Carotenoid Isomerase Research Articles

AtPDS overexpression in tomato: exposing unique patterns of carotenoid self-regulation and an alternative strategy for the enhancement of fruit carotenoid content.

SummaryThe 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.

Effect of ploidy level on expression of lycopene biosynthesis genes and accumulation of phytohormones during watermelon (Citrullus lanatus) fruit development and ripening

The difference between lycopene and phytohormone levels among diploid, triploid and tetraploid plants of two watermelon cultivars during fruit growth and ripening was studied. The expression pattern of five genes (phytoene synthase (PSY1), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), and lycopene β-cyclase (LCYB)) was analyzed in details. In red-fleshed cultivar Mimei, lycopene content increased rapidly from 25 to 35 days after pollination (DAP), and then decreased at 40 DAP. Triploid and tetraploid fruit had higher levels of lycopene than diploid. Moreover, triploid tended to contain more lycopene than tetraploid during fruit growth and ripening stages. However, little amount of lycopene (0–2 mg kg−1 fresh weight (FW)) in yellow-fleshed cultivar Huangmei was found during all fruit development stages. In Mimei, transcript level of PSY1 was generally higher than the other four genes, and LCYB gene expression was the lowest among all five genes being tested. PSY1, CRTISO and LCYB genes showed higher transcript levels in polyploid than in diploid fruit. By contrast, in Huangmei, transcript level of LCYB was not the lowest, but only lower than that of PSY1. PSY1, CRTISO and LCYB genes showed higher expression levels in diploid than in polyploid fruit. In Mimei, the negative correlation between gibberellane (GA) content and lycopene accumulation was determined in all three different ploidy fruits, while a positive correlation was observed between abscisic acid (ABA) content and lycopene accumulation only in diploid watermelon. These results indicated that different lycopene contents in different ploidy watermelons is regulated by the differential transcription expression of the lycopene metabolic genes and phytohormones.

Inferring the Genetic Determinants of Fruit Colors in Tomato by Carotenoid Profiling.

Carotenoids are essential for plant and animal nutrition, and are important factors in the variation of pigmentation in fruits, leaves, and flowers. Tomato is a model crop for studying the biology and biotechnology of fleshy fruits, particularly for understanding carotenoid biosynthesis. In commercial tomato cultivars and germplasms, visual phenotyping of the colors of ripe fruits can be done easily. However, subsequent analysis of metabolic profiling is necessary for hypothesizing genetic factors prior to performing time-consuming genetic analysis. We used high performance liquid chromatography (HPLC), employing a C30 reverse-phase column, to efficiently resolve nine carotenoids and isomers of several carotenoids in yellow, orange, and red colored ripe tomatoes. High content of lycopene was detected in red tomatoes. The orange tomatoes contained three dominant carotenoids, namely δ-carotene, β-carotene, and prolycopene. The yellow tomatoes showed low levels of carotenoids compared to red or orange tomatoes. Based on the HPLC profiles, genes responsible for overproducing δ-carotene and prolycopene were described as lycopene ε-cyclase and carotenoid isomerase, respectively. Subsequent genetic analysis using DNA markers for segregating population and germplasms were conducted to confirm the hypothesis. This study establishes the usefulness of metabolic profiling for inferring the genetic determinants of fruit color.

Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma.

Crocins, the glucosides of crocetin, are present at high concentrations in saffron stigmas and accumulate in the vacuole. However, the biogenesis of the saffron chromoplast, the changes during the development of the stigma and the transport of crocins to the vacuole, are processes that remain poorly understood. We studied the process of chromoplast differentiation in saffron throughout stigma development by means of transmission electron microscopy. Our results provided an overview of a massive transport of crocins to the vacuole in the later developmental stages, when electron dense drops of a much greater size than plastoglobules (here defined “crocinoplast”) were observed in the chromoplast, connected to the vacuole with a subsequent transfer of these large globules inside the vacuole. A proteome analysis of chromoplasts from saffron stigma allowed the identification of several well-known plastid proteins and new candidates involved in crocetin metabolism. Furthermore, expressions throughout five developmental stages of candidate genes responsible for carotenoid and apocarotenoid biogenesis, crocins transport to the vacuole and starch metabolism were analyzed. Correlation matrices and networks were exploited to identify a series of transcripts highly associated to crocetin (such as 1-Deoxy-d-xylulose 5-phosphate synthase (DXS), 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), carotenoid isomerase (CRTISO), Crocetin glucosyltransferase 2 (UGT2), etc.) and crocin (e.g., ζ-carotene desaturase (ZDS) and plastid-lipid-associated proteins (PLAP2)) accumulation; in addition, candidate aldehyde dehydrogenase (ADH) genes were highlighted.

Distinct Mechanisms of the ORANGE Protein in Controlling Carotenoid Flux.

β-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, β-carotene accumulation is governed by the Orange gene (CmOr) golden single-nucleotide polymorphism (SNP) through a yet to be discovered mechanism. In Arabidopsis (Arabidopsis thaliana), OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here, we identified a CmOr nonsense mutation (Cmor-lowβ) that lowered fruit β-carotene levels with impaired chromoplast biogenesis. Cmor-lowβ exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the golden SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblot analyses of selected melon genotypes and by using chemical pathway inhibitors. The high β-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of β-carotene. This was revealed by genetic studies with double mutants including carotenoid isomerase (yofi), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The yofi F2 segregants accumulated prolycopene independently of the golden SNP Moreover, Cmor-lowβ was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 d after anthesis until ripening, suggesting that CmOr regulates the chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels to maintain carotenoid biosynthesis metabolic flux but that the mechanism of the CmOr golden SNP involves an inhibited metabolism downstream of β-carotene to dramatically affect both carotenoid content and plastid fate.

Photo-Isomerization of Prolycopene in Chloroplast Occurs through the Triplet State: Implications on Carotenoid Biosynthesis

The carotenoid lycopene in photosynthetic organisms forms the critical precursor to the biosynthesis of reaction center carotenoids beta-carotene and lutein. In plants and cyanobacteria, a redox-controlled flavoenzyme carotenoid isomerase (CRTISO) catalyzes the four-step geometric isomerization of 7,9,9′,7′-tetra-cis-lycopene (prolycopene) to all-trans-lycopene. In chloroplasts, the functional loss of CRTISO has been shown to be rescued by a light-mediated isomerization pathway. In order to address the chloroplast-specificity and compare the efficiency of the photoisomerization reaction against redox-controlled enzyme catalysis, we need to track the excited state dynamics of prolycopene, and evaluate the nature of electronic states that lead to the photoisomerization. Using broadband femtosecond transient absorption spectroscopy, we observe ∼610 fs rise of the triplet state from the photoexcited S2 with a quantum yield of ∼0.19. The triplet state mediates the first C=C bond isomerization at symmetric 9 or 9′ position on the tetra-cis backbone to yield the tri-cis-lycopene with 15% quantum yield. However, direct sensitization of the photoreactive triplet state via meso-tetraphenyl porphyrin sensitizer under steady state illumination leads to all-trans-lycopene with 58% quantum efficiency. Our work implies that long-lived chlorophyll triplets activate the efficient isomerization of prolycopene in chloroplasts in absence of CRTISO activity. In presence of CRTISO, the role of redox equivalents in dark isomerization of prolycopene, and the light induced signaling mechanisms for the regulation of carotenoid biosynthesis will be discussed.

Fine mapping and characterization of the or gene in Chinese cabbage (Brassica rapa L. ssp pekinensis).

Orange inner leaves/heads is a qualitative trait in Chinese cabbage that is controlled by a single recessive gene. Orange head Chinese cabbage contain more carotenoids than its white head counterpart; hence, this trait is of interest to both researchers and consumers. In this study, we selected the orange head Chinese cabbage line 07A163 and the white head Chinese cabbage line Chiifu as test materials. We localized the target gene controlling the orange head trait to the A09 linkage group, with a physical distance of approximately 19.9 kb between the two markers, syau26 and syau28. This region contains six candidate genes, including Bra031539, which was predicted to encode CRTISO, a carotenoid isomerase specifically required for carotenoid biosynthesis. A comparison of the nucleic acid sequences of the two test materials revealed 88 and 7-bp deletions and 88 SNPs in the promoter region of Bra031539 in line 07A163, along with a 6-bp deletion in the first exon and early termination at the 3' end of this gene. BLAST analysis revealed that 22 amino acids were altered and 17 amino acids were lost in Bra031539 in the orange head line 07A163. We developed the BrPro1 molecular marker in the promoter region of Bra031539 that can be used for early identification of orange head materials, thereby accelerating the breeding process of orange head Chinese cabbage.

The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis

BackgroundStrigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis.ResultsWe provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2.ConclusionOur data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0651-x) contains supplementary material, which is available to authorized users.

Cloning and characterization of a Lycium chinense carotenoid isomerase gene enhancing carotenoid accumulation in transgenic tobacco

Carotenoid isomerase(CRTISO)is a key enzyme that catalyzes the conversion of cis-lycopene to alltrans lycopene. In this study, we isolated and characterized the CRTISO gene from Lycium chinense (LcCRTISO) for the first time. The open reading frame of LcCRTISO was 1 815 bp encoding a protein of 604 amino acids with a molecular mass of 66.24 kDa. Amino acid sequence analysis revealed that the LcCRTISO had a high level of similarity to other CRTISO. Phylogenetic analysis displayed that LcCRTISO kept a closer relationship with the CRTISO of plants than with those of other species. Semi-quantitative PCR analysis indicated that LcCRTISO gene was expressed in all tissues tested with the highest expression in maturing fruits. The overexpression of LcCRTISO gene in transgenic tobacco resulted in an increase of total carotenoids in the leaves with β-carotene and lutein being the predominants. The results obtained here clearly suggested that the LcCRTISO gene was a promising candidate for carotenoid production.

Comprehensive transcriptome analysis of Crocus sativus for discovery and expression of genes involved in apocarotenoid biosynthesis.

BackgroundCrocus sativus stigmas form rich source of apocarotenoids like crocin, picrocrocin and saffranal which besides imparting color, flavour and aroma to saffron spice also have tremendous pharmacological properties. Inspite of their importance, the biosynthetic pathway of Crocus apocarotenoids is not fully elucidated. Moreover, the mechanism of their stigma specific accumulation remains unknown. Therefore, deep transcriptome sequencing of Crocus stigma and rest of the flower tissue was done to identify the genes and transcriptional regulators involved in the biosynthesis of these compounds.ResultsTranscriptome of stigma and rest of the flower tissue was sequenced using Illumina Genome Analyzer IIx platform which generated 64,604,402 flower and 51,350,714 stigma reads. Sequences were assembled de novo using trinity resulting in 64,438 transcripts which were classified into 32,204 unigenes comprising of 9853 clusters and 22,351 singletons. A comprehensive functional annotation and gene ontology (GO) analysis was carried out. 58.5 % of the transcripts showed similarity to sequences present in public databases while rest could be specific to Crocus. 5789 transcripts showed similarity to transcription factors representing 76 families out of which Myb family was most abundant. Many genes involved in carotenoid/apocarotenoid pathway were identified for the first time in this study which includes zeta-carotene isomerase and desaturase, carotenoid isomerase and lycopene epsilon-cyclase. GO analysis showed that the predominant classes in biological process category include metabolic process followed by cellular process and primary metabolic process. KEGG mapping analysis indicated that pathways involved in ribosome, carbon and starch and sucrose metabolism were highly represented. Differential expression analysis indicated that key carotenoid/apocarotenoid pathway genes including phytoene synthase, phytoene desaturase and carotenoid cleavage dioxygenase 2 are enriched in stigma thereby providing molecular proof for stigma to be the site of apocarotenoid biosynthesis.ConclusionsThis data would provide a rich source for understanding the carotenoid/apocarotenoid metabolism in Crocus. The database would also help in investigating many questions related to saffron biology including flower development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1894-5) contains supplementary material, which is available to authorized users.

Ultrafast Triplet Generation and its Sensitization Drives Efficient Photoisomerization of Tetra-cis-lycopene to All-trans-lycopene.

Lycopene biosynthesis in photosynthetic organisms controls the metabolic flux of reaction center carotenoids like β-carotene and lutein through the geometric four-step isomerization of 7,9,9',7'-tetra-cis-lycopene (prolycopene) to its all-trans form. In plants and cyanobacteria, a redox-controlled flavoenzyme carotenoid isomerase catalyzes the prolycopene isomerization although its functional loss inside the chloroplast can be rescued by light. In order to address the chloroplast-specificity and efficiency of the light-induced isomerization reaction, we need to critically understand the excited state dynamics of prolycopene and the nature of electronic states that lead to the isomerization. Using broadband femtosecond transient absorption spectroscopy, we observe ∼610 fs rise of the long-lived triplet state from the photoexcited S2 with a quantum yield of ∼0.19. The triplet state eventually triggers the first C═C bond isomerization at the symmetric 9 or 9' position on the tetra-cis backbone to yield the tri-cis product with 15% quantum efficiency. However, direct sensitization of the photoreactive triplet state via meso-tetraphenyl porphyrin sensitizer under steady state illumination leads to an efficient production of all-trans-lycopene with 58% quantum yield. Our work implies that chlorophyll-enriched chloroplasts should form an optimized photoreaction vessel for prolycopene isomerization, and synthetic utilization of such cis-carotenoids can lead to efficient triplet harvesting photon-conversion devices.

The promoter from the Citrus unshiu carotenoid isomerase gene directs differential GUS expression in transgenic Arabidopsis

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated a promoter region of CuCRTISO from Citrus unshiu. We determined whether the promoter encoded organ-specific or developmental-specific expression and identified possible cis-acting promoter elements. The promoter and two truncated versions were fused to the β-glucuronidase (GUS) gene and transformed into Arabidopsis thaliana. Transgenic lines expressing the 2501-bp promoter (pCiso-Prom1) and truncated promoters (pCiso-Prom2 and pCiso-Prom3) showed the same developmental and organ-specific activity. GUS expression was detected in the cotyledon and root at 5 and 10 days after germination, mature leaf, and anther. The CuCRTISO promoter contained several cis-acting elements involved in hormonal and environmental stress. Drought stress or abscisic acid treatment did not induce GUS expression in any transgenic lines. Heat stress induced GUS expression in the pCiso-Prom1 line; this promoter construct contains the heat stress-responsive element. Ethylene and cold stress treatments induced GUS expression only in the pCiso-Prom3 line, although all transgenic lines contained the same cis-acting ethylene and low-temperature response elements, which could indicate the existence of unknown repressor element(s) in the CuCRTISO promoter. The CuCRTISO expression of Citrus unshiu during fruit maturation showed that in the green stage, it was highly expressed and then subsequently decreased after the stage. These studies indicate that CuCRTISO promoter activity is regulated in a developmental and organ-specific manner that responds to heat, cold, and ethylene. These results provide new insights into the role of cis-acting element(s) in CuCRTISO promoter activity.

Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway.

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.

Enhanced bioavailability of lycopene when consumed as cis-isomers from tangerine compared to red tomato juice, a randomized, cross-over clinical trial.

Tangerine tomatoes (Solanum lycopersicum) are rich in tetra-cis-lycopene resulting from natural variation in carotenoid isomerase. Our objective was to compare the bioavailability of lycopene from tangerine to red tomato juice, and elucidate physical deposition forms of these isomers in tomatoes by light and electron microscopy. Following a randomized cross-over design, subjects (n = 11, 6 M/5 F) consumed two meals delivering 10 mg lycopene from tangerine (94% cis) or red tomato juice (10% cis). Blood was sampled over 12 h and triglyceride-rich lipoprotein fractions of plasma were isolated and analyzed using HPLC-DAD-MS/MS. Lycopene was crystalline in red tomato chromoplasts and globular in tangerine tomatoes. With tangerine tomato juice we observed a marked 8.5-fold increase in lycopene bioavailability compared to red tomato juice (p < 0.001). Fractional absorption was 47.70 ± 8.81% from tangerine and 4.98 ± 1.92% from red tomato juices. Large heterogeneity was observed among subjects. Lycopene is markedly more bioavailable from tangerine than from red tomato juice, consistent with a predominance of cis-lycopene isomers and presence in chromoplasts in a lipid dissolved globular state. These results justify using tangerine tomatoes as a lycopene source in studies examining the potential health benefits of lycopene-rich foods.

Carotenoid content and root color of cultivated carrot: a candidate-gene association study using an original broad unstructured population.

Accumulated in large amounts in carrot, carotenoids are an important product quality attribute and therefore a major breeding trait. However, the knowledge of carotenoid accumulation genetic control in this root vegetable is still limited. In order to identify the genetic variants linked to this character, we performed an association mapping study with a candidate gene approach. We developed an original unstructured population with a broad genetic basis to avoid the pitfall of false positive detection due to population stratification. We genotyped 109 SNPs located in 17 candidate genes – mostly carotenoid biosynthesis genes – on 380 individuals, and tested the association with carotenoid contents and color components. Total carotenoids and β-carotene contents were significantly associated with genes zeaxanthin epoxydase (ZEP), phytoene desaturase (PDS) and carotenoid isomerase (CRTISO) while α-carotene was associated with CRTISO and plastid terminal oxidase (PTOX) genes. Color components were associated most significantly with ZEP. Our results suggest the involvement of the couple PDS/PTOX and ZEP in carotenoid accumulation, as the result of the metabolic and catabolic activities respectively. This study brings new insights in the understanding of the carotenoid pathway in non-photosynthetic organs.

Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis.

Strigolactones (SLs) are a class of phytohormones and rhizosphere signaling compounds with high structural diversity. Three enzymes, carotenoid isomerase DWARF27 and carotenoid cleavage dioxygenases CCD7 and CCD8, were previously shown to convert all-trans-β-carotene to carlactone (CL), the SL precursor. However, how CL is metabolized to SLs has remained elusive. Here, by reconstituting the SL biosynthetic pathway in Nicotiana benthamiana, we show that a rice homolog of Arabidopsis More Axillary Growth 1 (MAX1), encodes a cytochrome P450 CYP711 subfamily member that acts as a CL oxidase to stereoselectively convert CL into ent-2'-epi-5-deoxystrigol (B-C lactone ring formation), the presumed precursor of rice SLs. A protein encoded by a second rice MAX1 homolog then catalyzes the conversion of ent-2'-epi-5-deoxystrigol to orobanchol. We therefore report that two members of CYP711 enzymes can catalyze two distinct steps in SL biosynthesis, identifying the first enzymes involved in B-C ring closure and a subsequent structural diversification step of SLs.

Erratum to: Loss of Function of the Carotenoid Isomerase Gene BrCRTISO Confers Orange Color to the Inner Leaves of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Erratum to: Loss of Function of the Carotenoid Isomerase Gene BrCRTISO Confers Orange Color to the Inner Leaves of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Identification and characterization of a new allele for ZEBRA LEAF 2, a gene encoding carotenoid isomerase in rice

The Japonica rice cultivar ‘Nipponbare’ was mutagenized by ethyl methane sulfonate (EMS) to obtain a stable and heritable “zebra leaf” mutant zb2. Under natural conditions, yellow and green striped leaves appeared at the seedling stage of this mutant and the leaves gradually turned pale green towards the end of the tillering stage. Both temperature and illumination affected the traits of zb2 mutants. Compared with the wild type, concentrations of chlorophyll and carotenoid were significantly lower in zb2 mutants. Electron microscopic examination of the leaves revealed aberrant chloroplast structure in zb2 mutants. The zb2 mutant phenotype was found to be caused by recessive mutations in a single nuclear gene. By using map-based cloning, the target gene was mapped to a 111.9kb region on chromosome 11. Sequencing analysis revealed a G to T mutation in exon 9 of the ZB2 gene in the mutant. This mutation transformed the 413th Glu residue in the mRNA to a stop codon, resulting in early termination of translation. The ZB2 gene encodes carotenoid isomerase, a key enzyme in carotenoid synthesis, was designated a new allele of OsCRTISO. These results indicated that zb2 is a new allele of Oscrtiso/phs3. Real-time fluorescence quantitative PCR revealed that expression of ZB2 was highest in the leaves compared to the other organs of wild type plants. Furthermore, ZB2 expression was found to be significantly lower in the mutants compared to the wild type. Our findings further elucidate the function of the OsCRTISO gene and contribute to growing understanding of the metabolic pathway of carotenoids in rice.

Melatonin promotes ripening and improves quality of tomato fruit during postharvest life.

In this study, the effect of melatonin on the postharvest ripening and quality improvement of tomato fruit was carried out. The tomatoes were immersed in exogenous melatonin for 2h, and then the related physiological indicators and the expression of genes during post-harvest life were evaluated. Compared with control check (CK), the 50 µM melatonin treatment significantly increased lycopene levels by 5.8-fold. Meanwhile, the key genes involved in fruit colour development, including phytoene synthase1 (PSY1) and carotenoid isomerase (CRTISO), showed a 2-fold increase in expression levels. The rate of water loss from tomato fruit also increased 8.3%, and the expression of aquaporin genes, such as SlPIP12Q, SlPIPQ, SlPIP21Q, and SlPIP22, was up-regulated 2- to 3-fold under 50 µM melatonin treatment. In addition, 50 µM melatonin treatment enhanced fruit softening, increased water-soluble pectin by 22.5%, and decreased protopectin by 19.5%. The expression of the cell wall modifying proteins polygalacturonase (PG), pectin esterase1 (PE1), β-galactosidase (TBG4), and expansin1 (Exp1) was up-regulated under 50 µM melatonin treatment. Melatonin increased ethylene production by 27.1%, accelerated the climacteric phase, and influenced the ethylene signalling pathway. Alteration of ethylene production correlated with altered 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS4) expression. The expression of ethylene signal transduction-related genes such as NR, SlETR4, SlEIL1, SlEIL3, and SlERF2, was enhanced by 50 µM melatonin. The effect of melatonin on ethylene biosynthesis, ethylene perception, and ethylene signalling may contribute to fruit ripening and quality improvement in tomato. This research may promote the application of melatonin on postharvest ripening and quality improvement of tomato fruit as well as other horticultural productions in the future.

Loss of Function of the Carotenoid Isomerase Gene BrCRTISO Confers Orange Color to the Inner Leaves of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage varieties with orange inner leaves are of interest due to their attractive appearance and high carotenoid content. Previously, a recessive locus, Br-or, that confers this trait was mapped to linkage group A09 in Brassica rapa, and Bra031539 was identified as the candidate gene. We used a backcross population consisting of 1,392 individuals for fine mapping; three linked markers delimited the Br-or locus to a region of 9.47 kb, which contained only one predicted functional gene, Bra031539 (the carotenoid isomerase gene BrCRTISO). We identified two bacterial artificial chromosome (BAC) clones carrying Br-or and confirmed that this allele of BrCRTISO confers the mutant phenotype by sequencing. Many DNA sequence variations are present in Br-or, including a 90-bp DNA deletion in the promoter and a 501-bp insertion at the 3′ end of the gene. GUS staining showed that the spatial expression of BrCRTISO was perfectly coincident with the site of carotenoid synthesis and that the 90-bp deletion in the promoter slightly downregulated expression of the gene. Using an in vitro enzyme assay, we showed that the mutant Br-or protein cannot convert prolycopene to all-trans-lycopene. Light has a positive effect on the expression of carotenoid biosynthesis genes, except for beta-ring carotene hydroxylase (CHXB), and this coincided with the increase in total lycopene and β-carotene in the Br-or mutant. We also demonstrated that loss of BrCRTISO function, upregulation of the upstream genes, and downregulation of downstream genes lead to the accumulation of prolycopene and confer an orange color to the inner head leaves in Chinese cabbage.