Candidate gene analysis of organ pigmentation loci in the Solanaceae.
ABSTRACT: Ten structural genes from the Capsicum (pepper) carotenoid biosynthetic pathway have been localized on a (Capsicum annuum x Capsicum chinense)F(2) genetic map anchored in Lycopersicon (tomato). The positions of these genes were compared with positions of the same genes in tomato when known, and with loci from pepper, potato, and tomato that affect carotenoid levels in different tissues. C2, one of three phenotypically defined loci determining pepper fruit color, cosegregated with phytoene synthase. The capsanthin-capsorubin synthase (Ccs) locus, shown previously to cosegregate with Y, another pepper fruit color locus, mapped to pepper chromosome 6. Other structural genes in pepper corresponded to loci affecting carotenoid production as follows: Ccs in pepper and the B locus for hyperaccumulation of beta-carotene in tomato fruit mapped to homeologous regions; the position of the lycopene beta-cyclase gene in pepper may correspond to the lutescent-2 mutation in tomato; and the lycopene epsilon-cyclase locus in pepper corresponded to the lycopene epsilon-cyclase locus/Del mutation for hyperaccumulation of delta-carotene in tomato fruit. Additional associations were seen between the structural genes and previously mapped loci controlling quantitative variation in pepper and tomato fruit color. These results demonstrate that comparative analyses using candidate genes may be used to link specific metabolic phenotypes and loci that affect these phenotypes in related species.
Project description:Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases beta-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes beta-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene beta-cyclase, an enzyme that converts lycopene to beta-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that beta-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.
Project description:The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene β-cyclase, β-carotene hydroxylase, zeaxanthin epoxidase and capsanthin-capsorubin synthase (CCS) genes, in 94 pepper accessions by single-molecule real-time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra-performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.
Project description:Members of the tomato clade exhibit a wide diversity in fruit color, but the mechanisms governing inter-species diversity of coloration are largely unknown. The carotenoid profiles, carotenogenic gene expression and proteome profiles of green-fruited Solanum habrochaites (SH), orange-fruited S. galapagense, and red-fruited S. pimpinellifolium were compared with cultivated tomato [S. lycopersicum cv. Ailsa Craig (SL)] to decipher the molecular basis of coloration diversity. Green-fruited SH, though it showed normal expression of chromoplast-specific phytoene synthase1 and lycopene ?-cyclase genes akin to orange/red-fruited species, failed to accumulate lycopene and ?-carotene. The SH phytoene synthase1 cDNA encoded an enzymatically active protein, whereas the lycopene ?-cyclase cDNA was barely active. Consistent with its green-fruited nature, SH's fruits retained chloroplast structure and PSII activity, and had impaired chlorophyll degradation with high pheophorbide a levels. Comparison of the fruit proteomes with SL revealed retention of the proteome complement related to photosynthesis in SH. Targeted peptide monitoring revealed a low abundance of key carotenogenic and sequestration proteins in SH compared with tomato. The green-fruitedness of SH appears to stem from blocks at several critical steps regulating fruit-specific carotenogenesis namely the absence of chloroplast to chromoplast transformation, block in carotenoid biosynthesis, and a dearth of carotenoid sequestering proteins.
Project description:The diverse fruit colors of peppers (Capsicum spp.) are due to variations in carotenoid composition and content. Mature fruit color in peppers is regulated by three independent loci, C1, C2, and Y. C2 and Y encode phytoene synthase (PSY1) and capsanthin-capsorubin synthase (CCS), respectively; however, the identity of the C1 gene has been unknown. With the aim of identifying C1, we analyzed two pepper accessions with different fruit colors: Capsicum frutescens AC08-045 and AC08-201, whose fruits are light yellow and white, respectively. Ultra-performance liquid chromatography showed that the total carotenoid content was six times higher in AC08-045 than in AC08-201 fruits, with similar composition of main carotenoids and slight difference in minor components. These results suggest that a genetic factor in AC08-201 may down-regulate overall carotenoid biosynthesis. Analyses of candidate genes related to carotenoid biosynthesis and plastid abundance revealed that both accessions carry non-functional alleles of CCS, golden2-like transcription factor (GLK2), and PSY1. However, a nonsense mutation (C2571T) in PRR2, a homolog of Arabidopsis pseudo response regulator2-like (APRR2), was present in only AC08-201. In a population derived from a cross between AC08-045 and AC08-201, a SNP marker based on the nonsense mutation co-segregated fully with fruit color, implying that the mutation in PRR2 may cause the white color of AC08-201 fruits. Transmission electron microscopy (TEM) of AC08-201 fruit pericarp also showed less developed granum structure in chloroplast and smaller plastoglobule in chromoplast compared to those of AC08-045. Virus-induced gene silencing (VIGS) of PRR2 significantly reduced carotenoid accumulation in Capsicum annuum 'Micropep Yellow', which carries non-functional mutations in both PSY1 and CCS. Furthermore, sequence analysis of PSY1, CCS, and PRR2 in other white pepper accessions of C. annuum and Capsicum chinense showed that they commonly have non-functional alleles in PSY1, CCS, and PRR2. Thus, our data demonstrate that the fruit color locus C1 in Capsicum spp. corresponds to the gene PRR2.
Project description:BACKGROUND: Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. RESULTS: Total carotenoids progressively increased during fruit ripening up to ~55 ?g g(-1) fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. CONCLUSIONS: Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening.
Project description:The regulation of plant carotenogenesis is an active research area for both biological discovery and practical implementation. In tomato (Solanum lycopersicum), we demonstrate additional bottlenecks exist in the poly-cis-transformation of phytoene to lycopene in the context of ripening-induced PSY1 expression and activity and reveal phytoene desaturase (PDS), as a target for manipulation towards elevated lycopene content in maturing tomato fruit. Overexpression of Arabidopsis PDS, AtPDS, elevated PDS transcript abundance in all aerial tissues resulting in both altered carotenoid accumulation and associated pathway gene expression in a tissue-specific manner. Significant increases in downstream carotenoids (all-trans-lycopene and β-carotene) and minimal changes in carotenogenic gene expression (carotenoid isomerase-like 1, CRTIL1) suggest overexpression of heterologous AtPDS in tomato circumvents endogenous regulatory mechanism observed with previous strategies. In transgenic leaves, depletion of the PDS substrate, phytoene, was accompanied by minor, but significant increases in xanthophyll production. Alterations in the leaf carotenogenic transcript profile, including the upstream MEP pathway, were observed revealing unique feedback and feedforward regulatory mechanisms in response to AtPDS overexpression. AtPDS overexpression in the background of the tangerine (carotenoid isomerase, CRTISO) mutant exposes its potential in elevating downstream cis-lycopene accumulation in ripe tomato fruit, as cis-lycopene is more bioavailable yet less abundant than all-trans-lycopene in the wild-type control. In summary, we demonstrate the limitation of PDS in ripening fruit, its utility in modifying carotenoid profiles towards improved quality, and reveal novel carotenoid pathway feedback regulation.
Project description:Carotenoids, the widespread and structurally diverse class of pigments, accumulate in the fruits of tomato plants in a tissue specific manner. The carotenoid biosynthetic pathway genes have been cloned and characterized in tomato and other plants, however, its regulation is still obscure. We collected and analyzed forty different accessions of tomato for the present study. HPLC analysis revealed differential accumulation of major carotenoids (lycopene and ß-carotene) in the ripe fruit tissue. In order to understand the underlying regulatory mechanisms in carotenoid biosynthesis and accumulation, we sequenced the cis-acting elements i.e. promoter, 5' and 3' untranslated regions of the carotenoid pathway genes, in all accessions, followed by their in silico validation. Major differences observed in the CAAT Box, Opaque-2 Box and L-box in the promoters of carotenoid isomerase and lycopene-beta cyclase genes, respectively, along with the variations in musashi binding element of 5' untranslated regions of the carotenoid isomerase gene, suggest their differential role in regulating the carotenogenesis process in tomato. The binding sites for various transcription factors namely RIN, AGAMOUS, CRY, RAP2.2 and PIF1 on the promoters of important carotenoid pathway genes were predicted in silico. We propose that expression of carotenoid genes and also the formation of protein product in ripe tomato fruits, is regulated efficiently by the binding of these transcription factors at selected sites in the promoter region. Finally, the differential expression of the above-mentioned genes in different developmental tissues supports the possible involvement of promoters and untranslated regions in carotenoid biosynthesis and accumulation process. The present study has generated significant information concerning regulatory players involved in the carotenoid biosynthesis in tomato.
Project description:The MADS-box transcription factors play essential roles in many physiological and biochemical processes of plants, especially in fruit ripening. Here, a tomato MADS-box gene, SlCMB1, was isolated. SlCMB1 expression declined with the fruit ripening from immature green to B?+?7 (7 days after Breaker) fruits in the wild type (WT) and was lower in Nr and rin mutants fruits. Tomato plants with reduced SlCMB1 mRNA displayed delayed fruit ripening, reduced ethylene production and carotenoid accumulation. The ethylene production in SlCMB1-RNAi fruits decreased by approximately 50% as compared to WT. The transcripts of ethylene biosynthesis genes (ACS2, ACS4, ACO1 and ACO3), ethylene-responsive genes (E4, E8 and ERF1) and fruit ripening-related genes (RIN, TAGL1, FUL1, FUL2, LoxC and PE) were inhibited in SlCMB1-RNAi fruits. The carotenoid accumulation was decreased and two carotenoid synthesis-related genes (PSY1 and PDS) were down-regulated while three lycopene cyclase genes (CYCB, LCYB and LCYE) were up-regulated in transgenic fruits. Furthermore, yeast two-hybrid assay showed that SlCMB1 could interact with SlMADS-RIN, SlMADS1, SlAP2a and TAGL1, respectively. Collectively, these results indicate that SlCMB1 is a new component to the current model of regulatory network that regulates ethylene biosynthesis and carotenoid accumulation during fruit ripening.
Project description:Tomato (Solanum lycopersicum) fruit accumulate the red carotenoid pigment lycopene. The recessive mutation yellow-flesh (locus r) in tomato eliminates fruit carotenoids by disrupting the activity of the fruit-specific phytoene synthase (PSY1), the first committed step in the carotenoid biosynthesis pathway. Fruits of the recessive mutation tangerine (t) appear orange due to accumulation of 7,9,7',9'-tetra-cis-lycopene (prolycopene) as a result of a mutation in the carotenoid cis-trans isomerase. It was established 60 y ago that tangerine is epistatic to yellow-flesh. This uncharacteristic epistasis interaction defies a paradigm in biochemical genetics arguing that mutations that disrupt enzymes acting early in a biosynthetic pathway are epistatic to other mutations that block downstream steps in the same pathway. To explain this conundrum, we have investigated the interaction between tangerine and yellow-flesh at the molecular level. Results presented here indicate that allele r(2997) of yellow-flesh eliminates transcription of PSY1 in fruits. In a genetic background of tangerine, transcription of PSY1 is partially restored to a level sufficient for producing phytoene and downstream carotenoids. Our results revealed the molecular mechanism underlying the epistasis of t over r and suggest the involvement of cis-carotenoid metabolites in a feedback regulation of PSY1 gene expression.
Project description:Light is a major environmental factor affecting the regulation of secondary metabolites, such as pigments and flavor. The Solanaceae plant family has diverse patterns of fruit metabolisms that serve as suitable models to understand the molecular basis of its regulation across species. To investigate light-dependent regulation for fruit pigmentation and volatile flavors, major fruit pigments, their biosynthetic gene expression, and volatiles were analyzed in covered fruits of tomato and bell pepper. Immature covered fruits were found to be ivory in color and no chlorophyll was detected in both plants. The total carotenoid content was found to be reduced in ripe tomato and bell pepper under cover. Naringenin chalcone decreased more than 7-fold in ripe tomato and total flavonoids decreased about 10-fold in immature and ripe pepper fruit under light deficiency. Light positively impacts fruit pigmentation in tomato and bell pepper by regulating gene expression in carotenoid and flavonoid biosynthesis, especially phytoene synthase and chalcone synthase, respectively. Nineteen volatile flavors were detected, and seven of these exhibited light-dependent regulations for both ripe tomato and pepper. This study will help in improving fruit quality and aid future research works to understand the molecular mechanisms regulating the influence of light-dependency on pigments and flavor volatiles.