Carotenoid Cleavage Research Articles

Phylogeny of PmCCD Gene Family and Expression Analysis of Flower Coloration and Stress Response in Prunus mume.

The CCD gene family plays a crucial role in the cleavage of carotenoids, converting them into apocarotenoids. This process not only impacts the physiology and development of plants but also enhances their tolerance toward different stresses. However, the character of the PmCCD gene family and its role in ornamental woody Prunus mume remain unclear. Here, ten non-redundant PmCCD genes were identified from the P. mume genome, and their physicochemical characteristics were predicted. According to the phylogenetic tree, PmCCD proteins were classified into six subfamilies: CCD1, CCD4, CCD7, CCD8, NCED and CCD-like. The same subfamily possessed similar gene structural patterns and numbers of conserved motifs. Ten PmCCD genes were concentrated on three chromosomes. PmCCD genes exhibited interspecific collinearity with P. armeniaca and P. persica. Additionally, PmCCD genes had obvious specificity in different tissues and varieties. Compared with white-flowered 'ZLE', PmCCD1 and PmCCD4 genes were low-expressed in 'HJH' with yellow petals, which suggested PmCCD1 and PmCCD4 might be related to the formation of yellow flowers in P. mume. Nine PmCCD genes could respond to NaCl or PEG treatments. These genes might play a crucial role in salt and drought resistance in P. mume. Moreover, PmVAR3 and PmSAT3/5 interacted with PmCCD4 protein in yeast and tobacco leaf cells. This study laid a foundation for exploring the role of the PmCCD gene family in flower coloration and stress response in P. mume.

Comparison of carotenoid, chlorophyll concentrations and their biosynthetic transcript levels in different coloured cauliflower

Carotenoids and chlorophylls are among the most widely distributed pigments in nature that play essential roles in the photosynthetic apparatus and confer diverse colours in plants. Among all vegetables, cauliflower (Brassica oleracea L. ssp. var. botrytis) is rich in phytochemicals and is an important crop grown all over the world. This study investigates carotenoid and chlorophyll concentrations in differently pigmented cultivars and elucidates the role of transcriptional regulation of carotenoid accumulation including lutein and β-carotene. Here, we characterised changes in pigments by UHPLC-DAD-ToF-MS and changes in transcript levels of carotenoid metabolic genes by qRT-PCR in florets and leaves of orange (‘Jaffa’ and ‘Sunset’), purple (‘Di Sicilia Violetto’ and ‘Graffiti’), green (‘Trevi’) and white (‘Clapton’) cultivars. Transcript levels of all carotenoid metabolic genes showed different transcript level patterns in the leaves and florets. Compared to the other cultivars, the orange cultivars had the highest levels of β-carotene in the florets and lutein in the leaves resulting in changes lutein/β-carotene ratios. In the green cultivar, higher transcript levels were also found, especially for phytoene synthase and phytoene desaturase genes of the core biosynthesis pathway. However, no increased carotenoid concentrations were observed, possibly due to a higher carotenoid turnover induced by the carotenoid cleavage dioxygenase 4 in the green cultivar. In the white (‘Clapton’) and purple (‘Di Sicilia Violetto’ and ‘Graffiti’) cultivars the phytoene desaturase transcript levels as well as carotenoid concentrations were low. Chlorophyll concentrations changed in trend comparable to the carotenoid concentrations and were only significantly lower in the leaves of the orange cultivar ‘Jaffa’. Also, the chlorophyll a/b ratio changed in ‘Jaffa’. In florets the highest chlorophylls concentrations were observed for the green cultivar (‘Trevi’) and the purple cultivar (‘Di Sicilia Violetto’). Taken together, the study demonstrates the complex source-sink relationship of carotenoid accumulation in different coloured cauliflower.

Global hypermethylation of the N6-methyladenosine RNA modification associated with apple heterografting.

Grafting can facilitate better scion performance and is widely used in plants. Numerous studies have studied the involvement of mRNAs, small RNAs, and epigenetic regulations in the grafting process. However, it remains unclear whether the mRNA N6-methyladenosine (m6A) modification participates in the apple (Malus x domestica Borkh.) grafting process. Here, we decoded the landscape of m6A modification profiles in 'Golden delicious' (a cultivar, Gd) and Malus prunifolia 'Fupingqiuzi' (a unique rootstock with resistance to environmental stresses, Mp), as well as their heterografted and self-grafted plants. Interestingly, global hypermethylation of m6A occurred in both heterografted scion and rootstock compared with their self-grafting controls. Gene Ontology (GO) term enrichment analysis showed that grafting-induced differentially m6A-modified genes were mainly involved in RNA processing, epigenetic regulation, stress response, and development. Differentially m6A-modified genes harboring expression alterations were mainly involved in various stress responses and fatty acid metabolism. Furthermore, grafting-induced mobile mRNAs with m6A and gene expression alterations mainly participated in ABA synthesis and transport (e.g. carotenoid cleavage dioxygenase 1 [CCD1] and ATP-binding cassette G22 [ABCG22]) and abiotic and biotic stress responses, which might contribute to the better performance of heterografted plants. Additionally, the DNA methylome analysis also demonstrated the DNA methylation alterations during grafting. Downregulated expression of m6A methyltransferase gene MdMTA (ortholog of METTL3) in apples induced the global m6A hypomethylation and distinctly activated the expression level of DNA demethylase gene MdROS1 (REPRESSOR OF SILENCING 1) showing the possible association between m6A and 5mC methylation in apples. Our results reveal the m6A modification profiles in the apple grafting process and enhance our understanding of the m6A regulatory mechanism in plant biological processes.

Multiple loss-of-function mutations of carotenoid cleavage dioxygenase 4 reveal its major role in both carotenoid level and apocarotenoid composition in flue-cured mature tobacco leaves

Apocarotenoid volatiles contribute to the flavor of many agricultural products. In many flowering plants, carotenoid cleavage dioxygenase 4 (CCD4) is involved in the decomposition of carotenoids and resultant production of C13-apocarotenoids, such as β-ionone derived from β-carotene. To understand the possible role of tobacco CCD4 genes (NtCCD4-S, NtCCD4-T1 and NtCCD4-T2) in these processes, we analyzed loss-of-function phenotypes. RNA interference transgenic plants showed yellow color in mature (senescent) leaves. Mature leaves of chemically induced double-mutant plants showed a stronger yellow color, and those of triple-mutant plants showed a pronounced yellow color. Carotenoid analysis of the leaves from mutants showed that lutein and β-carotene increased in line with the degree of color change compared to wild type, whereas there was little change in green color in their young leaves. This result indicates that CCD4s are important for the decomposition of carotenoids in the tobacco leaf maturation process. Analysis of apocarotenoids in flue-cured leaves of the multiple-mutant plants showed that many compounds, including megastigmatrienones, were decreased in comparison to wild type, whereas intriguingly β-ionone and dihydroactinidiolide were increased. Our results suggest that CCD4s play a key role in both carotenoid level and apocarotenoid composition in flue-cured mature tobacco leaves.

Expression profile of the NCED/CCD genes in chickpea and lentil during abiotic stress reveals a positive correlation with increased plant tolerance

Carotenoid cleavage dioxygenase (CCD) gene family is organized in two subfamilies: (i) 9-cis epoxycarotenoid dioxygenase (NCED) genes and (ii) CCD genes. NCED genes are essential for catalyzing the first step of the abscisic-acid (ABA) biosynthesis, while CCD genes produce precursors of the strigolactones hormone. The functional characterization of these gene subfamilies has not been yet performed in chickpea and lentil. Herein, were identified and systematically characterized two NCED and five CCD genes in the chickpea and two NCED and six CCD genes in lentil. After in silico sequence analysis and phylogeny, the expression profile of the NCED/CCD genes was determined by meta-analysis and real-time PCR in plants under different stress conditions. Sequence data revealed that NCED/CCD genes are highly conserved between chickpea and lentil. This conservation was observed both at gene and protein sequence levels and phylogenetic relationships. Analysis of the promoter sequences revealed that all NCED/CCD genes have a considerable number of cis-regulatory elements responsive to biotic and abiotic stress. Protein sequence analysis evidenced that NCED/CCD genes share several conserved motifs and that they have a highly interconnected interaction network. Furthermore, the three-dimensional structure of these proteins was determined and indicated that some proteins have structures with considerable similarity. The meta-analysis revealed that NCED/CCD genes are dynamically modulated in different organs and under different stress conditions, but they have a positive correlation with plant tolerance. In accordance, real-time PCR data showed that both NCED and CCD genes are differentially modulated in plants under drought stress. In particular, CaNCED2, CaCCD5, LcNCED2, LcCCD1, and LcCCD2 genes have a positive correlation with improved plant tolerance to drought stress. Therefore, this study presented a detailed characterization of the chickpea and lentil NCED/CCD genes and provided new insights to improve abiotic stress tolerance in these two important crops.

Natural variations in Crocus sativus lycopene epsilon cyclase (CstLcyE) alter carotenoid/apocarotenoid content and stress tolerance

Crocus sativus is an economically and pharmaceutically important crop. The dried stigmas of C. sativus flowers constitute saffron, the only commercial source of apocarotenoids like crocin, picrocrocin, and safranal. Some elite clones of C. sativus with higher crocin content were identified from plant populations over three years. Sequencing approach demonstrated that mutations are present in CstLycE. The C. sativus apocarotenoids are formed by cleavage of β-branch carotenoids. Cyclization of lycopene is the key branch point in carotenoid/apocarotenoid pathway. While lycopene β-cyclase converts lycopene into β-carotene, combined action of β and ε-cyclases forms α-carotene. Towards this, the mutant populations with critical mutations in CstLycE result in loss of function, and hence the metabolic flux was diverted towards β-branch. In-silico analysis and site-directed mutagenesis confirmed the critical nature of these mutations. To ascertain the role of CstLycE, transient over-expression and RNAi lines of CstLcyE in C. sativus were generated, and we observed enhanced β-branch carotenoid and apocarotenoid content in RNAi lines. The effect of CstLcyB and CstLcyE was compared, and CstLcyE RNAi lines had a more profound impact on apocarotenoid accumulation than CstLcyB over-expression lines. CstLcyE-RNAi plants also accumulated more abscisic acid and were more tolerant to abiotic stresses.

The transcription factor SPL13 mediates strigolactone suppression of shoot branching by inhibiting cytokinin synthesis in Solanum lycopersicum.

Plant architecture imposes a large impact on crop yield. IDEAL PLANT ARCHITECTURE 1 (IPA1), which encodes a SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor, is a target of molecular design for improving grain yield. However, the roles of SPL transcription factors in regulating tomato (Solanum lycopersicum) plant architecture are unclear. Here, we show that the expression of SPL13 is down-regulated in the lateral buds of strigolactone (SL)-deficient ccd mutants and is induced by GR24 (a synthetic analog of SL). Knockout of SPL13 by CRISPR/Cas9 resulted in higher levels of cytokinins (CKs) and transcripts of the CK synthesis gene ISOPENTENYL TRANSFERASES 1 (IPT1) in the stem nodes, and more growth of lateral buds. GR24 suppresses CK synthesis and lateral bud growth in ccd mutants, but is not effective in spl13 mutants. On the other hand, silencing of the IPT1 gene inhibited bud growth of spl13 mutants. Interestingly, SL levels in root extracts and exudates are significantly increased in spl13 mutants. Molecular studies indicated that SPL13 directly represses the transcription of IPT1 and the SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and MORE AXILLARY GROWTH 1 (MAX1). The results demonstrate that SPL13 acts downstream of SL to suppress lateral bud growth by inhibiting CK synthesis in tomato. Tuning the expression of SPL13 is a potential approach for decreasing the number of lateral shoots in tomato.

Transcription factor VvWRKY70 inhibits both norisoprenoid and flavonol biosynthesis in grape.

Norisoprenoids and flavonols are important secondary metabolites in grape berries (Vitis vinifera L.). The former is a class of ubiquitous flavor and fragrance compounds produced by the cleavage of carotenoids, and the latter, which is derived from the flavonoid metabolic pathway, has been proposed as a general quality marker for red grapes. However, the transcriptional regulatory mechanisms underlying norisoprenoid and flavonol production are still not fully understood. In this study, we characterized a transcription factor, VvWRKY70, as a repressor of both norisoprenoid and flavonol biosynthesis in grape berries, and its expression was down-regulated by light and high-temperature treatment. Overexpressing VvWRKY70 in grape calli reduced norisoprenoid and flavonol production, particularly under light exposure or at high temperature, by repressing the expression of several related genes in the isoprenoid and flavonoid metabolic pathways. VvWRKY70 downregulated β-CAROTENE HYDROXYLASE 2 (VvBCH2) and CHALCONE SYNTHASE 3 (VvCHS3) expression based on yeast one-hybrid analysis combined with electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR. We discuss the role of VvWRKY70 in the coordinated regulatory network of isoprenoid and flavonoid metabolism. These findings provide a theoretical basis to improve flavor, color and other comprehensive qualities of fruit crops and their processing products.

CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase (CCD) Genes in Sorghum Alters Strigolactone Biosynthesis and Plant Biotic Interactions

Strigolactones are a group of small molecules that play critical roles in plant developmental processes and root biotic interactions. Strigolactones are agronomically important due to their role as a signal for the germination of a parasitic weed ( Striga spp.) that reduces yields of cereal crops worldwide. To identify the genes encoding strigolactones in sorghum and their function, we characterized two CRISPR/Cas9-mediated gene knockouts of carotenoid cleavage dioxygenase 8 ( CCD8) genes ( SbCCD8a and SbCCD8b), which have been shown in other plant species to be involved in strigolactone biosynthesis. Although strigolactones are important for the parasitization of sorghum in Africa, the functions of members of the CCD8 family have not been characterized. The impact of the knockouts on strigolactone production, plant growth and development, resistance to the parasitic weed Striga, and the root-associated microbiomes were investigated in this study. The results revealed that knockout of SbCCD8 genes in sorghum significantly reduced orobanchol production and Striga germination. Strigolactone deficiency altered the shoot and root architecture and reduced grain yield of sorghum. The knockout of the SbCCD8b gene significantly affected the rhizosphere bacterial diversity and community composition at sorghum plant grain-fill stage due to the abolition of orobanchol exudation from roots. Reduced amounts of orobanchol in root exudates also influenced root-associated fungal taxa abundance. Our findings provide new insights into potentially sustainable approaches for the recruitment of beneficial microbes and for parasitic weed control through manipulation of strigolactone production in sorghum.

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.

Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor.

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates.

Genome-wide identification of the NCED gene family and functional characterization of PavNCED5 related to bud dormancy in sweet cherry

Abscisic acid (ABA) is involved in responding to various stresses and regulating the dormancy of flower buds in fruit trees. The cleavage of carotenoids is catalyzed by 9-cis epoxy carotenoid dioxygenase (NCED), and is a key step in ABA biosynthesis. Although NCED genes have been identified in a variety of plants, the characteristics of these genes in sweet cherry (Prunus avium L.) trees have been seldom reported. In the present study, 10 PavNCED genes were identified in the sweet cherry genome and distributed on three chromosomes. Cis-element analysis of PavNCEDs indicated that they might be related to hormone regulation, stress responses, and plant development. Through co-expression network analysis, the transcription factors were identified, such as ERF, bHLH and TCP, which might be involved in the regulation of PavNCEDs. Expression pattern analysis of the PavNCED family genes showed that PavNCED1–5 genes were highly expressed during the flower bud dormancy. Importantly, the expression of PavNCED5 was decreased with the decline of ABA content in the sweet cherry. Additionally, the overexpression of PavNCED5 in Arabidopsis showed dormancy deficiency of seeds. This implies that PavNCED5 may play an important role during the dormancy stage of sweet cherry. In general, we characterized the NCED family in sweet cherry and revealed the essential role of PavNCED5 in flower bud dormancy. These findings open up future research directions and provide valuable information for the further functional study of PavNCEDs in sweet cherry.

Lycopene Accumulation in Transgenic Mice Lacking One or Both Carotenoid Cleaving Enzymes

Backgroundβ-carotene oxygenase 1 (BCO1) and β-carotene oxygenase 2 (BCO2) are responsible for the cleavage of carotenoids in mammals. Objective: The goals of this study were to (1) establish the relative contribution of each enzyme on lycopene accumulation in mice and (2) examine the role of lycopene on gene expression in the gut of wild type (WT) mice. MethodsWe utilized male and female WT, Bco1-/-, Bco2-/-, and Bco1-/-Bco2-/- double knockout (DKO) mice. We gavaged the mice with either 1 mg of lycopene resuspended in cottonseed oil or vehicle as a control group daily for 2 wk. In a second study, we evaluated the effect of dietary vitamin A on lycopene absorption and intestinal gene expression by RT-PCR. We also quantified lycopene concentration isomer distribution by high performance liquid chromatography. ResultsOf the 11 tissues measured, the liver accounted for 94 to 98% of the lycopene content across genotypes. We did not observe sex differences between genotypes, although hepatic lycopene levels in Bco1-/- mice were approximately half in comparison to the other genotypes; Bco1-/- verses Bco2-/- (P < 0.0001), DKO mice (P < 0.001), WT (ns). Analyses of mitochondrial lycopene content revealed a 3- to 5-fold enrichment compared with total hepatic content (P < 0.05) in all genotypes and sexes. In our second study, WT mice fed a vitamin A-deficient diet (VAD) accumulated greater amounts of lycopene in the liver than those fed a vitamin A-sufficient diet (VAS) (P < 0.01). These changes were accompanied by an upregulation of the vitamin A-responsive transcription factor intestine specific homeobox (ISX) in mice fed VAD + lycopene and VAS + lycopene diets compared with VAD control-fed mice (P < 0.05). ConclusionsOur data suggest that BCO2 is the primary lycopene cleavage enzyme in mice. Lycopene concentration was enriched in the mitochondria of hepatocytes independently of genotype, and lycopene stimulated vitamin A signaling in WT mice.

One-Pot Efficient Bioconversion of Crocetin from Zeaxanthin via a Dual-Enzyme System

Saffron contains pharmacologic active compounds and a large number of flavor compounds. Both originate from carotenoids by biocatalyzed oxidation. Crocetin in saffron exhibits various attractive bioactivities and could be considered in the treatment of various diseases including cancer, coronary heart disease, and hypertension. However, powerful strategies for the efficient and sustainable source of crocetin are lacking. To address this issue, we demonstrated a one-pot approach to bioconversion of crocetin from zeaxanthin via a dual-enzyme system consisting of carotenoid cleavage dioxygenase 2 (CsCCD2-M1 (R192F/S323A)) and aldehyde dehydrogenase (SynALD) with optimized protein expression and reaction system. Meanwhile, it was observed that the byproduct 3-hydroxy-β-apo-8′-carotenoic acid (3-HACA) severely accumulated due to the imbalance in the activity of the CsCCD2-M1 and SynALD. A suitable molar ratio of 6:1 and the optimized dual-enzyme reaction conditions were developed to eliminate 3-HACA completely. Consequently, the efficient biosynthesis of crocetin (1.48 mg/L/h) and an 80.8% conversion rate of crocetin within 2 h were accomplished after scaling up the optimized system by 10 000 times. Moreover, over 34% of enzyme activity was still retained in the residual enzyme system after three times of recycling. This study not only provides a sustainable means of crocetin production in vitro but also presents a new insight into the rapid construction of one-pot synthesis of high-value compounds with complex structures.

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.

Dynamic change of the carotenoid metabolic pathway profile during oolong tea processing with supplementary LED light

Carotenoid-derived volatiles are important contributors to tea aroma quality. However, the profile of the carotenoid pathway and carotenoid-derived volatiles (CDVs) artificial regulation in oolong tea processing has yet to be investigated. In the present work, the content and varieties of carotenoid-derived volatiles, the genome-wide identification of carotenoid cleavage dioxygenase (CsCCD) gene family, the expression level of CsCCD and other key genes in the carotenoid pathway, and the profile of carotenoid substances were analyzed by multi-omics and bioinformatics methods with innovative postharvest supplementary LED light during oolong tea processing. The results showed that during oolong tea processing, a total of 17 CDVs were identified. The content of β-ionone increased up to 26.07 times that of fresh leaves and its formation was significantly promoted with supplementary LED light from 0.54 μg/g to 0.83 μg/g in the third turning over treatment. A total of 11 CsCCD gene family members were identified and 119 light response cis-acting regulatory elements of CsCCD were found. However, the expression level of most genes in the carotenoid pathway including CsCCD were reduced due to mechanical stress. ‘Huangdan’ fresh tea leaves had a total of 1 430.46 μg/g 22 varieties of carotenoids, which mainly composed of lutein(78.10%), β-carotene(8.24%) and zeaxanthin(8.18%). With supplementary LED light, the content of antherxanthin and zeaxanthin in xanthophyll cycle was regulated and CDVs such as α-ionone, β-ionone, pseudoionone, damascenone, 6,10-dimethyl-5,9-undecadien-2-one, citral, geranyl acetate and α-farnesene were promoted significantly in different phases during oolong tea processing. Our results revealed the profile of the carotenoid metabolism pathway in oolong tea processing from the perspective of precursors, gene expression and products, and put forward an innovative way to improve CDVs by postharvest supplementary LED light.

Arbuscular mycorrhizal fungi-indicative blumenol-C-glucosides predict lipid accumulations and fitness in plants grown without competitors.

Hydroxy- and carboxyblumenol C-glucosides specifically accumulate in roots and leaves of plants harboring arbuscular mycorrhizal fungi (AMF). To explore blumenol function in AMF relationships, we silenced an early key-gene in blumenol biosynthesis, CCD1 (carotenoid cleavage dioxygenase 1), in the ecological model plant, Nicotiana attenuata, and analyzed whole-plant performance in comparison with control and CCaMK-silenced plants, unable to form AMF associations. Root blumenol accumulations reflected a plant's Darwinian fitness, as estimated by capsule production, and were positively correlated with AMF-specific lipid accumulations in roots, with relationships that changed as plants matured when grown without competitors. When grown with wild-type competitors, transformed plants with decreased photosynthetic capacity or increased carbon flux to roots had blumenol accumulations that predicted plant fitness and genotype trends in AMF-specific lipids, but had similar levels of AMF-specific lipids between competing plants, likely reflecting AMF-networks. We propose that when grown in isolation, blumenol accumulations reflect AMF-specific lipid allocations and plant fitness. When grown with competitors, blumenol accumulations predict fitness outcomes, but not the more complicated AMF-specific lipid accumulations. RNA-seq analysis provided candidates for the final biosynthetic steps of these AMF-indicative blumenol C-glucosides; abrogation of these steps will provide valuable tools for understanding blumenol function in this context-dependent mutualism.

Integrated metabolic and transcriptional analysis reveals the role of carotenoid cleavage dioxygenase 4 (IbCCD4) in carotenoid accumulation in sweetpotato tuberous roots

BackgroundPlant carotenoids are essential for human health, having wide uses in dietary supplements, food colorants, animal feed additives, and cosmetics. With the increasing demand for natural carotenoids, plant carotenoids have gained great interest in both academic and industry research worldwide. Orange-fleshed sweetpotato (Ipomoea batatas) enriched with carotenoids is an ideal feedstock for producing natural carotenoids. However, limited information is available regarding the molecular mechanism responsible for carotenoid metabolism in sweetpotato tuberous roots.ResultsIn this study, metabolic profiling of carotenoids and gene expression analysis were conducted at six tuberous root developmental stages of three sweetpotato varieties with different flesh colors. The correlations between the expression of carotenoid metabolic genes and carotenoid levels suggested that the carotenoid cleavage dioxygenase 4 (IbCCD4) and 9-cis-epoxycarotenoid cleavage dioxygenases 3 (IbNCED3) play important roles in the regulation of carotenoid contents in sweetpotato. Transgenic experiments confirmed that the total carotenoid content decreased in the tuberous roots of IbCCD4-overexpressing sweetpotato. In addition, IbCCD4 may be regulated by two stress-related transcription factors, IbWRKY20 and IbCBF2, implying that the carotenoid accumulation in sweeetpotato is possibly fine-tuned in responses to stress signals.ConclusionsA set of key genes were revealed to be responsible for carotenoid accumulation in sweetpotato, with IbCCD4 acts as a crucial player. Our findings provided new insights into carotenoid metabolism in sweetpotato tuberous roots and insinuated IbCCD4 to be a target gene in the development of new sweetpotato varieties with high carotenoid production.