Regulation Of Carotenoid Biosynthesis Research Articles

GATA transcription factor WC2 regulates the biosynthesis of astaxanthin in yeast Xanthophyllomyces dendrorhous.

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one‐hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two‐hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild‐type strain did not largely modify the carotenoid profile, indicating translational and/or post‐translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild‐type strain and the XdWC2‐disrupted strain apparently improved the production of monocyclic carotenoid 3‐hydroxy‐3′, 4′‐didehydro‐β, ψ‐carotene‐4‐one (HDCO) rather than β‐carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over‐producing strains.

SR5AL serves as a key regulatory gene in lycopene biosynthesis by Blakeslea trispora

BackgroundTrisporic acids are considered to be key regulators of carotenoid biosynthesis and sexual reproduction in zygomycetes, but the mechanisms underlying this regulation have not been fully elucidated.ResultsIn this study, the relationships between trisporic acids and lycopene synthesis were investigated in Blakeslea trispora. The lycopene concentration in single fermentation by the (−) strain with the addition of 24 μg/L trisporic acids was slightly higher than that observed in mated fermentation. After transcriptomic analysis, a steroid 5α-reductase-like gene, known as SR5AL in B. trispora, was first reported. 5α-Reductase inhibitors reduced lycopene biosynthesis and downregulated the expression of sex determination and carotenoid biosynthesis genes. Overexpression of the SR5AL gene upregulated these genes, regardless of whether trisporic acids were added.ConclusionThese findings indicated that the SR5AL gene is a key gene associated with the response to trisporic acids.

Transcriptome Analysis Reveals Key Genes and Pathways Associated with the Petal Color Formation in Cabbage (Brassica oleracea L. var. capitata).

Petal color is an important agronomic trait in cabbage (Brassica oleracea L. var. capitata). Although the key gene BoCCD4 has been functionally characterized, the underlying molecular regulatory mechanism of petal color formation in cabbage is still unclear. In this study, we applied the transcriptome analysis of yellow petals from the cabbage inbred line YL-1 and white petals from the Chinese kale inbred line A192-1 and the BoCCD4-overexpressing transgenic line YF-2 (YL-1 background), which revealed 1928 DEGs common to both the A192-1 vs. YL-1 and the YL-1 vs. YF-2 comparison groups. One key enzyme-encoding gene, BoAAO3, and two key TF-encoding genes, Bo2g151880 (WRKY) and Bo3g024180 (SBP), related to carotenoid biosynthesis were significantly up-regulated in both the A192-1 and YF-2 petals, which was consistent with the expression pattern of BoCCD4. We speculate that these key genes may interact with BoCCD4 to jointly regulate carotenoid biosynthesis in cabbage petals. This study provides new insights into the molecular regulatory mechanism underlying petal color formation in cabbage.

Correction to: Regulation of Carotenoid Biosynthesis by Shade Relies on Specific Subsets of Antagonistic Transcription Factors and Cofactors.

Correction to: Regulation of Carotenoid Biosynthesis by Shade Relies on Specific Subsets of Antagonistic Transcription Factors and Cofactors.

Methyl salicylate affects the lipophilic and hydrophilic antioxidant capacities of apricot by regulating carotenoid biosynthesis and phenolic metabolism

The carotenoid biosynthesis and phenolic metabolism were studied to explain the effect of methyl salicylate (MeSA) on the lipophilic antioxidant capacity (LAC) and hydrophilic antioxidant capacity (HAC) in apricot during postharvest storage. Our results indicated that the HAC of apricot was higher than LAC and mainly responsible for total antioxidant capacity of apricot. Preharvest spraying of MeSA (0.2 mmol L−1) could improve the value of HAC but declined LAC of apricot. The enhanced HAC in MeSA treated apricot was positively related to the increased content of phenolics, especially to (+)-catechin, which was catalyzed by the enzymes related to phenolic metabolism. While, the decline of LAC in apricot treated by MeSA could be attributed to the inhibition of carotenoids accumulation, which was regulated by carotenogenic genes. We concluded that MeSA could affect the lipophilic and hydrophilic antioxidant capacity of apricot by regulating carotenoid biosynthesis and phenolic metabolism.

Genome-Wide Comparative Analysis of the R2R3-MYB Gene Family in Five Solanaceae Species and Identification of Members Regulating Carotenoid Biosynthesis in Wolfberry.

The R2R3-MYB is a large gene family involved in various plant functions, including carotenoid biosynthesis. However, this gene family lacks a comprehensive analysis in wolfberry (Lycium barbarum L.) and other Solanaceae species. The recent sequencing of the wolfberry genome provides an opportunity for investigating the organization and evolutionary characteristics of R2R3-MYB genes in wolfberry and other Solanaceae species. A total of 610 R2R3-MYB genes were identified in five Solanaceae species, including 137 in wolfberry. The LbaR2R3-MYB genes were grouped into 31 subgroups based on phylogenetic analysis, conserved gene structures, and motif composition. Five groups only of Solanaceae R2R3-MYB genes were functionally divergent during evolution. Dispersed and whole duplication events are critical for expanding the R2R3-MYB gene family. There were 287 orthologous gene pairs between wolfberry and the other four selected Solanaceae species. RNA-seq analysis identified the expression level of LbaR2R3-MYB differential gene expression (DEGs) and carotenoid biosynthesis genes (CBGs) in fruit development stages. The highly expressed LbaR2R3-MYB genes are co-expressed with CBGs during fruit development. A quantitative Real-Time (qRT)-PCR verified seven selected candidate genes. Thus, Lba11g0183 and Lba02g01219 are candidate genes regulating carotenoid biosynthesis in wolfberry. This study elucidates the evolution and function of R2R3-MYB genes in wolfberry and the four Solanaceae species.

Damage response protein 1 (Dap1) functions in the synthesis of carotenoids and sterols in Xanthophyllomyces dendrorhous

Cytochrome P450s (P450s) are heme-containing proteins involved in several cellular functions, including biosynthesis of steroidal hormones, detoxification of xenobiotic compounds, among others. Damage response protein 1 (Dap1) has been described as a positive regulator of P450s through protein-protein interactions in organisms such as Schizosaccharomyces pombe. Three P450s in the carotenogenic yeast Xanthophyllomyces dendrorhous have thus far been characterized: Cyp51 and Cyp61, which are involved in ergosterol biosynthesis, and CrtS (astaxanthin synthase), which is involved in biosynthesis of the carotenoid astaxanthin. In this work, we describe the X. dendrorhous DAP1 gene, deletion of which affected yeast pigmentation by decreasing the astaxanthin fraction and increasing the β-carotene (a substrate of CrtS) fraction, which is consistent with the known role of CrtS. We found that the proportion of ergosterol was also decreased in the Δdap1 mutant. However, even though the fractions of the end products of these two pathways (the synthesis of carotenoids and sterols) were decreased in the Δdap1 mutant, the transcript levels of genes from the P450 systems involved were higher than those in the wild-type strain. We demonstrate that Dap1 coimmunoprecipitates with these three P450s, suggesting that Dap1 interacts with these three proteins. We propose that Dap1 regulates the synthesis of astaxanthin and ergosterol in X. dendrorhous, probably by regulating the P450s involved in both biosynthetic pathways at the protein level. This work suggests a new role for Dap1 in the regulation of carotenoid biosynthesis in X. dendrorhous.

Glycine-rich RNA-binding cofactor RZ1AL is associated with tomato ripening and development

Tomato ripening is a complex and dynamic process coordinated by many regulatory elements, including plant hormones, transcription factors, and numerous ripening-related RNAs and proteins. Although recent studies have shown that some RNA-binding proteins are involved in the regulation of the ripening process, understanding of how RNA-binding proteins affect fruit ripening is still limited. Here, we report the analysis of a glycine-rich RNA-binding protein, RZ1A-Like (RZ1AL), which plays an important role in tomato ripening, especially fruit coloring. To analyze the functions of RZ1AL in fruit development and ripening, we generated knockout cr-rz1al mutant lines via the CRISPR/Cas9 gene-editing system. Knockout of RZ1AL reduced fruit lycopene content and weight in the cr-rz1al mutant plants. RZ1AL encodes a nucleus-localized protein that is associated with Cajal-related bodies. RNA-seq data demonstrated that the expression levels of genes that encode several key enzymes associated with carotenoid biosynthesis and metabolism were notably downregulated in cr-rz1al fruits. Proteomic analysis revealed that the levels of various ribosomal subunit proteins were reduced. This could affect the translation of ripening-related proteins such as ZDS. Collectively, our findings demonstrate that RZ1AL may participate in the regulation of carotenoid biosynthesis and metabolism and affect tomato development and fruit ripening.

Overexpression of Orange Gene (OsOr-R115H) Enhances Heat Tolerance and Defense-Related Gene Expression in Rice (Oryza sativa L.).

In plants, the orange (Or) gene plays roles in regulating carotenoid biosynthesis and responses to environmental stress. The present study investigated whether the expression of rice Or (OsOr) gene could enhance rice tolerance to heat stress conditions. The OsOr gene was cloned and constructed with OsOr or OsOr-R115H (leading to Arg to His substitution at position 115 on the OsOr protein), and transformed into rice plants. The chlorophyll contents and proline contents of transgenic lines were significantly higher than those of non-transgenic (NT) plants under heat stress conditions. However, we found that the levels of electrolyte leakage and malondialdehyde in transgenic lines were significantly reduced compared to NT plants under heat stress conditions. In addition, the levels of expression of four genes related to reactive oxygen species (ROS) scavenging enzymes (OsAPX2, OsCATA, OsCATB, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) responded to abiotic stress was showed significantly higher in the transgenic lines than NT plants under heat stress conditions. Therefore, OsOr-R115H could be exploited as a promising strategy for developing new rice cultivars with improved heat stress tolerance.

Overexpression of native ORANGE (OR) and OR mutant protein in Chlamydomonas reinhardtii enhances carotenoid and ABA accumulation and increases resistance to abiotic stress

The carotenoid content of plants can be increased by overexpression of the regulatory protein ORANGE (OR) or a mutant variant known as the ‘golden SNP’. In the present study, a strong light-inducible promoter was used to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine in the microalgae Chlamydomonas reinhardtii. Overexpression of CrORWT and CrORHis roughly doubled and tripled, respectively, the accumulation of several different carotenoids, including β-carotene, α-carotene, lutein and violaxanthin in C. reinhardtii and upregulated the transcript abundance of nearly all relevant carotenoid biosynthetic genes. In addition, microscopic analysis revealed that the OR transgenic cells were larger than control cells and exhibited larger chloroplasts with a disrupted morphology. Moreover, both CrORWT and CrORHis cell lines showed increased tolerance to salt and paraquat stress. The levels of endogenous phytohormone abscisic acid (ABA) were also increased in CrORWT and CrORHis lines, not only in normal growth conditions but also in growth medium supplemented with salt and paraquat. Together these results offer new insights regarding the role of the native OR protein in regulating carotenoid biosynthesis and the accumulation of several carotenoids in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance potentially mediated by ABA.

Retraction Note: Light Harvesting-like Protein 3 Interacts with Phytoene Synthase and Is Necessary for Carotenoid and Chlorophyll Biosynthesis in Rice

Carotenoid biosynthesis is essential for the generation of photosynthetic pigments, phytohormone production, and flower color development. The light harvesting like 3 (LIL3) protein, which belongs to the light-harvesting complex protein family in photosystems, interacts with geranylgeranyl reductase (GGR) and protochlorophyllide oxidoreductase (POR) both of which are known to regulate terpenoid and chlorophyll biosynthesis, respectively, in both rice and Arabidopsis. In our study, a CRISPR-Cas9 generated 4-bp deletion mutant oslil3 showed aberrant chloroplast development, growth defects, low fertility rates and reduced pigment contents. A comparative transcriptomic analysis of oslil3 suggested that differentially expressed genes (DEGs) involved in photosynthesis, cell wall modification, primary and secondary metabolism are differentially regulated in the mutant. Protein-protein interaction assays indicated that LIL3 interacts with phytoene synthase (PSY) and in addition the gene expression of PSY genes are regulated by LIL3. Subcellular localization of LIL3 and PSY suggested that both are thylakoid membrane anchored proteins in the chloroplast. We suggest that LIL3 directly interacts with PSY to regulate carotenoid biosynthesis. This study reveals a new role of LIL3 in regulating pigment biosynthesis through interaction with the rate limiting enzyme PSY in carotenoid biosynthesis in rice presenting it as a putative target for genetic manipulation of pigment biosynthesis pathways in crop plants.

Characterization and functional analysis of phytoene synthase gene family in tobacco

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

Identification and characterization of carotenoid biosynthesis related genes in a novel dark skinned citrus mutant cultivar ‘Suneat’

We explored the relationship between skin color and the expression of genes related to carotenoid biosynthesis during fruit ripening in two citrus cultivars, ‘Shiranuhi’ (a hybrid mandarin cultivar) and ‘Suneat’ (mutant cultivar derived from a natural bud mutation of ‘Shiranuhi’). We identified 16 and 7 genes putatively involved in carotenoid biosynthesis and regulation via phylogenetic analysis in sweet orange. Further, we determined the expression pattern of these genes by quantitative real time reverse transcriptase PCR (qRT-PCR). The result showed that the expression levels of CsPSY (Cs6g15910), CsZDS (Cs3g11180), CsZ-ISO (Cs5g24730), CsBCH1 (Cs9g19270), CsFUL2 (Cs5g12280), CsTAGL1 (Cs7g16960), CsRIN1 (Cs6g19680), CsRIN2 (Cs7g10980), and CsHY5 (Cs7g05140) were up-regulated in the skin of mutant cultivar ‘Suneat’ than any other tissues of both citrus cultivars. Of these CsPSY, CsZDS, CsZ-ISO and CsBCH1 genes were related to carotenoid biosynthesis and CsFUL2, CsTAGL1, CsRIN1, CsRIN2, and CsHY5 were known to involve in the regulation of carotenoid biosynthesis. Our results reveal that the attractive dark orange skin color of ‘Suneat’ might be due to the up-regulation of carotenoid biosynthetic as well as regulatory genes and thereby higher levels carotenoids at ripening.

Arabidopsis ORANGE protein regulates plastid pre-protein import through interacting with Tic proteins.

Chloroplast-targeted proteins are actively imported into chloroplasts via the machinery spanning the double-layered membranes of chloroplasts. While the key translocons at the outer (TOC) and inner (TIC) membranes of chloroplasts are defined, proteins that interact with the core components to facilitate pre-protein import are continuously being discovered. A DnaJ-like chaperone ORANGE (OR) protein is known to regulate carotenoid biosynthesis as well as plastid biogenesis and development. In this study, we found that OR physically interacts with several Tic proteins including Tic20, Tic40, and Tic110 in the classic TIC core complex of the chloroplast import machinery. Knocking out or and its homolog or-like greatly affects the import efficiency of some photosynthetic and non-photosynthetic pre-proteins. Consistent with the direct interactions of OR with Tic proteins, the binding efficiency assay revealed that the effect of OR occurs at translocation at the inner envelope membrane (i.e. at the TIC complex). OR is able to reduce the Tic40 protein turnover rate through its chaperone activity. Moreover, OR was found to interfere with the interaction between Tic40 and Tic110, and reduces the binding of pre-proteins to Tic110 in aiding their release for translocation and processing. Our findings suggest that OR plays a new and regulatory role in stabilizing key translocons and in facilitating the late stage of plastid pre-protein translocation to regulate plastid pre-protein import.

A multifocal approach towards understanding the complexities of carotenoid biosynthesis and accumulation in rice grains.

Carotenoids are mostly C40 terpenoids that participate in several important functions in plants including photosynthesis, responses to various forms of stress, signal transduction and photoprotection. While the antioxidant potential of carotenoids is of particular importance for human health, equally important is the role of β-carotene as the precursor for vitamin A in the human diet. Rice, which contributes upto 40% of dietary energy for mankind, contains very low level of β-carotene, thereby making it an important crop for enhancing β-carotene accumulation in its grains and consequently targeting vitamin A deficiency. Biosynthesis of carotenoids in the endosperm of white rice is blocked at the first enzymatic step wherein geranylgeranyl diphosphate is converted to phytoene by the action of phytoene synthase (PSY). Strategies aimed at enhancing β-carotene levels in the endosperm of white rice identified Narcissus pseudonarcissus (npPSY) and bacterial CRT1 as the regulators of the carotenoid biosynthetic pathway in rice. Besides transcriptional regulation of PSY, posttranscriptional regulation of PSY expression by OR gene, molecular synergism between ε-LCY and β-LCY and epigenetic control of CRITSO through SET DOMAIN containing protein appear to be the other regulatory nodes which regulate carotenoid biosynthesis and accumulation in rice grains. In this review, we elucidate a comprehensive and deeper understanding of the regulatory mechanisms of carotenoid metabolism in crops that will enable us to identify an effective tool to alleviate carotenoid content in rice grains.

A Tetratricopeptide Repeat Protein Regulates Carotenoid Biosynthesis and Chromoplast Development in Monkeyflowers (Mimulus).

Little is known about the factors regulating carotenoid biosynthesis in flowers. Here, we characterized the REDUCED CAROTENOID PIGMENTATION2 (RCP2) locus from two monkeyflower (Mimulus) species, the bumblebee-pollinated species Mimulus lewisii and the hummingbird-pollinated species Mimulus verbenaceus We show that loss-of-function mutations of RCP2 cause drastic down-regulation of the entire carotenoid biosynthetic pathway. The causal gene underlying RCP2 encodes a tetratricopeptide repeat protein that is closely related to the Arabidopsis (Arabidopsis thaliana) REDUCED CHLOROPLAST COVERAGE proteins. RCP2 appears to regulate carotenoid biosynthesis independently of RCP1, a previously identified R2R3-MYB master regulator of carotenoid biosynthesis. We show that RCP2 is necessary and sufficient for chromoplast development and carotenoid accumulation in floral tissues. Simultaneous down-regulation of RCP2 and two closely related paralogs, RCP2-L1 and RCP2-L2, yielded plants with pale leaves deficient in chlorophyll and carotenoids and with reduced chloroplast compartment size. Finally, we demonstrate that M. verbenaceus is just as amenable to chemical mutagenesis and in planta transformation as the more extensively studied M. lewisii, making these two species an excellent platform for comparative developmental genetics studies of closely related species with dramatic phenotypic divergence.

A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium

The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75–80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expression.

Transcriptome analysis identifies key genes involved in carotenoid biosynthesis in the flesh of red pummelo (Citrus maxima)

Red-fleshed pummelo (Citrus grandis), a mutant of ‘Guanxi’, is an important economic plant rich in carotenoids. The transcriptome of ‘Guanxi’ pummelo was compared with red-fleshed pummelo, with the aim of identifying the key genes responsible for red flesh through RNA sequencing. In total, 44.11 and 47.71 Gb clean reads were respectively obtained after filtering in red and white flesh. Altogether 46,425 unigenes, with an average length of 1208 bp, were assembled. Compared with that of white flesh, 3722 genes were up-regulated, whereas 1421 genes appeared down-regulated in the red-fleshed pummelo. A GO analysis assigned 11,983 differentially expressed genes to 53 subcategories, and 3582, 4722 and 3679 differentially expressed genes were involved in biological processes, cellular components and molecular functions, respectively. The mevalonate pathway involved in terpenoid backbone biosynthesis was significantly different between ‘Guanxi’ pummelo and its red-fleshed mutant, and the differences were closely associated with carotenoid biosynthesis. The expression levels of the carotenoid biosynthetic pathway genes were different between ‘Guanxi’ pummelo and its red-fleshed mutant, with the upstream genes having higher expression levels in red-fleshed pummelo than in ‘Guanxi’ pummelo, whereas the downstream gene β-CHX was downregulated. Two bHLH transcription factors (CL4778.Contig1 and CL4778.Contig2) were of the same branch as CubHLH1, regulating carotenoid biosynthesis in Citrus unshiu Marc., and Unigene CL4778.Contig2 had expression differences in two kinds of pummelo, which suggested CL4778.Contig2 should be a candidate gene controlling carotenoid accumulation in red pummelo. The study would be beneficial to identify the major gene regulating the red flesh trait.

Modulation of Activity of a Carotenoid Pathway Through the Use of the TeT-on Regulatory System: Application in the Fungus Fusarium fujikuroi.

Carotenoids are widespread pigments in photosynthetic species, but they are also found in nonphotosynthetic microorganisms, such as bacteria and fungi. The amenability of fungi to genetic studies have made some fungal species advantageous models in the study of the genetics and biochemistry of carotenoid biosynthesis, while others have been used for biotechnological carotenoid production. The availability of molecular techniques that allow modulating the expression of target genes is a powerful tool in the manipulation of carotenoid synthesis. An example of an adjustable gene expression is based on the tetracycline-controlled transcriptional activation system, known as Tet-on. We describe here the material and protocols for the construction of a Tet-on regulated gene, its introduction in the filamentous fungus F. fujikuroi, and its use to modulate the expression of a negative regulator of carotenoid biosynthesis.

The Role of Carotenogenic Metabolic Flux in Carotenoid Accumulation and Chromoplast Differentiation: Lessons From the Melon Fruit.

Carotenoids have various roles in plant physiology. Plant carotenoids are synthesized in plastids and are highly abundant in the chromoplasts of ripening fleshy fruits. Considerable research efforts have been devoted to elucidating mechanisms that regulate carotenoid biosynthesis, yet, little is known about the mechanism that triggers storage capacity, mainly through chromoplast differentiation. The Orange gene (OR) product stabilizes phytoene synthase protein (PSY) and triggers chromoplast differentiation. OR underlies carotenoid accumulation in orange cauliflower and melon. The OR’s ‘golden SNP’, found in melon, alters the highly evolutionary conserved Arginine108 to Histidine and controls β-carotene accumulation in melon fruit, in a mechanism yet to be elucidated. We have recently shown that similar carotenogenic metabolic flux is active in non-orange and orange melon fruit. This flux probably leads to carotenoid turnover but known carotenoid turnover products are not detected in non-orange fruit. Arrest of this metabolic flux, using chemical inhibitors or mutations, induces carotenoid accumulation and biogenesis of chromoplasts, regardless of the allelic state of OR. We suggest that the ‘golden SNP’ induces β-carotene accumulation probably by negatively affecting the capacity to synthesize downstream compounds. The accumulation of carotenoids induces chromoplast biogenesis through a metabolite-induced mechanism. Carotenogenic turnover flux can occur in non-photosynthetic tissues, which do not accumulate carotenoids. Arrest of this flux by the ‘golden SNP’ or other flux-arrest mutations is a potential tool for the biofortification of agricultural products with carotenoids.