Carotenoid Cleavage Dioxygenase Research Articles

Dynamics of annatto pigment synthesis and accumulation in seeds of Bixa orellana L. revealed by integrated chemical, anatomical, and RNA-Seq analyses.

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3, and S4) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Identification of superior haplotypes for CCD8 regulating tiller number and grain yield in rice

Rice is one of the major food crops of the world. In the present day of increasing population there is an urgent need to increase the rice grain production. Yield is a complex trait and is mainly orchestrated by plant architecture. In the present study, a diverse set of 100 accessions from the 3K RG panel were evaluated for Carotenoid cleavage dioxygenase 8 (CCD8) loci which plays a role in altering tiller number which ultimately governs the crop yield. A normal phenotypic distribution for total tiller numbers and number of productive tillers was observed for the set of 100 accessions used in the study. Five non synonymous SNPs were present for CCD8 loci for the 100 rice 3K accessions in this study. Haplotype analysis was carried out using five non synonymous SNPs to understand the genetic diversity of the population and of which two significant SNPs in the positions 31223371 and 31223383 grouped the accessions into two haplotype H1 and H2 with the allelic combination of GC and AT respectively. Haplotype group H1 with 89 accessions and H2 with 8 accessions were formed. Among the two haplotype groups, H2 had the maximum mean value for both tiller number and productive tiller number indicating the superiority of the H2 allelic combination over H1. Hence H2 is considered as a superior haplotype that can be potentially explored for allele mining and can be used in future crop improvement programs. Keywords: CCD8, tiller number, genetic diversity, haplotype.

A carotenoid cleavage dioxygenase 4 from Paulownia tomentosa determines visual and aroma signals in flowers

Paulownia tomentosa is an economically important fast-growing tree, and its flowers and fruits are a rich source of biologically active secondary metabolites. In addition, the flowers of P. tomentosa are distinguished by a strong aroma and are also excellent nectariferous plants. The flowers are pale lilac and characterized by the presence of yellow nectar guides, whose color changes during the development of the flower, representing reliable signals to pollinators while enhancing reproductive success. The chemical analyses of the nectar guides revealed the presence of carotenoids as the pigments responsible for the observed coloration, with β-carotene levels determining the color changes observed after anthesis, with a reduction at anthesis and further increase and accumulation in post anthesis. To understand how β-carotene accumulation was controlled in the nectar guides, the expression of genes related to carotenoid biosynthesis and metabolism was analyzed. Carotenogenic gene expression was not associated with the observed changes in β-carotene during flower development. However, the expression of a gene encoding a carotenoid cleavage dioxygenase, CCD4–4, was co-related with the levels of β-carotene in the nectar guides. In addition, CCD4–4 cleavage β-carotene at C9-C10 and C9'-C10' positions, resulting in the generation of β-ionone, which was detected in flowers at anthesis. The obtained results indicated a developmental stage specific regulation of apocarotenoid formation through β-carotene cleavage, resulting in color changes and volatile production as key traits for plant-pollinator interactions. Data availabilityData will be made available on request.

Strigolactones positively regulate Verticillium wilt resistance in cotton via crosstalk with other hormones.

Verticillium wilt caused by Verticillium dahliae is a serious vascular disease in cotton (Gossypium spp.). V. dahliae induces the expression of the CAROTENOID CLEAVAGE DIOXYGENASE 7 (GauCCD7) gene involved in strigolactone (SL) biosynthesis in Gossypium australe, suggesting a role for SLs in Verticillium wilt resistance. We found that the SL analog rac-GR24 enhanced while the SL biosynthesis inhibitor TIS108 decreased cotton resistance to Verticillium wilt. Knock-down of GbCCD7 and GbCCD8b genes in island cotton (Gossypium barbadense) decreased resistance, whereas overexpression of GbCCD8b in upland cotton (Gossypium hirsutum) increased resistance to Verticillium wilt. Additionally, Arabidopsis (Arabidopsis thaliana) SL mutants defective in CCD7 and CCD8 putative orthologs were susceptible, whereas both Arabidopsis GbCCD7- and GbCCD8b-overexpressing plants were more resistant to Verticillium wilt than wild-type (WT) plants. Transcriptome analyses showed that several genes related to the jasmonic acid (JA)- and abscisic acid (ABA)-signaling pathways, such as MYELOCYTOMATOSIS 2 (GbMYC2) and ABA-INSENSITIVE 5, respectively, were upregulated in the roots of WT cotton plants in responses to rac-GR24 and V. dahliae infection but downregulated in the roots of both GbCCD7- and GbCCD8b-silenced cotton plants. Furthermore, GbMYC2 suppressed the expression of GbCCD7 and GbCCD8b by binding to their promoters, which might regulate the homeostasis of SLs in cotton through a negative feedback loop. We also found that GbCCD7- and GbCCD8b-silenced cotton plants were impaired in V. dahliae-induced reactive oxygen species (ROS) accumulation. Taken together, our results suggest that SLs positively regulate cotton resistance to Verticillium wilt through crosstalk with the JA- and ABA-signaling pathways and by inducing ROS accumulation.

Natural overexpression of CAROTENOID CLEAVAGE DIOXYGENASE 4 in tomato alters carotenoid flux.

Carotenoids and apocarotenoids function as pigments and flavor volatiles in plants that enhance consumer appeal and offer health benefits. Tomato (Solanum lycopersicum.) fruit, especially those of wild species, exhibit a high degree of natural variation in carotenoid and apocarotenoid contents. Using positional cloning and an introgression line (IL) of Solanum habrochaites "LA1777', IL8A, we identified carotenoid cleavage dioxygenase 4 (CCD4) as the factor responsible for controlling the dark orange fruit color. CCD4b expression in ripe fruit of IL8A plants was ∼8,000 times greater than that in the wild type, presumably due to 5' cis-regulatory changes. The ShCCD4b-GFP fusion protein localized in the plastid. Phytoene, ζ-carotene, and neurosporene levels increased in ShCCD4b-overexpressing ripe fruit, whereas trans-lycopene, β-carotene, and lutein levels were reduced, suggestive of feedback regulation in the carotenoid pathway by an unknown apocarotenoid. Solid-phase microextraction-gas chromatography-mass spectrometry analysis showed increased levels of geranylacetone and β-ionone in ShCCD4b-overexpressing ripe fruit coupled with a β-cyclocitral deficiency. In carotenoid-accumulating Escherichia coli strains, ShCCD4b cleaved both ζ-carotene and β-carotene at the C9-C10 (C9'-C10') positions to produce geranylacetone and β-ionone, respectively. Exogenous β-cyclocitral decreased carotenoid synthesis in the ripening fruit of tomato and pepper (Capsicum annuum), suggesting feedback inhibition in the pathway. Our findings will be helpful for enhancing the aesthetic and nutritional value of tomato and for understanding the complex regulatory mechanisms of carotenoid and apocarotenoid biogenesis.

Suppression of carotenoid cleavage dioxygenase 1 (NtCCD1) increases carotenoid contents and attenuates reactive oxygen species (ROS) in Tobacco Leaves

Suppression of carotenoid cleavage dioxygenase 1 (NtCCD1) increases carotenoid contents and attenuates reactive oxygen species (ROS) in Tobacco Leaves

Jasmonic Acid-Induced β-Cyclocitral Confers Resistance to Bacterial Blight and Negatively Affects Abscisic Acid Biosynthesis in Rice.

Jasmonic acid (JA) regulates the production of several plant volatiles that are involved in plant defense mechanisms. In this study, we report that the JA-responsive volatile apocarotenoid, β-cyclocitral (β-cyc), negatively affects abscisic acid (ABA) biosynthesis and induces a defense response against Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight in rice (Oryza sativa L.). JA-induced accumulation of β-cyc was regulated by OsJAZ8, a repressor of JA signaling in rice. Treatment with β-cyc induced resistance against Xoo and upregulated the expression of defense-related genes in rice. Conversely, the expression of ABA-responsive genes, including ABA-biosynthesis genes, was downregulated by JA and β-cyc treatment, resulting in a decrease in ABA levels in rice. β-cyc did not inhibit the ABA-dependent interactions between OsPYL/RCAR5 and OsPP2C49 in yeast cells. Furthermore, we revealed that JA-responsive rice carotenoid cleavage dioxygenase 4b (OsCCD4b) was localized in the chloroplast and produced β-cyc both in vitro and in planta. These results suggest that β-cyc plays an important role in the JA-mediated resistance against Xoo in rice.

Identification and Characterization of CCD Gene Family in Rose (Rosa chinensis Jacq. ‘Old Blush’) and Gene Co-Expression Network in Biosynthesis of Flower Scent

Rose (Rosa sp.) is a widely used raw material for essential oil extraction and fragrance production. The carotenoid cleavage dioxygenases pathway is one of the main metabolic pathways for the degradation of carotenoids, which is located downstream of the terpenoids biosynthesis pathway and is closely related to the biosynthesis of volatile compounds. We performed a comprehensive genome-wide analysis of the rose CCD family genes (RcCCDs) in terms of phylogeny, sequence characterization, gene structure, gene duplication events, and transcriptome. Finally, 15 CCD family members were identified from the rose genome, and they were classified into three clades: nine for the CCD clade, four for the NCED clade, and two for the CCD-LIKE clade. The RcCCDs were distributed on chromosomes 1, 4, 5, 6, and 7, and were concentrated on both ends of the chromosomes. RcCCDs did not have paralogous genes or whole genome duplication events (WGD), eleven of them were single-copy genes, and their repetitive sequences were mainly dispersed and tandem. Ten RcCCDs were differentially expressed in the transcriptomes of different flowering stages. The expression of four of them increased and then decreased, which was the same process as the accumulation of volatile compounds, and it was speculated that these genes might be involved in the biosynthesis of volatile compounds. A total of fifteen modules were obtained by weighted gene co-expression network analysis of eighteen volatile compounds-related genes, of which six modules were a highly significant positive correlation with volatile compounds, and 20 hub genes in the modules were predicted. These hub genes all exercised their functions in the early flowering stage with strict temporal specificity. This study provided a theoretical basis for further exploring the biological functions of RcCCDs and hub genes regulating the synthesis and metabolism of volatile compounds in rose.

Comparative transcriptional analysis of metabolic pathways and mechanisms regulating essential oil biosynthesis in four elite Cymbopogon spp.

Cymbopogon is an important aromatic and medicinal grass with several species of ethnopharmaceutical importance. The genus is extremely rich in secondary metabolites, monoterpenes like geraniol and citral being principal constituents, also used as biomarker for classification and identification of Cymbopogon chemotypes. In the light of this, present study involved RNA sequencing and comparison of expression profiles of four contrasting Cymbopogon species namely C. flexuosus var. Chirharit (citral rich and frost resistant), C. martinii var. PRC-1 (geraniol rich), C. pendulus var. Praman (the most stable and citral-rich genotype), and Jamrosa (a hybrid of C. nardus var. confertiflorus × C. jwarancusa (rich in geraniol and geranyl acetate). The transcriptome profiles revealed marked differences in gene expression patterns of 28 differentially expressed genes (DEGs) of terpenoid metabolic pathways between the four Cymbopogon sp. The major DEGs were Carotenoid Cleavage Dioxygenases (CCD), Aspartate aminotransferase (ASP amino), Mevalonate E-4 hydroxy, AKR, GGPS, FDPS, and AAT. In addition, few TFs related to different regulatory pathways were also identified. The gene expression profiles of DEGs were correlated to the EO yield and their monoterpene compositions. Overall, the PRC-1 (C. martinii) shows distinguished gene expression profiles from all other genotypes. Thus, the transcriptome sequence database expanded our understanding of terpenoid metabolism and its molecular regulation in Cymbopogon species. Additionally, this data also serves as an important source of knowledge for enhancing oil yield and quality in Cymbopogon and closely related taxa. Key messageUnfolding the new secretes surrounding EO biosynthesis and regulation in four contrasting Cymbopogon species.

Engineering a fermenting yeast able to produce the fragrant β-ionone apocarotenoid for enhanced aroma properties in wine.

Wine is composed of multitudinous flavour components and volatile organic compounds that provide this beverage with its attractive properties of taste and aroma. The perceived quality of a wine can be attributed to the absolute and relative concentrations of favourable aroma compounds; hence, increasing the detectable levels of an attractive aroma, such as β-ionone with its violet and berry notes, can improve the organoleptic qualities of given wine styles. We here describe the generation of a new grape-must fermenting strain of Saccharomyces cerevisiae that is capable of releasing β-ionone through the heterologous expression of both the enzyme carotenoid cleavage dioxygenase 1 (CCD1) and its substrate, β-carotene. Haploid laboratory strains of S. cerevisiae were constructed with and without integrated carotenogenic genes and transformed with a plasmid containing the genes of CCD1. These strains were then mated with a sporulated diploid wine industry yeast, VIN13, and four resultant crosses-designated MQ01-MQ04-which were capable of fermenting the must to dryness were compared for their ability to release β-ionone. Analyses of their fermentation products showed that the MQ01 strain produced a high level of β-ionone and offers a fermenting hybrid yeast with the potential to enhance the organoleptic qualities of wine.

Transcriptome profiling reveals the roles of pigment formation mechanisms in yellow Paeonia delavayi flowers

The yellow colour of ornamental varieties of tree peony originated from Paeonia delavayi. However, but P. delavayi and Paeonia suffruticosa belong to different subgroups, so hybridization is difficult and results in a long breeding cycle. However, no comprehensive transcriptomic profiling has focused on the colour formation mechanisms of yellow tree peony petals. Analysing the colour formation mechanism of yellow petals in P. delavayi is very important for directional molecular breeding. In this study, the transcriptional map of yellow pigment development in petals was used to analyse the mechanism of petal colour formation. We analysed the genes related to the metabolism of flavonoids and carotenoids and the transcription factors (TFs) involved in P. delavayi var. lutea (pure yellow individual) yellow pigment development using transcriptome sequence profiling. Transcriptome sequence profiles revealed three and four differentially expressed transcripts (DETs) involved in flavonoid biosynthesis and carotenoid biosynthesis, respectively. An analysis of DETs in the flavonoid pathway showed that chalcone synthase (CHS) and chalcone 2´-glucosyltransferases (THC2’GT) act in synergy to synthesize isosalipurposide (ISP). CHS and flavonol synthase (FLS) synergistically synthesize quercetin and kaempferol. DEG analysis of the carotenoid pathway revealed that phytoene synthase (PSY), carotenoid isomerase (CRTISO) and β-carotene hydroxylases (CHYB) play a key role in regulating lutein formation, and carotenoid cleavage dioxygenase (CCD) plays an important role in the degradation of carotenoids. These two pathways may be regulated by TF families such as bHLH, ARF, and MYB. The results of the transient overexpression of genes showed that CHS and CHI are regulated by PdMYB2. In this study, the molecular mechanism of ISP synthesis was analysed in depth, and the complete metabolic pathway of carotenoids in Paeonia L. was reported for the first time. By studying the formation mechanism of yellow pigment in P. delavayi petals, a breeding strategy for improving flavonol and carotenoid contents and reducing anthocyanin synthesis by genetic engineering was suggested.

Jasmonate activates a CsMPK6-CsMYC2 module that regulates the expression of β-citraurin biosynthetic genes and fruit coloration in orange (Citrus sinensis).

Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid β-citraurin is responsible for the peel color in "Newhall" orange (Citrus sinensis). Although jasmonates are known to regulate the biosynthesis and accumulation of carotenoids, their effects on β-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and β-citraurin production in "Newhall" orange. A MeJA treatment induced the expression of CsMYC2, which encodes a transcription factor that serves as a master regulator of jasmonate responses. CsMYC2 bound the promoter of the gene that encodes carotenoid cleavage dioxygenase 4b (CsCCD4b), the key gene for β-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene β-cyclase (CsLCYb), and β-carotene hydroxylase (CsBCH) and induced their expression. In addition, CsMYC2 promoted CsMPK6 expression. Notably, we found that CsMPK6 interacted with CsMYC2 and that this interaction decreased the stability and DNA-binding activity of CsMYC2. Thus, we conclude that negative feedback regulation attenuates JA signaling during the jasmonate-induced coloration of citrus fruit. Together, our findings indicate that jasmonates induce β-citraurin biosynthesis in citrus by activating a CsMPK6-CsMYC2 cascade, thereby affecting fruit coloration.

Enhanced Production of Apocarotenoids by Salicylic Acid Elicitation in Cell Suspension Cultures of Saffron (Crocus sativus L.)

A cell suspension culture of saffron (Crocus sativus L.) was previously established from style-derived calli to obtain an in vitro system for crocin, an uncommon and valuable water-soluble apocarotenoid, and carotenoid production suitable for future scaling up. To shed more light on the correlation between apocarotenoid biosynthesis and key-gene expression, in this study, SA was used at 0.5 mM concentration to elicit crocin production and the effects on carotenoid production were analyzed after 6, 12, 24, and 48 h. HPLC-DAD analysis was used for total crocin quantification as well as the other carotenoids zeaxanthin, β-carotene and lutein. Quantitative RT-PCR was used to analyze the transcript levels of saffron apocarotenoid biosynthetic key genes PSY (phytoene synthase), BCH1 (β-carotene hydroxylase), and CCD2 (carotenoid cleavage dioxygenase) after SA elicitation. In saffron suspension-cultured cells elicited by SA, the carotenoid biosynthetic pathway was mostly enhanced toward crocin biosynthesis, known to exert strong biological activity and therapeutic effects, rather than lutein or xanthins. SA increased BCH1 and CCD2 gene expression 15.6 and 3.3 times, respectively, compared to the control at 24 h after elicitation. Although a dynamic change of metabolite contents and gene expression was observed during the 48 h time course in response to SA elicitation, the changes of zeaxanthin and crocin were consistent with the regulation of the corresponding genes BCH and CCD2 during the time course. In conclusion, the effects of SA on regulation of gene expression in the apocarotenoid pathway could be successfully applied for the biotechnological production of crocin.

Differences in commercial quality and carotenoids profile of yellow- and white-fleshed nectarine fruit during low temperature storage and the regulation of carotenoids by sugar

The aim of this study was to assess commercial quality parameters, carotenoids and sugars in two different nectarine cultivars (‘Zijinhong’, yellow flesh, and ‘Ruiguang’, white flesh) to gain insight into their commercial quality differences and the interaction among carotenoid, glucose, sucrose and fructose in nectarines during low temperature storage. In comparison with the white-fleshed cultivar ‘Ruiguang’, higher storability by higher soluble solid content and slower decrease of fruit firmness were exhibited in yellow-fleshed ‘Zijinhong’ nectarine. The increased carotenoids accumulation and inhibition of glucose and fructose degradation occurred in both cultivars, and glucose were positively correlated with total carotenoid contents (0.9825). It was confirmed through an in vitro test that glucose and fructose induced the biosynthesis of carotenoids. In addition, the gene expression of phytoene synthase and carotenoid cleavage dioxygenase were highly correlated with carotenoid content in ‘Zijinhong’ nectarine flesh.

Characteristics of a new carotenoid cleavage dioxygenase NtCCD10 derived from Nicotiana tabacum.

A new carotenoid cleavage dioxygenase NtCCD10 from tobacco was characterized. There is some difference between NtCCD10 and CCD1 in structure. NtCCD10 can cleave the C5-C6 (C5'-C6') and C9-C10 (C9'-C10') double bonds of carotenoids and has high catalytic activity. Carotenoid cleavage dioxygenases (CCDs) cleave carotenoids to produce a variety of apocarotenoids, which have important biological functions for organisms in nature. There are eleven CCDs subfamilies in the plant kingdom, many of which have been extensively characterized in their functions. However, as a newly classified subfamily, the function of CCD10 has rarely been studied. In this work, the function of an NtCCD10 gene from dicotyledonous Nicotiana tabacum was cloned and characterized, and its phylogeny, molecular structural modeling and protein structure were also systematically analyzed. Like other CCDs, NtCCD10 also possesses a seven bladed β-propeller with Fe2+ cofactor in its center constituting the active site of the enzyme. The Fe2+ is also coordinated bonding with four conserved histidine residues. Meanwhile, NtCCD10 also has many unique features, such as its α1 and α3 helixes are not anti-parallel, a special β-sheet and a longer access tunnel for substrates. When expressed in engineered Escherichia coli (producing phytoene, lycopene, β-carotene, and zeaxanthin) and Saccharomyces cerevisiae (producing β-carotene), NtCCD10 could symmetrically cleave phytoene and β-carotene at the C9-C10 and C9'-C10' positions to produce geranylacetone and β-ionone, respectively. In addition, NtCCD10 could also cleave the C5-C6 and C5'-C6' double bonds of lycopene to generate 6-methyl-5-heptene-2-one (MHO). NtCCD10 has higher catalytic activity than PhCCD1 in yeast, which provides a good candidate CCD for biosynthesis of β-ionone and has potential applications in biotechnological industry. This study identified the taxonomic position and catalytic activity of the first NtCCD10 in dicotyledonous plants. This will provide a reference for the discovery and functional identification of CCD10 enzymes in dicotyledons.

ZAXINONE SYNTHASE 2 regulates growth and arbuscular mycorrhizal symbiosis in rice.

Carotenoid cleavage, catalyzed by CAROTENOID CLEAVAGE DIOXYGENASEs (CCDs), provides signaling molecules and precursors of plant hormones. Recently, we showed that zaxinone, a apocarotenoid metabolite formed by the CCD ZAXINONE SYNTHASE (ZAS), is a growth regulator required for normal rice (Oryza sativa) growth and development. The rice genome encodes three OsZAS homologs, called here OsZAS1b, OsZAS1c, and OsZAS2, with unknown functions. Here, we investigated the enzymatic activity, expression pattern, and subcellular localization of OsZAS2 and generated and characterized loss-of-function CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and associated protein 9)-Oszas2 mutants. We show that OsZAS2 formed zaxinone in vitro. OsZAS2 was predominantly localized in plastids and mainly expressed under phosphate starvation. Moreover, OsZAS2 expression increased during mycorrhization, specifically in arbuscule-containing cells. Oszas2 mutants contained lower zaxinone content in roots and exhibited reduced root and shoot biomass, fewer tillers, and higher strigolactone (SL) levels. Exogenous zaxinone application repressed SL biosynthesis and partially rescued the growth retardation of the Oszas2 mutant. Consistent with the OsZAS2 expression pattern, Oszas2 mutants displayed a lower frequency of arbuscular mycorrhizal colonization. In conclusion, OsZAS2 is a zaxinone-forming enzyme that, similar to the previously reported OsZAS, determines rice growth, architecture, and SL content, and is required for optimal mycorrhization.

Anthocyanin pigmentation as a quantitative visual marker for arbuscular mycorrhizal fungal colonization of Medicago truncatula roots.

Visualization of root colonization by arbuscular mycorrhizal fungi (AMF) is the most elementary experiment in the field of mycorrhizal symbiosis. The most widely used approach for evaluating levels of AMF colonization is staining with trypan blue or ink, which is scored using the time-consuming grid intersection method. Here we demonstrate the use of an anthocyanin-based visual marker system for visualizing AMF colonization of Medicago truncatula roots. Expression of MtLAP1, a transcription factor which regulates the production of anthocyanins, from the AMF-induced Kunitz Protease Inhibitor 106 promoter, allowed the visualization of arbuscules in live plant tissues without microscopy or staining. This marker system allowed straightforward qualitative evaluation of the ram1, vpy and dmi3 AMF phenotypes using Agrobacterium rhizogenes hairy-root transformation. For the strigolactone biosynthesis mutant carotenoid cleavage dioxygenase 8a and a novel mutant scooby, which show quantitative AMF symbiotic phenotypes, the amount of anthocyanins in the roots estimated by spectrophotometry correlated very well with colonization levels estimated by staining and scoring using the grid intersection method. The LAP1-based marker system therefore provides a highly efficient approach for mutant screening and monitoring of AMF colonization in live tissues by eye, or for quantitative assessment using a simple and quick photometric assay.

Characteristics of NtCCD1-3 from tobacco, and protein engineering of the CCD1 to enhance β -ionone production in yeast.

Biosynthesis of β-ionone by microbial cell factories has become a promising way to obtain natural β-ionone. The catalytic activity of carotenoid cleavage dioxygenase 1 (CCD1) in cleavage of β-carotene to β-ionone severely limits its biosynthesis. In this study, NtCCD1-3 from Nicotiana tabacum with high ability to cleave β-carotene was screened. Multiple strategies for improving the β-ionone yield in Saccharomyces cerevisiae were performed. The results showed that NtCCD1-3 could cleave a variety of caroteniods at the 9,10 (9′,10′) double bonds and lycopene at the 5,6 (5′,6′) positions. The insertion site delta for NtCCD1-3 gene was more suitable for enhancing the yield of β-ionone, showing 19.1-fold increase compared with the rox1 site. More importantly, mutant K38A of NtCCD1-3 in membrane-bonding domains could greatly promote β-ionone production by more than 3-fold. We also found that overexpression of the NADH kinase Pos5 could improve β-ionone yield up to 1.5 times. These results may provide valuable references for biosynthesis of β-ionone.

Efficient production of the β-ionone aroma compound from organic waste hydrolysates using an engineered Yarrowia lipolytica strain.

This study demonstrates the feasibility of establishing a natural compound supply chain in a biorefinery. The process starts with the biological or chemical hydrolysis of food and agricultural waste into simple and fermentative sugars, followed by their fermentation into more complex molecules. The yeast strain, Yarrowia lipolytica, was modified by introducing high membrane affinity variants of the carotenoid cleavage dioxygenase enzyme, PhCCD1, to increase the production of the aroma compound, β-ionone. The initial hydrolysis process converted food waste or sugarcane bagasse into nutrient-rich hydrolysates containing 78.4 g/L glucose and 8.3 g/L fructose, or 34.7 g/L glucose and 20.1 g/L xylose, respectively. During the next step, engineered Y. lipolytica strains were used to produce β-ionone from these feedstocks. The yeast strain YLBI3120, carrying a modified PhCCD1 gene was able to produce 4 g/L of β-ionone with a productivity of 13.9 mg/L/h from food waste hydrolysate. This is the highest yield reported for the fermentation of this compound to date. The integrated process described in this study could be scaled up to achieve economical large-scale conversion of inedible food and agricultural waste into valuable aroma compounds for a wide range of potential applications.

Aster la vista: Unraveling the biochemical basis of carotenoid homeostasis in the human retina.

Carotenoids play pivotal roles in vision as light filters and precursor of chromophore. Many vertebrates also display the colorful pigments as ornaments in bare skin parts and feathers. Proteins involved in the transport and metabolism of these lipids have been identified including class B scavenger receptors and carotenoid cleavage dioxygenases. Recent research implicates members of the Aster protein family, also known as GRAM domain-containing (GRAMD), in carotenoid metabolism. These multi-domain proteins facilitate the intracellular movement of carotenoids from their site of cellular uptake by scavenger receptors to the site of their metabolic processing by carotenoid cleavage dioxygenases. We provide a model how the coordinated interplay of these proteins and their differential expression establishes carotenoid distribution patterns and function in tissues, with particular emphasis on the human retina.