endogenous hormones and gene expression dynamics during petal colour transitions, i.e., green bud petals (GB_Pe), white flower petals (WF_Pe) and yellow flower petals (YF_Pe).ResultsMetabolome analysis showed that YF_Pe contained a much higher content of carotenoids than GB_Pe and WF_Pe, with α-carotene, zeaxanthin, violaxanthin and γ-carotene identified as the major carotenoid compounds in YF_Pe. Comparative transcriptome analysis revealed that the key differentially expressed genes (DEGs) involved in carotenoid biosynthesis, such asphytoene synthase,phytoene desaturaseandζ-carotene desaturase, were significantly upregulated in YF_Pe. The results indicated that upregulated carotenoid concentrations and carotenoid biosynthesis-related genes predominantly promote colour transition. Meanwhile, two anthocyanins (pelargonidin and cyanidin) were significantly increased in YF_Pe, and the expression level of ananthocyanidin synthasegene was significantly upregulated, suggesting that anthocyanins may contribute to vivid yellow colour in YF_Pe. Furthermore, analyses of changes in indoleacetic acid, zeatin riboside, gibberellic acid, brassinosteroid (BR), methyl jasmonate and abscisic acid (ABA) levels indicated that colour transitions are regulated by endogenous hormones. The DEGs involved in the auxin, cytokinin, gibberellin, BR, jasmonic acid and ABA signalling pathways were enriched and associated with petal colour transitions.ConclusionOur results provide global insight into the pigment accumulation and the regulatory mechanisms underlying petal colour transitions during the flower development process inL. japonica.

Carotenoids are natural pigments that are indispensable to plants and humans, whereas the regulation of carotenoid biosynthesis by post-translational modification remains elusive. Here, we show that a tomato E3 ubiquitin ligase, Plastid Protein Sensing RING E3 ligase 1 (PPSR1), is responsible for the regulation of carotenoid biosynthesis. PPSR1 exhibits self-ubiquitination activity and loss of PPSR1 function leads to an increase in carotenoids in tomato fruit. PPSR1 affects the abundance of 288 proteins, including phytoene synthase 1 (PSY1), the key rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY1 contains two ubiquitinated lysine residues (Lys380 and Lys406) as revealed by the global analysis and characterization of protein ubiquitination. We provide evidence that PPSR1 interacts with PSY1 precursor protein and mediates its degradation via ubiquitination, thereby affecting the steady-state level of PSY1 protein. Our findings not only uncover a regulatory mechanism for controlling carotenoid biosynthesis, but also provide a strategy for developing carotenoid-enriched horticultural crops., Wang et al. report on the role of a novel E3 ubiquitin ligase, Plastid Protein Sensing RING E3 ligase 1 (PPSR1), during tomato fruit ripening and find that it interacts with phytoene synthase 1 (PSY1) precursor protein and mediates its degradation via ubiquitination. This affects the steady-state level of PSY1 protein, the key rate-limiting enzyme in the carotenoid biosynthetic pathway. This study may provide a strategy for developing carotenoid-enriched horticultural crops.

Lycopene is the most potent antioxidant among all carotenoids and is beneficial to human health. A ripe fruit of autumn olive (

Genes homologous to PSY1 and PSY2 that encode phytoene synthase isoforms in Capsicum species C. baccatum, C. chinense, C. frutescens, C. tovarii, C. eximium, and C. chacoense were identified. High conservatism of functionally significant sites of phytoene synthases of the analyzed accessions was revealed. It was found that only PSY1-based clustering of pepper species corresponds to the traditional Capsicum phylogeny; C. eximium was a part of the Purple corolla complex, and C. chacoense was equidistant from Annuum and Baccatum clades. The absence of significant differences between PSY1 and PSY2 of yellow-fruited C. chinense and red-fruited pepper accessions was shown. The yellow color of C. chinense fruit may be the result of both decreased PSY1 expression and increased PSY2 transcription. Thus, it was demonstrated that the acquired fruit pigmentation retains strict phylogenetic limitations, which, however, can be overcome using artificial selection for the activity of phytoene synthase PSY1.

Hypercarotenogenesis in green algae evolved by mutation of PSY that increased its transcription at high light, disintegration of the eyespot in Dunaliella and acquisition of the capacity to export carotenoids from chloroplasts in Haematococcus. Carotenoids (Car) are lipid-soluble pigments synthesized in plants, algae, bacteria and fungi. Car have strong antioxidative properties and as such are utilized to reduce the danger of different diseases in humans. Two green microalgae are utilized as rich natural sources for Car: Dunaliella salina/bardawil accumulates 10% (w/w) β-carotene (βC), which is also pro-vitamin A, and Haematococcus pluvialis accumulates 4% (w/w) astaxanthin (Ast), the strongest antioxidant among Car. D. bardawil accumulates βC in plastoglobules within the chloroplast, whereas H. pluvialis deposits Ast in cytoplasmic lipid droplets (CLD). In this review we compare the hypercarotenogenic responses (HCR) in Dunaliella and in Haematococcus and try to outline hypothetical evolutionary pathways for its origin. We propose that a mutation in phytoene synthetase that increased its transcription level in response to high light stress had a pivotal role in the evolution of the HCR. Proteomic analyses indicated that in D. bardawil/salina the HCR evolved from dissociation and amplification of eyespot lipid globules. The more robust HCR in algae that accumulate carotenoids in CLD, such as H. pluvialis, required also acquisition of the capacity to export βC out of the chloroplast and its enzymatic conversion into Ast.

Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 μM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS showing an IC50 of 0.31 μM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some representative novel bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even better than zoledronic acid as a anti-resorptive agent.

Post-translational prenylation of the small GTP-binding proteins (GTPases) is vital to a plethora of biological processes, including cellular proliferation. We have identified a new class of thienopyrimidine-based bisphosphonate (ThP-BP) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) that block protein prenylation in multiple myeloma (MM) cells leading to cellular apoptosis. These inhibitors are also effective in blocking the proliferation of other types of cancer cells. We confirmed intracellular target engagement, demonstrated the mechanism of action leading to apoptosis, and determined a direct correlation between apoptosis and intracellular inhibition of hGGPPS. Administration of a ThP-BP inhibitor to a MM mouse model confirmed in vivo downregulation of Rap1A geranylgeranylation and reduction of monoclonal immunoglobulins (M-protein, a biomarker of disease burden) in the serum. These results provide the first proof-of-principle that hGGPPS is a valuable therapeutic target in oncology and more specifically for the treatment of multiple myeloma.

Although remarkable progress has been made toward understanding carotenoid biosynthesis, the mechanisms that regulate the transcription of carotenogenic genes remain poorly understood. Lycopene β-cyclases (LCYb) are critical enzymes located at the branch point of the carotenoid biosynthetic pathway. Here, we used the promoter sequence of LCYb1 as bait in a yeast one-hybrid screen for promoter-binding proteins from sweet orange (Citrus sinensis). This screen identified a MADS transcription factor, CsMADS6, that was coordinately expressed with fruit development and coloration. Acting as a nucleus-localized transcriptional activator, CsMADS6 directly bound the promoter of LCYb1 and activated its expression. Overexpression of CsMADS6 in citrus calli increased carotenoid contents and induced the expression of LCYb1 and other carotenogenic genes, including phytoene synthase (PSY), phytoene desaturase (PDS), and carotenoid cleavage dioxygenase1 (CCD1). CsMADS6 up-regulated the expression of PSY, PDS, and CCD1 by directly binding to their promoters, which suggested the multitargeted regulation of carotenoid metabolism by CsMADS6. In addition, the ectopic expression of CsMADS6 in tomato (Solanum lycopersicum) affected carotenoid contents and the expression of carotenogenic genes. The sepals of CsMADS6-overexpressing tomato lines exhibited dramatic changes in carotenoid profiles, accompanied by changes in plastid ultrastructure. Global transcriptome analysis of transgenic sepals revealed that CsMADS6 regulates a series of pathways that promote increases in flux through the carotenoid pathway. Overall, these findings establish that CsMADS6 directly regulates LCYb1 and other carotenogenic genes to coordinately and positively modulate carotenoid metabolism in plants, which may provide strategies to improve the nutritional quality of crops.

Phytoene (C40H64) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08–0.75 mg L−1 medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

Carotenogenesis has been intensively studied in carrot roots, and transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about the transcriptional regulation of carotenoid biosynthetic genes concerning carotenoid accumulation during infestation by the obligate parasite Phelipanche aegyptiaca. HPLC analysis revealed a decrease in carotenoid levels of the different carrot cultivars when parasitized by P. aegyptiaca. Besides, we isolated and analyzed P. aegyptiaca tubercles parasitizing the various carrot root cultivars and show that they accumulate different carotenoids compared to those in non-infested carrot roots. Expression analysis of PHYTOENE SYNTHASE (PSY1) and CAROTENOID ISOMERASE (CRTISO) as well as the strigolactone apocarotenoid biosynthetic genes DWARF27 (D27), CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 revealed that their transcript levels showed significant variation in P. aegyptiaca infested carrot roots. After parasite infestation, the expression of these genes was strongly reduced, as were the carotenoid levels and this was more pronounced in the uncommon non-orange varieties. We also analyzed the parasite genes encoding D27, CCD7 and CCD8 and show that they are expressed in tubercles. This raises important questions of whether the parasite produces its carotenoids and apocarotenoids including strigolactones and whether the latter might have a role in tubercle development.

Phytoene synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate (GGPP) synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The activity assay requires

Haematococcus pluvialis is widely distributed in the world and well known as the richest natural source of astaxanthin that is a strong antioxidant with excellent commercial value. The pathway of astaxanthin biosynthesis in H. pluvialis has been documented as an enzymatic reaction. Several enzymes have been reported, but their isoforms or homologs have not been investigated genome-wide. To better understand the astaxanthin biosynthesis pathway in H. pluvialis, eight candidates of the geranylgeranyl pyrophosphate synthase gene (HpGGPPS) predicted from Iso-seq data were isolated in this study. The length of coding region of these candidates varied from 960 bp to 1272 bp, composing of 7–9 exons. The putative amino acids of all candidates composed the signature domain of GGPPS gene. However, the motifs in the domain region are varied, indicating different bio-functions. Phylogenetic analysis revealed eight candidates can be clustered into three groups. Only two candidates in Group1 encode the synthase participating in the astaxanthin formation. The yield of astaxanthin from these two candidates, 7.1 mg/g (DW) and 6.5 mg/g (DW) respectively, is significant higher than that from CrtE (2.4 mg/g DW), a GGPPS gene from Pantoea ananatis. This study provides a potential productive pathway for astaxanthin synthesis.

The California poppy (Eschscholzia californica) is renowned for its brilliant golden-orange flowers, though white petal variants have been described. By whole-transcriptome sequencing, we have discovered in multiple white petal varieties a single deletion leading to altered splicing and C-terminal truncation of phytoene synthase (PSY), a key enzyme in carotenoid biosynthesis. Our findings underscore the diverse roles of phytoene synthase in shaping horticultural traits, and resolve a longstanding mystery of the regaled golden poppy.

The growth and astaxanthin production of Chlorella zofingiensis were examined under both heterotrophic and photoautotrophic conditions, and it was found that, in comparison to the photoautotrophic mode, the heterotrophic mode led to high algal densities but attenuated intracellular astaxanthin accumulation. Following the heterotrophy-photoautotrophy transition, a considerable increase in the astaxanthin content was observed, accompanied by the upregulation of key carotenogenic genes, including phytoene synthase (PSY), β-carotenoid hydroxylase (CHYb), β-carotenoid ketolase 1 (BKT1), and β-carotenoid ketolase 2 (BKT2). In contrast, the astaxanthin content and carotenogenic genes underwent an opposite change following the photoautotrophy-heterotrophy transition, suggesting the key role of light in stimulating astaxanthin biosynthesis. To improve the astaxanthin production by C. zofingiensis, a novel heterotrophy-photoinduction culture strategy without dilution was developed and evaluated. The astaxanthin content and productivity reached 2.7 mg g-1 of dry weight and 9.9 mg L-1 day-1, respectively, which were 4.0- and 2.5-fold higher than that obtained under the heterotrophic condition.

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue–specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.

Background Farnesol is an acyclic sesquiterpene alcohol present in the essential oils of various plants in nature. It has been reported to be valuable in medical applications, such as alleviation of allergic asthma, gliosis, and edema as well as anti-cancerous and anti-inflammatory effects. Coenzyme Q10 (CoQ10), an essential cofactor in the aerobic respiratory electron transport chain, has attracted growing interest owing to its clinical benefits and important applications in the pharmaceutical, food, and health industries. In this work, co-production of (E,E)-farnesol (FOH) and CoQ10 was achieved by combining 3 different exogenous terpenes or sesquiterpene synthase with the RNA interference of psy (responsible for phytoene synthesis in Rhodobacter sphaeroides GY-2). Results FOH production was significantly increased by overexpressing exogenous terpene synthase (TPS), phosphatidylglycerophosphatase B (PgpB), and sesquiterpene synthase (ATPS), as well as RNAi-mediated silencing of psy coding phytoene synthase (PSY) in R. sphaeroides strains. Rs-TPS, Rs-ATPS, and Rs-PgpB respectively produced 68.2%, 43.4%, and 21.9% higher FOH titers than that of the control strain. Interestingly, the CoQ10 production of these 3 recombinant R. sphaeroides strains was exactly opposite to that of FOH. However, CoQ10 production was almost unaffected in R. sphaeroides strains modified by psy RNA interference. The highest FOH production of 40.45 mg/L, which was twice as high as that of the control, was obtained from the TPS-PSYi strain, where the exogenous TPS was combined with the weakening of the phytoene synthesis pathway via psy RNA interference. CoQ10 production in TPS-PSYi, ATPS-PSYi, and PgpB-PSYi was decreased and lower than that of the control strain. Conclusions The original flux that contributed to phytoene synthesis was effectively redirected to provide precursors toward FOH or CoQ10 synthesis via psy RNA interference, which led to weakened carotenoid synthesis. The improved flux that was originally involved in CoQ10 production and phytoene synthesis was redirected toward FOH synthesis via metabolic modification. This is the first reported instance of FOH and CoQ10 co-production in R. sphaeroides using a metabolic engineering strategy. Electronic supplementary material The online version of this article (10.1186/s12934-019-1145-6) contains supplementary material, which is available to authorized users.

Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) β-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of β-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of β-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between β-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures.

Banana is one of the most important fruit crops consumed globally owing to its high nutritional value. Previously, we demonstrated that the ripe pulp of the banana cultivar (cv.) Nendran (AAB) contained a high amount of pro-vitamin A carotenoids. However, the molecular factors involved in the ripening process in Nendran fruit are unexplored. Hence, we commenced a transcriptome study by using the Illumina HiSeq 2500 at two stages i.e. unripe and ripe fruit-pulp of Nendran. Overall, 3474 up and 4727 down-regulated genes were obtained. A large number of identified transcripts were related to genes involved in ripening, cell wall degradation and aroma formation. Gene ontology analysis highlighted differentially expressed genes that play a key role in various pathways. These pathways were mainly linked to cellular, molecular and biological processes. The present transcriptome study also reveals a crucial role of up-regulated carotenoid biosynthesis pathway genes namely, lycopene beta cyclase and geranylgeranyl pyrophosphate synthase at the ripening stage. Genes related to the ripening and other processes like aroma and flavor were highly expressed in the ripe pulp. Expression of numerous transcription factor family genes was also identified. This study lays a path towards understanding the ripening, carotenoid accumulation and other related processes in banana.

β-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, β-carotene accumulation is governed by the Orange gene (CmOr) golden single-nucleotide polymorphism (SNP) through a yet to be discovered mechanism. In Arabidopsis (Arabidopsis thaliana), OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here, we identified a CmOr nonsense mutation (Cmor-lowβ) that lowered fruit β-carotene levels with impaired chromoplast biogenesis. Cmor-lowβ exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the golden SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblot analyses of selected melon genotypes and by using chemical pathway inhibitors. The high β-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of β-carotene. This was revealed by genetic studies with double mutants including carotenoid isomerase (yofi), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The yofi F2 segregants accumulated prolycopene independently of the golden SNP Moreover, Cmor-lowβ was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 d after anthesis until ripening, suggesting that CmOr regulates the chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels to maintain carotenoid biosynthesis metabolic flux but that the mechanism of the CmOr golden SNP involves an inhibited metabolism downstream of β-carotene to dramatically affect both carotenoid content and plastid fate.

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.

InNostocPCC 7120, two different ketolases, CrtW and CrtO are involved in the formation of keto carotenoids from β-carotene. In contrast to other cyanobacteria, CrtW catalyzes the formation of monoketo echinenone whereas CrtO is the only enzyme for the synthesis of diketo canthaxanthin. This is the major photo protective carotenoid in this cyanobacterium. Under high-light conditions, basic canthaxanthin formation was transcriptionally up-regulated. Upon transfer to high light, the transcript levels of all investigated carotenogenic genes including those coding for phytoene synthase, phytoene desaturase and both ketolases were increased. These transcription changes proceeded via binding of the transcription factor NtcA to the promoter regions of the carotenogenic genes. The binding was absolutely dependent on the presence of reductants and oxo-glutarate. Light-stimulated transcript formation was inhibited by DCMU. Therefore, photosynthetic electron transport is proposed as the sensor for high-light and a changing redox state as a signal for NtcA binding.

Tomato (Solanum lycopersicum L.) is one of the model plant to study carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes, viz. IIHR-249-1 and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1 (19.45 mg/100 g fresh weight) compared to IIHR-2866 (1.88 mg/100 g fresh weight) at the ripe stage. qPCR was performed for genes that are involved in the carotenoid biosynthetic pathway to study the difference in lycopene content in fruits of both the genotypes. The expression of Phytoene synthase (PSY) increased by 36-fold and Phytoene desaturase (PDS) increased by 14-fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta cyclase (CYC-B) decreased gradually from the initial stage to the ripe stage in IIHR-249-1. IIHR 249-1 showed 3- and 1.8-fold decrease in gene expression for Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analysed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) in high and low lycopene lines from F2 progenies also showed the decrease in transcript levels of both the genes in high lycopene F2 lines. We wish to suggest that the differential expression of lycopene beta-cyclases can be used in marker-assisted breeding.

Objective To investigate the relationship between geranylgeranyl pyrophosphate synthase (GGPPS) gene polymorphisms and bone response to alendronate in Chinese osteoporotic women.Methods A total of 639 postmenopausal women with osteoporosis or osteopenia were included and randomly received treatment of low dose (70 mg per two weeks) or standard dose (70 mg weekly) of alendronate for one year. The six tag single nucleotide polymorphisms of GGPPS gene were identified. Bone mineral density (BMD), serum cross-linked C-telopeptide of type I collagen (β-CTX), and total alkaline phosphatase (ALP) were measured before and after treatment. GGPPS gene polymorphisms and the changes of BMD and bone turnover markers after treatment were analyzed.Results rs10925503 polymorphism of GGPPS gene was correlated to serum β-CTX levels at baseline, and patients with TT genotype had significantly higher serum β-CTX level than those with TC or CC genotype (all P0.05). No correlation was found between polymorphisms of GGPPS gene and serum total ALP levels, as well as BMD at baseline. After 12 months of treatment, lumbar spine and hip BMD increased and serum bone turnover markers decreased significantly (P0.01), and without obvious differences between the low dose and standard dose groups (all P0.05). However, GGPPS gene polymorphisms were uncorrelated to percentage changes of BMD, serum total ALP, and β-CTX levels (all P0.05).Conclusion GGPPS gene polymorphisms are correlated to osteoclasts activity, but all tag single nucleotide polymorphisms of GGPPS gene have no influence on the skeletal response to alendronate treatment.

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l(-1) compared to 21 g ± 0.4 g l(-1) in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C

Background A major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels. Results Total carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24–72 h was only 14–17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose. Conclusion We conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

Red-peeled huyou has a distinct red peel color due mainly to the presence of red apocarotenoid β-citraurin as well as the increase in amount of total carotenoids. The expression level of carotenoid cleavage dioxygenase 4b1 (CCD4b1) accounted for 99.0% of total transcript abundance of CCD4s in red-peeled huyou peel and was nearly 100 times higher than that in ordinary huyou. β-Citraurin accumulation and peel coloration was mostly favored at 15 °C but strongly inhibited at moderately high temperatures 20 °C and 25 °C. Exogenous ethylene application for 3 d had no obvious effect on β-citraurin accumulation in red-peeled huyou but holding fruit at moderately higher temperatures (20 °C and 25 °C) for 3 d had a significant adverse effect on β-citraurin accumulation. The expression of phytoene synthase 1 (PSY1) and CCD4b1 was higher at 10 °C and 15 °C and significantly lower at 20 °C and 25 °C. The mechanisms governing the accumulation of β-citraurin are discussed.

The green microalga, Tetraselmis suecica, is commonly used in scientific, industrial, and aquacultural purposes because of its high stress tolerance and ease of culture in wide spectrums of environments. We hypothesized that carotenoids help to protect Tetraselmis cells from environmental stress by regulating genes in biosynthetic pathways. Here, we determined three major carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), and β-lycopene cyclase (LCY-B) in T. suecica, and examined the physiological parameters and gene expression responses when exposed to redox-active metals and non-redox-active metals. Phylogenetic analyses of each gene indicated that T. suecica clustered well with other green algae. Real-time PCR analysis showed that TsPSY, TsPDS, and TsLCY-B genes greatly responded to the redox-active metals in CuSO