Carotenoid Cleavage Oxygenases Research Articles

NinaB and BCO Collaboratively Participate in the β-Carotene Catabolism in Crustaceans: A Case Study on Chinese Mitten Crab Eriocheir sinensis.

Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and β, β-carotene 15, 15'-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro β-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with β-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its β-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the β-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically β-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid.

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.

Genome wide analysis of carotenoid cleavage oxygenases (CCO) gene family in Arachis hypogaea (peanut) under biotic stress

Carotenoid cleavage oxygenases (CCOs) enzymes play a vital role in plant growth and development through the synthesis of apocarotenoids and their derivative. These chemicals are necessary for flower and fruit coloration, as well as the manufacture of plant hormones such as abscisic acid (ABA) and strigolactones, which control a variety of physiological processes. The CCOs gene family has not been characterized in Arachis hypogaea. Genome mining of A. hypogaea identifies 24 AhCCO gene members. The AhCCO gene family was divided into two subgroups based on the recent study of the Arabidopsis thaliana CCO gene family classification system. Twenty-three AhCCO genes, constituting 95.8% of the total, were regulated by 29 miRNAs, underscoring the significance of microRNAs (miRNAs) in governing gene expression in peanuts. AhCCD19 is the only gene that lacks a miRNA target site. The physicochemical characteristics of CCO genes and their molecular weights and isoelectric points were studied further. The genes were then characterized regarding chromosomal distribution, structure, and promoter cis-elements. Light, stress development, drought stress, and hormone responsiveness were discovered to be associated with AhCCO genes, which can be utilized in developing more resilient crops. The investigation also showed the cellular location of the encoded proteins and discovered that the peanut carotenoid oxygenase gene family’s expansion was most likely the result of tandem, segmental, and whole-genome duplication events. The localization expresses the abundance of genes mostly in the cytoplasm and chloroplast. Expression analysis shows that AhCCD7 and AhCCD14 genes show the maximum expression in the apical meristem, lateral leaf, and pentafoliate leaf development, while AhNCED9 and AhNCED13 express in response to Aspergillus flavus resistance. This knowledge throws light on the evolutionary history of the AhCCO gene family and may help researchers better understand the molecular processes behind gene duplication occurrences in plants. An integrated synteny study was used to find orthologous carotenoid oxygenase genes in A. hypogaea, whereas Arabidopsis thaliana and Beta vulgaris were used as references for the functional characterization of peanut CCO genes. These studies provide a foundation for future research on the regulation and functions of this gene family. This information provides valuable insights into the genetic regulation of AhCCO genes. This technology could create molecular markers for breeding programs to develop new peanut lines.

Functional Analysis of β-Carotene Oxygenase 2 (BCO2) Gene in Yesso Scallop (Patinopecten yessoensis).

Carotenoids are essential nutrients for humans and animals, and carotenoid coloration represents an important meat quality parameter for many farmed animals. Increasingly, studies have demonstrated that vertebrate carotenoid cleavage oxygenases (CCOs) are essential enzymes in carotenoid metabolism and are therefore potential candidate genes for improving carotenoid deposition. However, our understanding of carotenoid bioavailability and CCOs functions in invertebrates, particularly marine species, is currently quite limited. We previously identified that a CCO homolog, PyBCO-like 1, was the causal gene for carotenoid coloration in the 'Haida golden scallop', a variety of Yesso scallop (Patinopecten yessoensis) characterized by carotenoid enrichment. Here, we found that another CCO-encoding gene named PyBCO2 (β-carotene oxygenase 2) was widely expressed in P. yessoensis organs/tissues, with the highest expression in striated muscle. Inhibiting BCO2 expression in P. yessoensis through RNA interference led to increased carotenoid (pectenolone and pectenoxanthin) deposition in the striated muscle, and the color of the striated muscle changed from white to light orange. Our results indicate that PyBCO2 might be a candidate gene used for improving carotenoid content in normal Yesso scallops, and also in 'Haida golden scallops'.

Genome-wide identification and in-silico expression analysis of CCO gene family in sunflower (Helianthus annnus) against abiotic stress

Carotenoid cleavage oxygenases (CCOs) enzymes play an important role in plant growth and development by producing a wide array of apocarotenoids and their derivatives. These compounds are vital for colouring flowers and fruits and synthesizing plant hormones such as abscisic acid and strigolactones. Despite their importance, the gene family responsible for CCO enzymes in sunflowers has not been identified. In this study, we identify the CCO genes of the sunflower plant to fill this knowledge gap. Phylogenetic and synteny analysis indicated that the Helianthus annnus CCO (HaCCO) genes were conserved in different plant species and they could be divided into three subgroups based on their conserved domains. Analysis using MEME tool and multiple sequence alignment identified conserved motifs in the HaCCO gene sequence. Cis-regulatory elements (CREs) analysis of the HaCCO genes indicated the presence of various responsive elements related to plant hormones, development, and responses to both biotic and abiotic stresses. This implies that these genes may respond to plant hormones, developmental cues, and drought stress, offering potential applications in the development of more resistant crops. Genes belonging to the 9-cis-epoxy carotenoid dioxygenases (NCED) subgroups predominantly exhibited chloroplast localization, whereas the genes found in other groups are primarily localized in the cytoplasm. These 21 identified HaCCOs were regulated by 60 miRNAs, indicating the crucial role of microRNAs in gene regulation in sunflowers. Gene expression analysis under drought stress revealed significant up-regulation of HaNCED16 and HaNCED19, genes that are pivotal in ABA hormone biosynthesis. During organ-specific gene expression analysis, HaCCD12 and HaCCD20 genes exhibit higher activity in leaves, indicating a potential role in leaf pigmentation. This study provides a foundation for future research on the regulation and functions of the CCO gene family in sunflower and beyond. There is potential for developing molecular markers that could be employed in breeding programs to create new sunflower lines resistant to biotic and abiotic stresses.

Genome-wide identification of CCO gene family in cucumber (Cucumis sativus) and its comparative analysis with A. thaliana

Carotenoid cleavage oxygenase (CCO) is an enzyme capable of converting carotenoids into volatile, aromatic compounds and it plays an important role in the production of two significant plant hormones, i.e., abscisic acid (ABA) and strigolactone (SL). The cucumber plant genome has not been mined for genomewide identification of the CCO gene family. In the present study, we conducted a comprehensive genome-wide analysis to identify and thoroughly examine the CCO gene family within the genomic sequence of Cucumis sativus L. A Total of 10 CCO genes were identified and mostly localized in the cytoplasm and chloroplast. The CCO gene is divided into seven subfamilies i.e. 3 NCED, 3 CCD, and 1 CCD-like (CCDL) subfamily according to phylogenetic analysis. Cis-regulatory elements (CREs) analysis revealed the elements associated with growth and development as well as reactions to phytohormonal, biotic, and abiotic stress conditions. CCOs were involved in a variety of physiological and metabolic processes, according to Gene Ontology annotation. Additionally, 10 CCO genes were regulated by 84 miRNA. The CsCCO genes had substantial purifying selection acting upon them, according to the synteny block. In addition, RNAseq analysis indicated that CsCCO genes were expressed in response to phloem transportation and treatment of chitosan oligosaccharides. CsCCD7 and CsNCED2 showed the highest gene expression in response to the exogenous application of chitosan oligosaccharides to improve cold stress in cucumbers. We also found that these genes CsCCD4a and CsCCDL-a showed the highest expression in different plant organs with respect to phloem content. The cucumber CCO gene family was the subject of the first genome-wide report in this study, which may help us better understand cucumber CCO proteins and lay the groundwork for the gene family's future cloning and functional investigations.

Genome-wide analysis of carotenoid cleavage oxygenases and identification of ripening-associated DzNCED5a in durian (Durio zibethinus) fruit

Durian (Durio zibethinus L.), popularly known as the “King of fruits,” holds significant economic importance in Southeast Asia, including Thailand. During its ripening process, the phytohormone abscisic acid (ABA) content has been reported to increase. However, a comprehensive understanding of ABA's specific role in durian fruit ripening remains elusive. Furthermore, little is known about the molecular aspects of the carotenoid cleavage pathway in this iconic fruit. Therefore, we performed genome-wide identification of the carotenoid cleavage oxygenase (CCO) family in durian. This family includes the nine-cis-epoxycarotenoid dioxygenases (NCEDs) responsible for ABA production and the carotenoid cleavage dioxygenases exhibiting diverse substrate specificities. Through phylogenetic analysis, we classified 14 CCOs in durian into 8 distinct subfamilies. Notably, each DzCCO subfamily displayed a conserved motif composition. Cis-acting element prediction showed that cis-elements related to plant hormones and environmental stress responses were distributed in the DzCCO promoter. In addition, transcriptome analysis was performed to examine the expression pattern during the fruit development and ripening stages. Interestingly, DzNCED5a, a ripening-associated gene, exhibited the highest expression level at the ripe stage, outperforming other CCOs. Its expression markedly correlated with increased ABA contents during the ripening stages of both the “Monthong” variety and other durian cultivars. Transiently expressed DzNCED5a in Nicotiana benthamiana leaves confirmed its function in ABA biosynthesis. These findings highlight the involvement of DzNCED5a in ABA production and its potential importance in durian fruit ripening. Overall, this study provides insights into the significance of CCOs in durian fruit ripening.

Evolution and expression analysis of carotenoid cleavage oxygenase gene family in Chinese mitten crab Eriocheir sinensis

Carotenoid cleavage oxygenase (CCO) plays a pivotal role in various biological activities, including antioxidant and immune functions in animals. This paper investigates the evolution and expression of CCO genes based on three chordates and 27 arthropods. Aquatic animals exhibit a higher abundance of CCO genes. Despite this, research on CCO in crustaceans has been notably limited, with a complete absence of any previous studies on the CCO genes for the Chinese mitten crab (Eriocheir sinensis). In this study, six CCO genes were identified in the E. sinensis genome database. Results reveal that the evolution of the CCO gene family in Crustacea is primarily characterized by purifying selection, with a preference for employing similar codons. EsCCO1 and EsCCO3 were mainly expressed in the epidermal layer, and EsCCO4 was mainly expressed in the hindgut. Meanwhile, EsCCO5 and EsCCO6 were mainly expressed in the hepatopancreas and endometrium. A notable detail that different EsCCO genes demonstrate distinct expression patterns within various tissues of E. sinensis. The findings of this study offer fundamental insights that could serve as a basis for further exploration into the functions and regulatory mechanisms of CCO genes in crustacean species.

Genome-wide identification and in-silico expression analysis of carotenoid cleavage oxygenases gene family in Oryza sativa (rice) in response to abiotic stress.

Rice constitutes a foundational cereal and plays a vital role in the culinary sector. However, the detriments of abiotic stress on rice quality and productivity are noteworthy. Carotenoid cleavage oxygenases (CCO) hold vital importance as they enable the particular breakdown of carotenoids and significantly contribute towards the growth and response to abiotic stress in rice. Due to the insufficient information regarding rice CCOs and their potential role in abiotic stress, their utilization in stress-resistant genetic breeding remains limited. The current research identified 16 CCO genes within the Oryza sativa japonica group. These OsCCO genes can be bifurcated into three categories based on their conserved sequences: NCEDs (9-Cis-epoxycarotenoid dioxygenases), CCDs (Carotenoid cleavage dioxygenases) and CCD-like (Carotenoid cleavage dioxygenases-like). Conserved motifs were found in the OsCCO gene sequence via MEME analysis and multiple sequence alignment. Stress-related cis-elements were detected in the promoter regions of OsCCOs genes, indicating their involvement in stress response. Additionally, the promoters of these genes had various components related to plant light, development, and hormone responsiveness, suggesting they may be responsive to plant hormones and involved in developmental processes. MicroRNAs play a pivotal role in the regulation of these 16 genes, underscoring their significance in rice gene regulation. Transcriptome data analysis suggests a tissue-specific expression pattern for rice CCOs. Only OsNCED6 and OsNCED10 significantly up-regulated during salt stress, as per RNA seq analyses. CCD7 and CCD8 levels were also higher in the CCD group during the inflorescence growth stage. This provides insight into the function of rice CCOs in abiotic stress response and identifies possible genes that could be beneficial for stress-resistant breeding.

In vitro effects of astaxanthin on bacterial and cell viability, cell migration and mitochondrial activities in four fish cell lines

Astaxanthin (AX) is a pigment commonly used in aquaculture. It is added to salmonid, shrimp, and crayfish feeds to produce the appropriate pink coloration to the flesh of these animals. Different in vitro effects of AX were studied to understand its mode of action in fish. The antioxidant activity and the potential bactericidal effects of AX on three fish pathogenic bacteria (Vibrio harveyi, V anguillarum and Photobacterium damselae) were determined. After 24 h of incubation with AX, no significant effects on the viability of V. harveyi were observed, while the viability of V. anguillarum and P. damselae was increased and decreased, respectively. These results are discussed according to the presence or absence of bacterial carotenoid cleavage oxygenases. Fish cell lines (SAF-1, DLB-1, FuB-1 and PLHC-1) were cultured and incubated for 24 h with different concentrations of AX (0, control, 0.1, 1 and 10 µM) and then cell viability was assessed. No significant effects or increased viability were observed in cells incubated with AX. To determine the effects of AX on cell migration, the scratch assay was performed. While no significant differences were detected when using SAF-1 or PLHC-1 cells, the inclusion of AX, at any of the concentrations tested, significantly (p < 0.05) increased the forward velocity in DLB-1 and FuB-1 cells. In addition, several parameters of mitochondrial metabolism were evaluated using the Agilent Seahorse XF mitochondrial stress assay kit. Different results were obtained depending on the cell line used and the mitochondrial parameter assayed but, in general, no significant effects or increases were detected after incubation of cells with AX. In conclusion, the results revealed that the effects of AX depend on the bacterium and cell line used in the assays and these findings improve our understanding of the mode of action of AX in fish and its potential applications in aquaculture.

Characterization of Carotenoid Cleavage Oxygenase Genes in Cerasus humilis and Functional Analysis of ChCCD1.

Carotenoid cleavage oxygenases (CCOs) are key enzymes that function in degrading carotenoids into a variety of apocarotenoids and some other compounds. In this study, we performed genome-wide identification and characterization analysis of CCO genes in Cerasus humilis. Totally, nine CCO genes could be classified into six subfamilies, including carotenoid cleavage dioxygenase 1 (CCD1), CCD4, CCD7, CCD8, CCD-like and nine-cis-epoxycarotenoid dioxygenase (NCED), were identified. Results of gene expression analysis showed that ChCCOs exhibited diverse expression patterns in different organs and in fruits at different ripening stages. To investigate the roles of ChCCOs in carotenoids degradation, enzyme assays of the ChCCD1 and ChCCD4 were performed in Escerichia coli BL21(DE3) that can accumulate lycopene, β-carotene and zeaxanthin. The prokaryotic expressed ChCCD1 resulted in obvious degradation of lycopene, β-carotene and zeaxanthin, but ChCCD4 did not show similar functions. To further determine the cleaved volatile apocarotenoids of these two proteins, headspace gas chromatography/mass spectrometer analysis was performed. Results showed that ChCCD1 could cleave lycopene at 5, 6 and 5', 6' positions to produce 6-methy-5-hepten-2-one and could catalyze β-carotene at 9, 10 and 9', 10' positions to generate β-ionone. Our study will be helpful for clarifying the roles of CCO genes especially ChCCD1 in regulating carotenoid degradation and apocarotenoid production in C. humilis.

Genome-Wide Identification and Expression Analysis of CCO Gene Family in Liriodendron chinense.

Carotenoid cleavage oxygenase (CCO) is an enzyme that can catalyze carotenoids to volatile aromatic substances and participate in the biosynthesis of two important phytohormones, i.e., abscisic acid (ABA) and strigolactone (SL). However, the genome-wide identification and analysis of the CCO gene family in the rare and endangered woody plant Liriodendron chinense has not been reported. Here, we performed a genome-wide analysis of the CCO gene family in the L. chinense genome and examined its expression pattern during different developmental processes and in response to various abiotic stresses. A total of 10 LcCCO genes were identified and divided into 6 subfamilies according to the phylogenetic analysis. Subcellular localization prediction showed that most of the LcCCO proteins were located in the cytoplasm. Gene replication analysis showed that segmental and tandem duplication contributed to the expansion of this gene family in the L. chinense genome. Cis-element prediction showed that cis-elements related to plant hormones, stress and light response were widely distributed in the promoter regions of LcCCO genes. Gene expression profile analysis showed that LcNCED3b was extensively involved in somatic embryogenesis, especially the somatic embryo maturation, as well as in response to heat and cold stress in leaves. Furthermore, qRT-PCR analysis showed that LcNCED3b obviously responded to drought stress in roots and leaves. This study provides a comprehensive overview of the LcCCO gene family and a potential gene target for the optimization of the somatic embryogenesis system and resistance breeding in the valuable forest tree L. chinense.

Identification and Expression Pattern of the Carotenoid Cleavage Oxygenase Gene Family in Lycium Suggest CCOs Respond to Abiotic Stress and Promote Carotenoids Degradation

Carotenoids are key metabolites in goji (Lycium), a traditional Chinese medicine plant; however, the carotenoid content varies in fruits of different goji species, and the mechanism of this variation is not clear. Carotenoids participate in signal transduction and photosynthesis, and function as colorants and photoprotectors. Members of the carotenoid cleavage oxygenase (CCO) gene family are involved in the regulation of phytohormones, pigments, and aromatic substances, such as abscisic acid (ABA), β-carotenoid, and α-ionone, by degrading carotenoids. Some CCO genes are also related to an abiotic stress response. Here, a total of 12 LbCCO genes were identified and analyzed from the L. barbarum genome. CCO genes were divided into six subfamilies based on the constructed phylogenetic tree, including LbNCEDs, LbCCD1, LbCCD3, LbCCD4, LbCCD7, and LbCCD8. Among them, CCD3 was reported for the first time. The gene structure and motif analysis revealed the conservation of CCO subfamilies. Pseudogene generation and the importance of each subfamily in CCOs were revealed by collinearity analysis. The spatiotemporal transcriptomes of L. barbarum and L. ruthenicum were compared, suggesting that CCD4-1 may dominate carotenoid degradation in goji fruits. Cis-acting elements prediction and environment responsive gene expression analyses indicated that salt-alkali stress and photothermal conditions might influence the expression of CCOs in goji. The results of this study enhance our understanding of the carotenoid degradation pathway, and the functions and responses of CCOs in goji species.

Identification and expression of the CCO family during development, ripening and stress response in banana.

Plant hormone abscisic acid (ABA) plays an important role in plant growth, development and response to biotic / abiotic stressors. Thus, it is necessary to investigate the crucial genes associated with ABA synthesis. Currently, the carotenoid cleavage oxygenases (CCOs) family that function as the key step for ABA synthesis are not well understood in banana. In this study, 13 MaCCO genes and 12 MbCCO genes, divided into NCED subgroup and CCD subgroup, were identified from the banana genome, and their evolutionary relationship, protein motifs, and gene structures were also determined. Transcriptomic analysis suggested the involvement of CCO genes in banana development, ripening, and response to abiotic and biotic stressors, and homologous gene pairs showed homoeologue expression bias in the A or B subgenome. Our results identified MaNCED3A, MaCCD1, and MbNCED3B as the genes with the highest expression during fruit development and ripening. MaNCED5 / MbNCED5 and MaNCED9A might respond to abiotic stress, and MaNCED3A, 3B, 6A, 9A, and MbNCED9A showed transcriptional changes that could be a response to Foc4 infection. These findings may contribute to the characterization of key enzymes involved in ABA biosynthesis, as well as to identify potential targets for the genetic improvement of banana.

Engineering and linker-mediated co-immobilization of carotenoid cleavage oxygenase with phenolic acid decarboxylase for efficiently converting ferulic acid into vanillin

The artificial cascade system with a coenzyme-independent decarboxylase and oxygenase was a very attractive approach for bioconversion of ferulic acid into vanillin. However, its potential was impeded by the low activity and thermostability of oxygenase. In this study, a combined strategy including the protein engineering of a carotenoid cleavage oxygenase from Thielavia terrestris (TtCCO) and its linker-mediated co-immobilization with a decarboxylase from Bacillus atrophaeus (BaPAD) was explored with aims to improve the catalytic efficiency and robustness of this artificial cascade system. We proved that engineering the enzyme by inserting an extra short peptide between E450 to E476 greatly affected TtCCO substrate specificity, catalytic activity, and thermostability. Among three variants, TtCCO2 displayed better thermostability and had 1.4-, 55- and 1.3-fold higher activity than TtCCO on 4-vinylguaiacol, resveratrol, and isoeugenol, respectively. The excellent protein and enzyme activity yield of immobilization was achieved by linker-mediated immobilization of 4lp-TtCCO2 @ mesoporous Y zeolite. A 100% conversion rate of ferulic acid and a 55% of the molar conversion yield of vanillin were obtained by co-immobilized 4lp-TtCCO2/4lp-BaPAD@mesoporous Y zeolite. This work demonstrated an effective engineering strategy for more efficient and robust oxygenases and the potential of this bi-enzyme cascade system for converting ferulic acid into vanillin.

Genome-wide identification and expression analysis of carotenoid cleavage oxygenase genes in Litchi (Litchi chinensis Sonn.)

BackgroundCarotenoid cleavage oxygenases (CCOs) include the carotenoid cleavage dioxygenase (CCD) and 9-cis-epoxycarotenoid (NCED), which can catalize carotenoid to form various apocarotenoids and their derivatives, has been found that play important role in the plant world. But little information of CCO gene family has been reported in litchi (Litchi chinensis Sonn.) till date.ResultsIn this study, a total of 15 LcCCO genes in litchi were identified based on genome wide lever. Phylogeny analysis showed that LcCCO genes could be classified into six subfamilies (CCD1, CCD4, CCD7, CCD8, CCD-like, and NCED), which gene structure, domain and motifs exhibited similar distribution patterns in the same subfamilies. MiRNA target site prediction found that there were 32 miRNA target sites in 13 (86.7%) LcCCO genes. Cis-elements analysis showed that the largest groups of elements were light response related, following was plant hormones, stress and plant development related. Expression pattern analysis revealed that LcCCD4, LcNCED1, and LcNCED2 might be involving with peel coloration, LcCCDlike-b might be an important factor deciding fruit flavor, LcNCED2 and LcNCED3 might be related to flower control, LcNCED1 and LcNCED2 might function in fruitlet abscission, LcCCD4a1, LcCCD4a2, LcCCD1, LcCCD4, LcNCED1, and LcNCED2 might participate in postharvest storage of litchi.ConclusionHerein, Genome-wide analysis of the LcCCO genes was conducted in litchi to investigate their structure features and potential functions. These valuable and expectable information of LcCCO genes supplying in this study will offer further more possibility to promote quality improvement and breeding of litchi and further function investigation of this gene family in plant.

A nuclear receptor heterodimer, CgPPAR2-CgRXR, acts as a regulator of carotenoid metabolism in Crassostrea gigas

Nuclear receptors (NRs) are mostly ligand-activated transcription factors in animals and play essential roles in metabolism and homeostasis. The NR heterodimer composed of PPAR/RXR (peroxisome proliferator-activated receptor/retinoidXreceptor) is considered a key regulator of lipid metabolism in vertebrate. However, in molluscs, how this heterodimer is involved in carotenoid metabolism remains unclear. To elucidate how this heterodimer regulates carotenoid metabolism, we identified a PPAR gene in C. gigas, designated as CgPPAR2 (LOC105323212), and functionally characterized it using two-hybrid and reporter systems. CgPPAR2 is a direct orthologue of vertebrate PPARs and the second PPAR gene identified in C. gigas genome in addition to CgPPAR1 (LOC105317849). The results demonstrated that CgPPAR2 protein can form heterodimer with C. gigas RXR (CgRXR), and then regulate carotenoid metabolism by controlling carotenoid cleavage oxygenases with different carotenoid cleavage efficiencies. This regulation can be affected by retinoid ligands, i.e., carotenoid derivatives, validating a negative feedback regulation mechanism of carotenoid cleavage for retinoid production. Besides, organotins may disrupt this regulatory process through the mediation of CgPPAR2/CgRXR heterodimer. This is the first report of PPAR/RXR heterodimer regulating carotenoid metabolism in mollusks, contributing to a better understanding of the evolution and conservation of this nuclear receptor heterodimer.

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases.

Here, the choice of the first coordination shell of the metal center is analyzed from the perspective of charge maintenance in a binary enzyme-substrate complex and an O2-bound ternary complex in the nonheme iron oxygenases. Comparing homogentisate 1,2-dioxygenase and gentisate dioxygenase highlights the significance of charge maintenance after substrate binding as an important factor that drives the reaction coordinate. We then extend the charge analysis to several common types of nonheme iron oxygenases containing either a 2-His-1-carboxylate facial triad or a 3-His or 4-His ligand motif, including extradiol and intradiol ring-cleavage dioxygenases, thiol dioxygenases, α-ketoglutarate-dependent oxygenases, and carotenoid cleavage oxygenases. After forming the productive enzyme-substrate complex, the overall charge of the iron complex at the 0, +1, or +2 state is maintained in the remaining catalytic steps. Hence, maintaining a constant charge is crucial to promote the reaction of the iron center beginning from the formation of the Michaelis or ternary complex. The charge compensation to the iron ion is tuned not only by protein-derived carboxylate ligands but also by substrates. Overall, these analyses indicate that charge maintenance at the iron center is significant when all the necessary components form a productive complex. This charge maintenance concept may apply to most oxygen-activating metalloenzymes systems that do not draw electrons and protons step-by-step from a separate reactant, such as NADH, via a reductase. The charge maintenance perception may also be useful in proposing catalytic pathways or designing prototypical reactions using artificial or engineered enzymes for biotechnological applications.

Evolutionary Origin of the Carotenoid Cleavage Oxygenase Family in Plants and Expression of Pepper Genes in Response to Abiotic Stresses.

Plant carotenoid cleavage oxygenase (CCO) is an enzyme that catalyzes the synthesis of carotenoids and participates in many important physiological functions. The plant CCOs exist in two forms, namely carotenoid cleavage dioxygenase (CCD) and nine-cis epoxide carotenoid dioxygenase (NCED). Although studies have shown that this gene family has been identified in many species, such as Arabidopsis, grape, and tomato, the evolutionary origin of the CCO family and the expression pattern of pepper genes in response to H2O2 and other abiotic stresses are still unclear. In this study, we used the bioinformatics method to identify and analyze the members of the CCO gene family from pepper and other 13 plants from lower to higher plant species based on the whole genome sequence. A total of 158 CCO genes were identified in different plant species and further divided into two groups (e.g., groups I and II). The former was subdivided into CCD7 and CCD8 and have independent evolutionary origins, respectively, while the latter was subdivided into CCD1, CCD4, CCD-like, and NCED, which may have come from a common ancestor. In addition, the results of RNA-seq showed that the expression patterns of pepper CaCCO genes were different in the tissues tested, and only few genes were expressed at high levels such as CaCCD1a, CaCCD4a, CaNCED3, and CaCCD1b. For hydrogen peroxide (H2O2) and other abiotic stresses, such as plant hormones, heat, cold, drought, and NaCl treatments, induction of about half of the CaCCO genes was observed. Moreover, the expression patterns of CaCCOs were further investigated under heat, cold, drought, and NaCl treatments using quantitative real-time PCR (qRT-PCR), and most members were responsive to these stresses, especially some CaCCOs with significant expression changes were identified, such as CaCCD4c, CaCCD-like1, CaCCD8, and CaCCD1b, suggesting the important roles of CaCCOs in abiotic stress responses. All these results will provide a valuable analytical basis for understanding the evolution and functions of the CCO family in plants.

Vitamin A Transporters in Visual Function: A Mini Review on Membrane Receptors for Dietary Vitamin A Uptake, Storage, and Transport to the Eye.

Vitamins are essential compounds obtained through diet that are necessary for normal development and function in an organism. One of the most important vitamins for human physiology is vitamin A, a group of retinoid compounds and carotenoids, which generally function as a mediator for cell growth, differentiation, immunity, and embryonic development, as well as serving as a key component in the phototransduction cycle in the vertebrate retina. For humans, vitamin A is obtained through the diet, where provitamin A carotenoids such as β-carotene from plants or preformed vitamin A such as retinyl esters from animal sources are absorbed into the body via the small intestine and converted into all-trans retinol within the intestinal enterocytes. Specifically, once absorbed, carotenoids are cleaved by carotenoid cleavage oxygenases (CCOs), such as Beta-carotene 15,15’-monooxygenase (BCO1), to produce all-trans retinal that subsequently gets converted into all-trans retinol. CRBP2 bound retinol is then converted into retinyl esters (REs) by the enzyme lecithin retinol acyltransferase (LRAT) in the endoplasmic reticulum, which is then packaged into chylomicrons and sent into the bloodstream for storage in hepatic stellate cells in the liver or for functional use in peripheral tissues such as the retina. All-trans retinol also travels through the bloodstream bound to retinol binding protein 4 (RBP4), where it enters cells with the assistance of the transmembrane transporters, stimulated by retinoic acid 6 (STRA6) in peripheral tissues or retinol binding protein 4 receptor 2 (RBPR2) in systemic tissues (e.g., in the retina and the liver, respectively). Much is known about the intake, metabolism, storage, and function of vitamin A compounds, especially with regard to its impact on eye development and visual function in the retinoid cycle. However, there is much to learn about the role of vitamin A as a transcription factor in development and cell growth, as well as how peripheral cells signal hepatocytes to secrete all-trans retinol into the blood for peripheral cell use. This article aims to review literature regarding the major known pathways of vitamin A intake from dietary sources into hepatocytes, vitamin A excretion by hepatocytes, as well as vitamin A usage within the retinoid cycle in the RPE and retina to provide insight on future directions of novel membrane transporters for vitamin A in retinal cell physiology and visual function.