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.
Carotenoids; Citrus fruit; gene expression; grapefruit; lycopene; lycopene β-cyclase; orange fruit
Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (CrtB). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the β-carotene hydroxylase gene (HYD), which was suppressed in the calli. Co-expression of CrtB and HYD in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of β-carotene. In addition, a model is proposed to interpret the common appearance of a favoured β,β-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.
Amyloplast; carotenogenesis; chromoplast; citrus; CrtB; crystal; engineered cell model; flavedo; phytoene synthase
The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-ϵ) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene.
Beta-carotene; carotenoid biosynthesis; gene expression; kiwifruit; lycopene beta-cyclase
Most studies of the biochemical and regulatory pathways that are associated with, and control, fruit expansion and ripening are based on homogenized bulk tissues, and do not take into consideration the multiplicity of different cell types from which the analytes, be they transcripts, proteins or metabolites, are extracted. Consequently, potentially valuable spatial information is lost and the lower abundance cellular components that are expressed only in certain cell types can be diluted below the level of detection. In this study, laser microdissection (LMD) was used to isolate epidermal and subepidermal cells from green, expanding Citrus clementina fruit and their transcriptomes were compared using a 20k citrus cDNA microarray and quantitative real-time PCR. The results show striking differences in gene expression profiles between the two cell types, revealing specific metabolic pathways that can be related to their respective organelle composition and cell wall specialization. Microscopy provided additional evidence of tissue specialization that could be associated with the transcript profiles with distinct differences in organelle and metabolite accumulation. Subepidermis predominant genes are primarily involved in photosynthesis- and energy-related processes, as well as cell wall biosynthesis and restructuring. By contrast, the most epidermis predominant genes are related to the biosynthesis of the cuticle, flavonoids, and defence responses. Furthermore, the epidermis transcript profile showed a high proportion of genes with no known function, supporting the original hypothesis that analysis at the tissue/cell specific levels can promote gene discovery and lead to a better understanding of the specialized contribution of each tissue to fruit physiology.
Cell wall; citrus; cuticle; epidermis; fruit; laser microdissection; microarray; transcriptome
Research on citrus fruit ripening has received considerable attention because of the importance of citrus fruits for the human diet. Organic acids are among the main determinants of taste and organoleptic quality of fruits and hence the control of fruit acidity loss has a strong economical relevance. In citrus, organic acids accumulate in the juice sac cells of developing fruits and are catabolized thereafter during ripening. Aconitase, that transforms citrate to isocitrate, is the first step of citric acid catabolism and a major component of the citrate utilization machinery. In this work, the citrus aconitase gene family was first characterized and a phylogenetic analysis was then carried out in order to understand the evolutionary history of this family in plants. Gene expression analyses of the citrus aconitase family were subsequently performed in several acidic and acidless genotypes to elucidate their involvement in acid homeostasis.
Analysis of 460,000 citrus ESTs, followed by sequencing of complete cDNA clones, identified in citrus 3 transcription units coding for putatively active aconitate hydratase proteins, named as CcAco1, CcAco2 and CcAco3. A phylogenetic study carried on the Aco family in 14 plant species, shows the presence of 5 Aco subfamilies, and that the ancestor of monocot and dicot species shared at least one Aco gene. Real-time RT-PCR expression analyses of the three aconitase citrus genes were performed in pulp tissues along fruit development in acidic and acidless citrus varieties such as mandarins, oranges and lemons. While CcAco3 expression was always low, CcAco1 and CcAco2 genes were generally induced during the rapid phase of fruit growth along with the maximum in acidity and the beginning of the acid reduction. Two exceptions to this general pattern were found: 1) Clemenules mandarin failed inducing CcAco2 although acid levels were rapidly reduced; and 2) the acidless "Sucreña" orange showed unusually high levels of expression of both aconitases, an observation correlating with the acidless phenotype. However, in the acidless "Dulce" lemon aconitase expression was normal suggesting that the acidless trait in this variety is not dependent upon aconitases.
Phylogenetic studies showed the occurrence of five different subfamilies of aconitate hydratase in plants and sequence analyses indentified three active genes in citrus. The pattern of expression of two of these genes, CcAco1 and CcAco2, was normally associated with the timing of acid content reduction in most genotypes. Two exceptions to this general observation suggest the occurrence of additional regulatory steps of citrate homeostasis in citrus.
In the present study, to investigate the mechanisms regulating carotenoid accumulation in citrus, a culture system was set up in vitro with juice sacs of three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). The juice sacs of all the three varieties enlarged gradually with carotenoid accumulation. The changing patterns of carotenoid content and the expression of carotenoid metabolic genes in juice sacs in vitro were similar to those ripening on trees in the three varieties. Using this system, the changes in the carotenoid content and the expression of carotenoid metabolic genes in response to environmental stimuli were investigated. The results showed that carotenoid accumulation was induced by blue light treatment, but was not affected by red light treatment in the three varieties. Different regulation of CitPSY expression, which was up-regulated by blue light while unaffected by red light, led to different changes in carotenoid content in response to these two treatments in Satsuma mandarin and Valencia orange. In all three varieties, increases in carotenoid content were observed with sucrose and mannitol treatments. However, the accumulation of carotenoid in the two treatments was regulated by distinct mechanisms at the transcriptional level. With abscisic acid (ABA) treatment, the expression of the genes investigated in this study was up-regulated in Satsuma mandarin and Lisbon lemon, indicating that ABA induced its own biosynthesis at the transcriptional level. This feedback regulation of ABA led to decreases in carotenoid content. With gibberellin (GA) treatment, carotenoid content was significantly decreased in the three varieties. Changes in the expression of genes related to carotenoid metabolism varied among the three varieties in response to GA treatment. These results provided insights into improving carotenoid content and composition in citrus during fruit maturation.
Carotenoid; citrus; in vitro; juice sacs; regulatory mechanism
Banana (Musa sp var ‘Robusta’) fruits harvested at 75–80% maturity were dip treated with different concentrations of ethrel (250–1,000 ppm) solution for 5 min. Ethrel at 500 ppm induced uniform ripening without impairing taste and flavour of banana. Untreated control banana fruits remained shriveled, green and failed to ripen evenly even after 8 days of storage. Fruits treated with 500 ppm of ethrel ripened well in 6 days at 20 ± 1 °C. Changes in total soluble solids, acidity, total sugars and total carotenoids showed increasing trends up to 6 days during ripening whereas fruit shear force values, pulp pH and total chlorophyll in peel showed decreasing trends. Sensory quality of ethrel treated banana fruits (fully ripe) were excellent with respect to external colour, taste, flavour and overall quality.
Robusta banana; Musa sp; 2-Chloro ethyl phosphonic acid; Ripening
The disappearance of chlorophyll is a visual sign of fruit ripening. Yet, chlorophyll breakdown in fruit has hardly been explored; its non-green degradation products are largely unknown. Here we report the analysis and structure elucidation of colorless tetrapyrrolic chlorophyll breakdown products in commercially available, ripening bananas (Musa acuminata, Cavendish cultivar). In banana peels, chlorophyll catabolites were found in an unprecedented structural richness: a variety of new fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs) were detected. As a rule, FCCs exist only "fleetingly" and are hard to observe. However, in bananas several of the FCCs (named Mc-FCCs) were persistent and carried an ester function at the propionate side-chain. NCCs were less abundant, and exhibited a free propionic acid group, but functional modifications elsewhere. The modifications of NCCs in banana peels were similar to those found in NCCs from senescent leaves. They are presumed to be introduced by enzymatic transformations at the stage of the mostly unobserved, direct FCC-precursors. The observed divergent functional group characteristics of the Mc-FCCs versus those of the Mc-NCCs indicated two major "late" processing lines of chlorophyll breakdown in ripening bananas. The "last common precursor" at the branching point to either the persistent FCCs, or towards the NCCs, was identified as a temporarily abundant "secondary" FCC. The existence of two "downstream" branches of chlorophyll breakdown in banana peels, and the striking accumulation of persistent Mc-FCCs call for attention as to the still-elusive biological roles of the resulting colorless linear tetrapyrroles.
catabolite; chlorophyll; fruit; luminescence; pigment; structure elucidation
Background and Aims
There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase.
Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices.
At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts.
These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.
Chloroplast; chromoplast; confocal; pigment fluorescence; Solanum lycopersicum; tomato
Capsicum species produce fruits that synthesize and accumulate carotenoid pigments, which are responsible for the fruits’ yellow, orange and red colors. Chili peppers have been used as an experimental model for studying the biochemical and molecular aspects of carotenoid biosynthesis. Most reports refer to the characterization of carotenoids and content determination in chili pepper fruits from different species, cultivars, varieties or genotypes. The types and levels of carotenoids differ between different chili pepper fruits, and they are also influenced by environmental conditions. Yellow-orange colors of chili pepper fruits are mainly due to the accumulation of α- and β-carotene, zeaxanthin, lutein and β-cryptoxanthin. Carotenoids such as capsanthin, capsorubin and capsanthin-5,6-epoxide confer the red colors. Chromoplasts are the sites of carotenoid pigment synthesis and storage. According to the most accepted theory, the synthesis of carotenoids in chili peppers is controlled by three loci: c1, c2 and y. Several enzymes participating in carotenoid biosynthesis in chili pepper fruits have been isolated and characterized, and the corresponding gene sequences have been reported. However, there is currently limited information on the molecular mechanisms that regulate this biosynthetic pathway. Approaches to gain more knowledge of the regulation of carotenoid biosynthesis are discussed.
Capsicum; carotenoids; chili pepper; biosynthesis; regulation
Carotenoid accumulation confers distinct colouration to plant tissues, with effects on plant response to light and as well as health benefits for consumers of plant products. The carotenoid pathway is controlled by flux of metabolites, rate-limiting enzyme steps, feed-back inhibition, and the strength of sink organelles, the plastids, in the cell. In apple (Malus × domestica Borkh), fruit carotenoid concentrations are low in comparison with those in other fruit species. The apple fruit flesh, in particular, begins development with high amounts of chlorophylls and carotenoids, but in all commercial cultivars a large proportion of this is lost by fruit maturity. To understand the control of carotenoid concentrations in apple fruit, metabolic and gene expression analysis of the carotenoid pathway were measured in genotypes with varying flesh and skin colour. Considerable variation in both carotenoid concentrations and compound profile was observed between tissues and genotypes, with carotenes and xanthophylls being found only in fruit accumulating high carotenoid concentrations. The study identified potential rate-limiting steps in carotenogenesis, which suggested that the expression of ZISO, CRTISO,and LCY-ε, in particular, were significant in predicting final carotenoid accumulation in mature apple fruit.
Apple; carotenoid isomerase; carotenoids; gene expression; Malus × domestica; Apple
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.
Carotenoid; chloroplast; chromoplast; colour; gene expression; fruit development; fruit ripening; loquat; plastid
The pericarp of Capsicum fruit is a rich dietary source of carotenoids. Accumulation of these compounds may be controlled, in part, by gene transcription of biosynthetic enzymes. The carotenoid composition in a number of orange-coloured C. annuum cultivars was determined using HPLC and compared with transcript abundances for four carotenogenic enzymes, Psy, LcyB, CrtZ-2, and Ccs determined by qRT-PCR. There were unique carotenoid profiles as well as distinct patterns of transcription of carotenogenic enzymes within the seven orange-coloured cultivars. In one cultivar, ‘Fogo’, carrying the mutant ccs-3 allele, transcripts were detected for this gene, but no CCS protein accumulated. The premature stop termination in ccs-3 prevented expression of the biosynthetic activity to synthesize the capsanthin and capsorubin forms of carotenoids. In two other orange-coloured cultivars, ‘Orange Grande’ and ‘Oriole’, both with wild-type versions of all four carotenogenic enzymes, no transcripts for Ccs were detected and no red pigments accumulated. Finally, in a third case, the orange-coloured cultivar, Canary, transcripts for all four of the wild-type carotenogenic enzymes were readily detected yet no CCS protein appeared to accumulate and no red carotenoids were synthesized. In the past, mutations in Psy and Ccs have been identified as the loci controlling colour in the fruit. Now there is evidence that a non-structural gene may control colour development in Capsicum.
Capsanthin; β-carotene; carotenoid biosynthesis; chromoplast; peppers; pericarp; qRT-PCR; xanthophylls
The characteristics of fruit ripening and expression of ripening-related genes were investigated in epi, an ethylene overproduction mutant of tomato (Lycopersicon esculentum Mill.). The epi produces apparently more ethylene than its wild type VFN8 at every stage of vegetative and fruit growth and ripening; compared to VFN8, the epi fruit showed higher CO2 evolution, faster descending of chlorophyll, slightly quicker increase of carotenoid and lycopene, and faster reduction in pericarp firmness during maturation and ripening; and the mRNAs of three ripening-related genes including E8, pTOM5 and pTOM6 were at higher levels in epi. The ripening-related characteristics changing of the fruit are consistent with the increase of ethylene production and ripening-related genes expression. These results suggest that epi mutation possibly did not affect the ethylene perception and signaling during fruit ripening, and that the modified characteristics of fruit ripening possibly resulted from the ethylene overproduction and increased expression of ripening-related genes.
Epinastic (epi) mutant; Ethylene overproduction; Ethylene signaling; Fruit ripening
Gardenia fruit (fruit of Gardenia jasminoides Ellis) is used as a natural pigment resource and a Chinese traditional medicine. The white mesocarp turning orange or red that occurs during gardenia fruit maturation arises from the production and accumulation of the apocarotenoids, especially crocin-1, which is derived from carotenoid. Meanwhile, the major medical component geniposide is accumulated in gardenia fruit. To further our understanding of the synthetic and accumulation mechanism for crocin-1 and geniposide in gardenia fruit, the contents of crocin-1 and geniposide and the transcripts of phytoene synthase (GjPSY) profiles in gardenia fruits were examined at various stages of maturation. The concentration of crocin-1 and geniposide in gardenia fruit was determined by reversed-phase high-performance liquid chromatography (HPLC). The results showed that the concentration of crocetin-1 was increased during fruit development and the concentration of geniposide does not change significantly during maturing. The expression levels of GjPSY mRNA were examined by RT-PCR. It was revealed that GjPSY was constitutively expressed during fruit development, suggesting that the primary mechanism that controls crocin accumulation in G. jasminoides fruits during development is not correlated to the differential regulation of transcript levels of GjPSY gene.
Huanglongbing (HLB) or “citrus greening” is the most destructive citrus disease worldwide. In this work, we studied host responses of citrus to infection with Candidatus Liberibacter asiaticus (CaLas) using next-generation sequencing technologies. A deep mRNA profile was obtained from peel of healthy and HLB-affected fruit. It was followed by pathway and protein-protein network analysis and quantitative real time PCR analysis of highly regulated genes. We identified differentially regulated pathways and constructed networks that provide a deep insight into the metabolism of affected fruit. Data mining revealed that HLB enhanced transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. Activation of protein degradation and misfolding processes were observed at the transcriptomic level. Transcripts for heat shock proteins were down-regulated at all disease stages, resulting in further protein misfolding. HLB strongly affected pathways involved in source-sink communication, including sucrose and starch metabolism and hormone synthesis and signaling. Transcription of several genes involved in the synthesis and signal transduction of cytokinins and gibberellins was repressed while that of genes involved in ethylene pathways was induced. CaLas infection triggered a response via both the salicylic acid and jasmonic acid pathways and increased the transcript abundance of several members of the WRKY family of transcription factors. Findings focused on the fruit provide valuable insight to understanding the mechanisms of the HLB-induced fruit disorder and eventually developing methods based on small molecule applications to mitigate its devastating effects on fruit production.
The present work investigates the effect of ripening stage on the chemical composition of essential oil extracted from peel of four citrus: bitter orange (Citrus aurantium), lemon (Citrus limon), orange maltaise (Citrus sinensis), and mandarin (Citrus reticulate) and on their antibacterial activity. Essential oils yields varied during ripening from 0.46 to 2.70%, where mandarin was found to be the richest. Forty volatile compounds were identified. Limonene (67.90–90.95%) and 1,8-cineole (tr-14.72%) were the most represented compounds in bitter orange oil while limonene (37.63–69.71%), β-pinene (0.63–31.49%), γ-terpinene (0.04–9.96%), and p-cymene (0.23–9.84%) were the highest ones in lemon. In the case of mandarin, the predominant compounds were limonene (51.81–69.00%), 1,8-cineole (0.01–26.43%), and γ-terpinene (2.53–14.06%). However, results showed that orange peel oil was dominated mainly by limonene (81.52–86.43%) during ripening. The results showed that ripening stage influenced significantly the antibacterial activity of the oils against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This knowledge could help establish the optimum harvest date ensuring the maximum essential oil, limonene, as well as antibacterial compounds yields of citrus.
In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutations in the Psy1 gene. The first mutation is a knockout allele (W180*) and the second mutation leads to an amino acid substitution (P192L). Plants carrying the Psy1 knockout allele show fruit with a yellow flesh colour similar to the r, r mutant, with no further change in colour during ripening. In the line with P192L substitution, fruit remain yellow until 3 days post-breaker and eventually turn red. Metabolite profiling verified the absence of carotenoids in the W180* line and thereby confirms that PSY1 is the only enzyme introducing substrate into the carotenoid pathway in ripening fruit. More subtle effects on carotenoid accumulation were observed in the P192L line with a delay in lycopene and β-carotene accumulation clearly linked to a very slow synthesis of phytoene. The observation of lutein degradation with ripening in both lines showed that lutein and its precursors are still synthesised in ripening fruit. Gene expression analysis of key genes involved in carotenoid biosynthesis revealed that expression levels of genes in the pathway are not feedback-regulated by low levels or absence of carotenoid compounds. Furthermore, protein secondary structure modelling indicated that the P192L mutation affects PSY1 activity through misfolding, leading to the low phytoene accumulation.
Carotenoids; Point mutation; Psy1; Tomato; TILLING
Citrus species constitute one of the major tree fruit crops of the subtropical regions with great economic importance. However, their peculiar reproductive characteristics, low genetic diversity and the long-term nature of tree breeding mostly impair citrus variety improvement. In woody plants, genomic science holds promise of improvements and in the Citrus genera the development of genomic tools may be crucial for further crop improvements. In this work we report the characterization of three BAC libraries from Clementine (Citrus clementina), one of the most relevant citrus fresh fruit market cultivars, and the analyses of 46.000 BAC end sequences. Clementine is a diploid plant with an estimated haploid genome size of 367 Mb and 2n = 18 chromosomes, which makes feasible the use of genomics tools to boost genetic improvement.
Three genomic BAC libraries of Citrus clementina were constructed through EcoRI, MboI and HindIII digestions and 56,000 clones, representing an estimated genomic coverage of 19.5 haploid genome-equivalents, were picked. BAC end sequencing (BES) of 28,000 clones produced 28.1 Mb of genomic sequence that allowed the identification of the repetitive fraction (12.5% of the genome) and estimation of gene content (31,000 genes) of this species. BES analyses identified 3,800 SSRs and 6,617 putative SNPs. Comparative genomic studies showed that citrus gene homology and microsyntheny with Populus trichocarpa was rather higher than with Arabidopsis thaliana, a species phylogenetically closer to citrus.
In this work, we report the characterization of three BAC libraries from C. clementina, and a new set of genomic resources that may be useful for isolation of genes underlying economically important traits, physical mapping and eventually crop improvement in Citrus species. In addition, BAC end sequencing has provided a first insight on the basic structure and organization of the citrus genome and has yielded valuable molecular markers for genetic mapping and cloning of genes of agricultural interest. Paired end sequences also may be very helpful for whole-genome sequencing programs.
One of the main characteristics of tomato (Solanum lycopersicum) fruit ripening is a massive accumulation of carotenoids (mainly lycopene), which may contribute to the nutrient quality of tomato fruit and its role in chemoprevention. Previous studies have shown that ethylene (ET) plays a central role in promoting fruit ripening. In this study, the role of jasmonic acid (JA) in controlling lycopene accumulation in tomato fruits was analysed by measuring fruit lycopene content and the expression levels of lycopene biosynthetic genes in JA-deficient mutants (spr2 and def1) and a 35S::prosystemin transgenic line (35S::prosys) with increased JA levels and constitutive JA signalling. The lycopene content was significantly decreased in the fruits of spr2 and def1, but was enhanced in 35S::prosys fruits. Simultaneously, the expression of lycopene biosynthetic genes followed a similar trend. Lycopene synthesis in methyl jasmonate (MeJA) vapour-treated fruits showed an inverted U-shaped dose response, which significantly enhanced the fruit lycopene content and restored lycopene accumulation in spr2 and def1 at a concentration of 0.5 µM. The results indicated that JA plays a positive role in lycopene biosynthesis. In addition, the role of ET in JA-induced lycopene accumulation was also examined. Ethylene production in tomato fruits was depressed in spr2 and def1 while it increased in 35S::prosys. However, the exogenous application of MeJA to Never ripe (Nr), the ET-insensitive mutant, significantly promoted lycopene accumulation, as well as the expression of lycopene biosynthetic genes. Based on these results, it is proposed that JA might function independently of ethylene to promote lycopene biosynthesis in tomato fruits.
Ethylene (ET); fruit; jasmonic acid (JA); lycopene; mutant; tomato
Pepper, Capsicum spp., is a worldwide crop valued for heat, nutrition, and rich pigment content. Carotenoids, the largest group of plant pigments, function as antioxidants and as vitamin A precursors. The most abundant carotenoids in ripe pepper fruits are β-carotene, capsanthin, and capsorubin. In this study, the carotenoid composition of orange fruited Capsicum lines was defined along with the allelic variability of the biosynthetic enzymes. The carotenoid chemical profiles present in seven orange pepper varieties were determined using a novel UPLC method. The orange appearance of the fruit was due either to the accumulation of β-carotene, or in two cases, due to only the accumulation of red and yellow carotenoids. Four carotenoid biosynthetic genes, Psy, Lcyb, CrtZ-2, and Ccs were cloned and sequenced from these cultivars. This data tested the hypothesis that different alleles for specific carotenoid biosynthetic enzymes are associated with specific carotenoid profiles in orange peppers. While the coding regions within Psy and CrtZ-2 did not change in any of the lines, the genomic sequence contained introns not previously reported. Lcyb and Ccs contained no introns but did exhibit polymorphisms resulting in amino acid changes; a new Ccs variant was found. When selectively breeding for high provitamin A levels, phenotypic recurrent selection based on fruit color is not sufficient, carotenoid chemical composition should also be conducted. Based on these results, specific alleles are candidate molecular markers for selection of orange pepper lines with high β-carotene and therefore high pro-vitamin A levels.
fruit pigmentation; carotenes; xanthophylls; capsanthin; capsorubin
The transcriptome of the fruit pulp of the sweet orange variety Anliu (WT) and that of its red fleshed mutant Hong Anliu (MT) were compared to understand the dynamics and differential expression of genes expressed during fruit development and ripening.
The transcriptomes of WT and MT were sampled at four developmental stages using an Illumina sequencing platform. A total of 19,440 and 18,829 genes were detected in MT and WT, respectively. Hierarchical clustering analysis revealed 24 expression patterns for the set of all genes detected, of which 20 were in common between MT and WT. Over 89% of the genes showed differential expression during fruit development and ripening in the WT. Functional categorization of the differentially expressed genes revealed that cell wall biosynthesis, carbohydrate and citric acid metabolism, carotenoid metabolism, and the response to stress were the most differentially regulated processes occurring during fruit development and ripening.
A description of the transcriptomic changes occurring during fruit development and ripening was obtained in sweet orange, along with a dynamic view of the gene expression differences between the wild type and a red fleshed mutant.
In recent years, there has been increasing public interest in plant antioxidants, thanks to the potential anticarcinogenic and cardioprotective actions mediated by their biochemical properties. The red (or blood) orange (Citrus sinensis (L.) Osbeck) is a pigmented sweet orange variety typical of eastern Sicily (southern Italy), California, and Spain. In this paper, we discuss the main health-related properties of the red orange that include anticancer, anti-inflammatory, and cardiovascular protection activities. Moreover, the effects on health of its main constituents (namely, flavonoids, carotenoids, ascorbic acid, hydroxycinnamic acids, and anthocyanins) are described. The red orange juice demonstrates an important antioxidant activity by modulating many antioxidant enzyme systems that efficiently counteract the oxidative damage which may play an important role in the etiology of numerous diseases, such as atherosclerosis, diabetes, and cancer. The beneficial effects of this fruit may be mediated by the synergic effects of its compounds. Thus, the supply of natural antioxidant compounds through a balanced diet rich in red oranges might provide protection against oxidative damage under differing conditions and could be more effective than, the supplementation of an individual antioxidant.
Citrus, as one of the globally important fruit trees, has been an object of interest for understanding genetics and evolutionary process in fruit crops. Meta-analyses of 19 Citrus species, including 4 globally and economically important Citrus sinensis, Citrus clementina, Citrus reticulata, and 1 Citrus relative Poncirus trifoliata, were performed. We observed that codons ending with A- or T- at the wobble position were preferred in contrast to C- or G- ending codons, indicating a close association with AT richness of Citrus species and P. trifoliata. The present study postulates a large repertoire of a set of optimal codons for the Citrus genus and P. trifoliata and demonstrates that GCT and GGT are evolutionary conserved optimal codons. Our observation suggested that mutational bias is the dominating force in shaping the codon usage bias (CUB) in Citrus and P. trifoliata. Correspondence analysis (COA) revealed that the principal axis [axis 1; COA/relative synonymous codon usage (RSCU)] contributes only a minor portion (∼10.96%) of the recorded variance. In all analysed species, except P. trifoliata, Gravy and aromaticity played minor roles in resolving CUB. Compositional constraints were found to be strongly associated with the amino acid signatures in Citrus species and P. trifoliata. Our present analysis postulates compositional constraints in Citrus species and P. trifoliata and plausible role of the stress with GC3 and coevolution pattern of amino acid.
Citrus; coevolution; mutational bias; Poncirus trifoliata; stress; GC3 biology
Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background.
Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues.
In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement.