Chromoplast-specific Lycopene Β-cyclase Research Articles

Targeted disruption of tomato chromoplast-specific lycopene β-cyclase (CYC-B) gene promotes early accumulation of lycopene in fruits and enhanced postharvest cold tolerance.

Targeted disruption of tomato chromoplast-specific lycopene β-cyclase (CYC-B) gene promotes early accumulation of lycopene in fruits and enhanced postharvest cold tolerance.

Transcription factor CsMADS3 coordinately regulates chlorophyll and carotenoid pools in Citrus hesperidium.

Citrus, one of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and down-regulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene β-cyclase (CsLCYb2), two key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

Identification of QTLs for root color and carotenoid contents in Japanese orange carrot F2 populations

Carrot is a major source of provitamin A in a human diet. Two of the most important traits for carrot breeding are carotenoid contents and root color. To examine genomic regions related to these traits and develop DNA markers for carrot breeding, we performed an association analysis based on a general liner model using genome-wide single nucleotide polymorphism (SNPs) in two F2 populations, both derived from crosses of orange root carrots bred in Japan. The analysis revealed 21 significant quantitative trait loci (QTLs). To validate the detection of the QTLs, we also performed a QTL analysis based on a composite interval mapping of these populations and detected 32 QTLs. Eleven of the QTLs were detected by both the association and QTL analyses. The physical position of some QTLs suggested two possible candidate genes, an Orange (Or) gene for visual color evaluation, and the α- and β-carotene contents and a chromoplast-specific lycopene β-cyclase (CYC-B) gene for the β/α carotene ratio. A KASP marker developed on the Or distinguished a quantitative color difference in a different, related breeding line. The detected QTLs and the DNA marker will contribute to carrot breeding and the understanding of carotenoid biosynthesis and accumulation in orange carrots.

Ethylene activation of carotenoid biosynthesis by a novel transcription factor CsERF061.

Chromoplast-specific lycopene β-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids, in citrus. Its regulatory metabolism is largely unknown. Here, we identified a group I ethylene response factor, CsERF061, in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, and increased the number of chromoplasts needed to sequester the elevated concentrations of carotenoids, which was accompanied by changes in the concentrations of abscisic acid and gibberellin. Electrophoretic mobility shift and dual-luciferase assays verified that CsERF061 activates the promoters of nine other key carotenoid pathway genes, PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which will be useful for manipulating carotenoid accumulation in citrus and other plants.

Building the Synthetic Biology Toolbox with Enzyme Variants to Expand Opportunities for Biofortification of Provitamin A and Other Health-Promoting Carotenoids.

Carotenoids are a large class of structures that are important in human health and include both provitamin A and nonprovitamin A compounds. Vitamin A deficiency is a global health problem that can be alleviated by enriching provitamin A carotenoids in a range of food crops. Suitable plants for biofortification are those with high levels of the provitamin A biosynthetic precursor, lycopene, which is enzymatically converted by lycopene β-cyclase (LCYB) to β-carotene, a provitamin A carotenoid. Crops, such as citrus, naturally accumulate high levels of provitamin A and other health-promoting carotenoids. Such plants may have useful genes to expand the synthetic biology toolbox for producing a range of phenotypes, including both high provitamin A crops and crops with unique compositions of health-promoting carotenoids. To examine enzyme variants having different activity levels, we introduced two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene. Overexpression in tomato of the stronger allele of the citrus chromoplast-specific lycopene β-cyclase (CsLCYb2a) produced "golden" transgenic tomato fruits with 9.3-fold increased levels of β-carotene at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of β-carotene but in the context of a more heterogeneous composition of carotenoids. From a synthetic biology standpoint, these allelic differences have value for producing cultivars with unique carotenoid profiles. Overexpression of the citrus LCYB genes was accompanied by increased expression of other genes encoding carotenoid biosynthetic enzymes and increased size and number of chromoplasts needed to sequester the elevated levels of carotenoids in the transgenic tomato fruits. The overexpression of the citrus LCYB genes also led to a pleiotropic effect on profiles of phytohormones and primary metabolites. Our findings show that enzyme variants are essential synthetic biology parts needed to create a wider range of metabolic engineering products. In this case, strong and weak variants of LCYB proved useful in creating dietary sources to alleviate vitamin A deficiency or, alternatively, to create crops with a heterogeneous composition including provitamin A and healthful, nonprovitamin A carotenoids.

Expression of a Chromoplast-Specific Lycopene β-Cyclase Gene (CYC-B) Is Implicated in Carotenoid Accumulation and Coloration in the Loquat.

Carotenoids are the principal pigments in the loquat. Although the metabolic pathway of plant carotenoids has been extensively investigated, few studies have been explored the regulatory mechanisms of loquat carotenoids because knowledge of the loquat genome is incomplete. The chromoplast-specific lycopene β-cyclase gene (CYC-B) could catalyze cyclization of lycopene to β-carotene. In this study, the differential accumulation patterns of loquat with different colors were analyzed and virus-induced gene silencing (VIGS) was utilized in order to verify CYC-B gene function. Using a cloning strategy of homologous genes, a CYC-B gene orthologue was successfully identified from the loquat. At a later stage of maturation, CYC-B gene expression and carotenoids concentrations in the ‘Dawuxing’ variety were higher than in ‘Chuannong 1-5-9′, possibly leading to the difference in pulp coloration of loquat. Interference of CYC-B gene expression in the loquat demonstrated clear visual changes. The green color in negative control fruits became yellow, while TRV2-CYC-B silenced fruits remained green. CYC-B gene expression and total carotenoid content in the pulp decreased by 32.5% and 44.1%, respectively. Furthermore, multiple key genes in the carotenoid metabolic pathway synergistically responded to downregulation of CYC-B gene expression. In summary, we provide direct evidences that CYC-B gene is involved in carotenoid accumulation and coloration in the loquat.

Antioxidant Balance and Regulation in Tomato Genotypes of Different Color

Antioxidants, antioxidant capacity, and the expression of isoprenoid metabolism–related genes and two pigmentation-related transcription factors were studied in four native and four hybrid tomato (Solanum lycopersicum) genotypes with different-colored fruit. Red fruit genotypes were associated with greater lycopene, β-carotene, lipophilic antioxidant capacity, and greater chromoplast-specific lycopene β-cyclase (CYC-B) transcript levels. Orange fruit genotypes had greater concentrations of tocopherols and greater transcript levels of homogentisate phytyl transferase (VTE-2), 1-deoxy-D-xylulose phosphate synthase (DXS), and 4-hydroxyphenylpyruvate dioxygenase (HPPD). The yellow fruit genotype was greater in total polyphenol and hydrophilic antioxidant capacity with greater expression of geranylgeranyl reductase (GGDR), phytol kinase (VTE-5), phytoene synthase (PSY) 2, lycopene β-cyclase (LCY-B), SlNAC1, and SINAC4. Greater levels of individual antioxidants were associated with specific coloration of tomato fruit. Moreover, the negative correlations between the expression of PSY1 and VTE-5, and between lycopene and chlorophyll, suggest a balance between carotenoids, tocopherols, and chlorophylls. The results of this study support either the direct commercialization of tomatoes with different color fruit or use of their genotypes in breeding programs to increase antioxidant levels among existing cultivars.

Transcriptomic changes triggered by carotenoid biosynthesis inhibitors and role of Citrus sinensis phosphate transporter 4;2 (CsPHT4;2) in enhancing carotenoid accumulation.

Carotenoid accumulation and chromoplast development in orange were perturbed by carotenoid inhibitors, and candidate genes were identified via transcriptomic analysis. The role of CsPHT4;2 in enhancing carotenoid accumulation was revealed. Carotenoids are important plant pigments and their accumulation can be affected by biosynthesis inhibitors, but the genes involved were largely unknown. Here, application of norflurazon (NFZ), 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) and clomazone for 30days to in vitro cultured sweet orange juice vesicles caused over-accumulation of phytoene (over 1000-fold), lycopene (2.92μgg-1FW, none in control), and deficiency in total carotenoids (reduced to 22%), respectively. Increased carotenoids were associated with bigger chromoplasts with enlarged plastoglobules or a differently crystalline structure in NFZ, and CPTA-treated juice vesicles, respectively. Global transcriptomic changes following inhibitor treatments were profiled. Induced expression of 1-deoxy-D-xylulose 5-phosphate synthase 1 by CPTA, hydroxymethylbutenyl 4-diphosphate reductase by both NFZ and CPTA, and reduced expression of chromoplast-specific lycopene β-cyclase by CPTA, as well as several downstream genes by at least one of the three inhibitors were observed. Expression of fibrillin 11 (CsFBN11) was induced following both NFZ and CPTA treatments. Using weighted correlation network analysis, a plastid-type phosphate transporter 4;2 (CsPHT4;2) was identified as closely correlated with high-lycopene accumulation induced by CPTA. Transient over-expression of CsPHT4;2 significantly enhanced carotenoid accumulation over tenfold in 'Cara Cara' sweet orange juice vesicle-derived callus. The study provides a valuable overview of the underlying mechanisms for altered carotenoid accumulation and chromoplast development following carotenoid inhibitor treatments and sheds light on the relationship between carotenoid accumulation and chromoplast development.

Metabolic and transcriptional elucidation of the carotenoid biosynthesis pathway in peel and flesh tissue of loquat fruit during on-tree development

BackgroundCarotenoids are the main colouring substances found in orange-fleshed loquat fruits. The aim of this study was to unravel the carotenoid biosynthetic pathway of loquat fruit (cv. ‘Obusa’) in peel and flesh tissue during distinct on-tree developmental stages through a targeted analytical and molecular approach.ResultsSubstantial changes regarding colour parameters, both between peel and flesh and among the different developmental stages, were monitored, concomitant with a significant increment in carotenoid content. Key genes and individual compounds that are implicated in the carotenoid biosynthetic pathway were further dissected with the employment of molecular (RT-qPCR) and advanced analytical techniques (LC-MS). Results revealed significant differences in carotenoid composition between peel and flesh. Thirty-two carotenoids were found in the peel, while only eighteen carotenoids were identified in the flesh. Trans-lutein and trans-β-carotene were the major carotenoids in the peel; the content of the former decreased with the progress of ripening, while the latter registered a 7.2-fold increase. However, carotenoid profiling of loquat flesh indicated trans-β-cryptoxanthin, followed by trans-β-carotene and 5,8-epoxy-β-carotene to be the most predominant carotenoids. High amounts of trans-β-carotene in both tissues were supported by significant induction in a chromoplast-specific lycopene β-cyclase (CYCB) transcript levels. PSY1, ZDS, CYCB and BCH were up-regulated and CRTISO, LCYE, ECH and VDE were down-regulated in most of the developmental stages compared with the immature stage in both peel and flesh tissue. Overall, differential regulation of expression levels with the progress of on-tree fruit development was more evident in the middle and downstream genes of carotenoid biosynthetic pathway.ConclusionsCarotenoid composition is greatly affected during on-tree loquat development with striking differences between peel and flesh tissue. A link between gene up- or down-regulation during the developmental stages of the loquat fruit, and how their expression affects carotenoid content per tissue (peel or flesh) was established.

Cytological and molecular characterization of carotenoid accumulation in normal and high-lycopene mutant oranges

Ripe Cara Cara sweet orange contains 25 times as much carotenoids in flesh as Newhall sweet orange, due to high accumulation of carotenes, mainly phytoene, lycopene and phytofluene. Only yellow globular chromoplasts were observed in Newhall flesh. Distinct yellow globular and red elongated crystalline chromoplasts were found in Cara Cara but only one type of chromoplast was present in each cell. The red crystalline chromoplasts contained lycopene as a dominant carotenoid and were associated with characteristic carotenoid sequestering structures. The increased accumulation of linear carotenes in Cara Cara is not explained by differences in expression of all 18 carotenogenic genes or gene family members examined, or sequence or abundance of mRNAs from phytoene synthase (PSY) and chromoplast-specific lycopene β-cyclase (CYCB) alleles. 2-(4-Chlorophenylthio)-triethylamine hydrochloride (CPTA) enhanced lycopene accumulation and induced occurrence of red crystalline chromoplasts in cultured Newhall juice vesicles, indicating that carotenoid synthesis and accumulation can directly affect chromoplast differentiation and structure. Norflurazon (NFZ) treatment resulted in high accumulation of phytoene and phytofluene in both oranges, and the biosynthetic activity upstream of phytoene desaturase was similar in Newhall and Cara Cara. Possible mechanisms for high carotene accumulation and unique development of red crystalline chromoplasts in Cara Cara are discussed.

Characterization of Carotenoid Accumulation and Carotenogenic Gene Expression During Fruit Development in Yellow and White Loquat Fruit

Accumulation of carotenoids in peel and pulp of the yellow-fleshed loquat ‘Zaozhong 6’ (ZZ6) and the white-fleshed loquat ‘Baiyu’ (BY) were tracked during different fruit development stages, and the expression of 15 carotenogenic genes were analyzed. During loquat fruit ripening the fresh weight content of β-carotene in peel and pulp of ZZ6 increased gradually and peaked at the fully ripe stage, reaching 68.53 µg⋅g−1 FW in the peel and 11.92 µg⋅g−1 FW in the pulp. In BY, the content of β-carotene in the peel increased and peaked at the fully ripe stage, reaching 38.89 µg⋅g−1 FW, while it decreased in the pulp from the original 0.47 µg⋅g−1 FW and reduced to 0.29 µg⋅g−1 FW. The content of β-cryptoxanthin in the peel and pulp of ZZ6 and BY both increased steadily, and peaked at the fully ripe stage; however, the content of lutein decreased in the peel of ZZ6 and increased in the pulp, but in BY, it dropped and then rose in the peel. There was no significant change of β-cryptoxanthin in the pulp of BY. After the breaker stage, the mRNA levels of phytoene synthase (PSY) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, while CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of ZZ6, compared with BY. The results showed that the expression level of PSY, CYCB, and BCH appeared to cooperatively regulate the accumulation of carotenoids.

An increase of lycopene content in tomato fruit is associated with a novel Cyc-B allele isolated through TILLING technology

Tomato Cyc-B gene encodes a chromoplast-specific lycopene β-cyclase that converts lycopene to β-carotene during ripening of the fruit. By screening the tomato Red Setter mutant population with the TILLING method, we identified eight new alleles at the Cyc-B locus. Results of greenhouse phenotypic analysis revealed that the novel A949G Cyc-B allele produced modifications in the carotenoid profile and content of tomato petals and fruit. The cyc-b7 genotype, carrying the A949G Cyc-B allele, was therefore evaluated in an open field trial for standard agronomic traits as well as carotenoid content of the fruit. Results of the field trial confirmed that the induced A949G missense mutation favored the accumulation of lycopene in the fruit with no detrimental effects on the yield or on other agronomic and technological properties such as fruit firmness and Brix degree of fruit juice. On the basis of these results, it can be affirmed that the A949G Cyc-B allele constitutes a useful new genetic variant which can be used for improving carotenoid content in tomato fruit and for the development of new tomato commercial lines. Finally, the results presented here furthermore demonstrate that TILLING is a powerful methodology not only as a confirmatory system for gene functional analysis but also for selecting new gene variants useful for genetic improvement of important crops.

Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g−1 and 13 μg g−1 fresh weight (FW), respectively, and for BS 19 μg g−1 and 0.27 μg g−1 FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.

Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

BackgroundCarotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061.ResultsA 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to significant increase in the activity of GUS in the transgenic plants. Promoter deletion analysis led to the identification of a short promoter region (-436 bp to ATG) that exhibited a higher promoter strength but similar developmental expression pattern as compared with the full-length ShCYC-B promoter.ConclusionFunctional characterization of the full-length ShCYC-B promoter and its deletion fragments in transient expression system in fruto as well as in stable transgenic tomato revealed that the promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits. Our study identified a short promoter region with functional activity and developmental expression pattern similar to that of the full-length ShCYC-B promoter. This 436 bp promoter region can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in CYC-B expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. Moreover, this short promoter region of ShCYC-B may be useful in genetic engineering of carotenoid content and other agronomic traits in tomato fruits.

Molecular and functional characterization of a novel chromoplast-specific lycopene β-cyclase from Citrus and its relation to lycopene accumulation

Carotenoids are the main pigments responsible of the colouration of Citrus fruits. The β-cyclization of lycopene, catalysed by the lycopene β-cyclases (β-LCY), seems to be a key regulatory step of the carotenoid pathway. In the present study, two β-LCYs from orange fruits (Citrus sinensis), named Csβ-LCY1 and Csβ-LCY2 have been isolated and the activity of the encoded proteins was demonstrated by functional analysis. Csβ-LCY1 was expressed at low levels and remained relatively constant during fruit ripening while Csβ-LCY2 showed a chromoplast-specific expression and a marked induction in both peel and pulp of orange fruits in parallel with the accumulation of β,β-xanthophylls. The potential involvement of Csβ-LCY2 in the accumulation of lycopene, characteristic of some Citrus species such as red grapefruits, was investigated. Expression of Csβ-LCY2 and another seven carotenoid biosynthetic genes were studied in the peel and pulp of the high lycopene-accumulating grapefruit, Star Ruby, and compared with those of ordinary Navel orange. In Star Ruby, the accumulation of lycopene during fruit maturation was associated with a substantial reduction in the expression of both β-LCY2 and β-CHX genes with respect to Navel orange. Moreover, two different alleles of β-LCY2: β-LCY2a and β-LCY2b were isolated from both genotypes, and functional assays demonstrated that the lycopene β-cyclase activity of the allele b was almost null. Interestingly, Star Ruby grapefruit predominantly expressed the unfunctional β-LCY2b allele during fruit ripening whereas Navel oranges preferably expressed the functional allele. It is suggested that the presence of diverse alleles of the β-LCY2 gene, encoding enzymes with altered activity, with different transcript accumulation may be an additional regulatory mechanism of carotenoid synthesis involved in the accumulation of lycopene in red grapefruits.