Carotenoids are a complex class of isoprenoid pigments playing diverse roles in plants and providing nutritional value. Metabolic engineering of the biosynthetic pathway has been of interest to specifically address global vitamin A deficiency by breeding cereal crop staples in the Poaceae (Grass family) for elevated levels of provitamin A carotenoids. However, there remain open questions about the rate-controlling steps that limit predictability of metabolic engineering in plants, whether by transgenic or nontransgenic means. We decided to focus on the first committed biosynthetic step which is mediated by phytoene synthase. Our studies revealed that in the Grasses, PSY is encoded by three genes. Maize transcript profiling, together with carotenoid and ABA analysis, revealed that the three PSY copies have subfunctionalized and provide the Grasses with a fine tine control of carotenogenesis in response to various developmental and external cues. Promoter analysis supports subfunctionalization; cis-element analysis of maize PSY1 alleles and comparison with Grass orthologs suggests that man's selection of yellow maize endosperm has occurred at the expense of a change of gene regulation in photosynthetic tissue as compared to the progenitor white endosperm PSY1 allele.
carotenoid biosynthesis; phytoene synthase; gene subfunctionalization; ABA; abiotic stress; transcriptional regulation
The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species.
We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5′ UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system.
The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.
Carotenoids are plastidial isoprenoid pigments essential for plant life. High carotenoid levels are found in chloroplasts and chromoplasts, but they are also produced in the etioplasts of seedlings that germinate in the dark. Our recent work has shown that an enhanced production of carotenoids in plastids of dark-grown Arabidopsis thaliana seedlings results in an improved transition to photosynthetic development (greening) upon illumination, illustrating the relevance of regulating etioplast carotenoid biosynthesis for plant fitness. We showed that the biosynthesis of etioplast carotenoids is controlled at the level of phytoene synthase (PSY), the enzyme catalyzing the first committed step of the pathway. Upregulation of PSY is necessary and sufficient to increase the production of carotenoids in dark-grown seedlings, in part because it triggers a feedback mechanism leading to the post-transcriptional accumulation of flux-controlling enzymes of the methylerythritol 4-phosphate (MEP) pathway, which synthesizes the substrates for PSY activity. Based on these and other recent data on the molecular mechanisms controlling deetiolation, we propose a model for the regulation of carotenoid biosynthesis in etioplasts.
carotenoid; deetiolation; etioplast; feedback regulation; MEP pathway
The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.
Carotenoids; Chlorella zofingiensis; Phytoene synthase; Transgenic microalgae; Chlamydomonas reinhardtii
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
The wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae.
Twelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2Hch, 3Hch and 7Hch in H. chilense, the former being the most significant one.
The results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2Hch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the Hch genome may constitute a valuable tool for YPC studies in the Triticeae.
Yellow pigment content (YPC); Macrocolinearity; Candidate genes; H. chilense; H. vulgare
As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.
In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.
The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.
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 carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.
A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of chlorophyll biosynthesis genes in a manner that is consistent with the increased synthesis of carotenoid precursors for ABA biosynthesis. In all tissues examined, induction of β-carotene hydroxylase transcript levels are linked to an increased demand for ABA.
This analysis provides compelling evidence to suggest that coordinated transcriptional regulation of isoprenoid-related biosynthesis pathway genes plays a major role in coordinating the synthesis of functionally related chloroplast localized isoprenoid-derived compounds.
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
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.
The green alga Haematococcus pluvialis produces large amounts of the pink carotenoid astaxanthin under high photon flux density (PFD) and other oxidative stress conditions. However, the regulation and physiological role of carotenogenesis leading to astaxanthin formation is not well understood. Comparative transcriptional expression of five carotenoid genes along with growth and pigment composition as a function of PFD was studied using a wild-type and an astaxanthin-overproduction mutant of H. pluvialis NIES144. The results indicate that astaxanthin biosynthesis was mainly under transcriptional control of the gene encoding carotenoid hydroxylase, and to a lesser extent, the genes encoding isopentenyl isomerase and phytoene desaturase, and to the least extent, the genes encoding phytoene synthase and carotenoid oxygenase. The expression of a plastid terminal oxidase (PTOX) gene ptox2 underwent transient up-regulation under elevated PFDs, suggesting that PTOX may be functionally coupled with phytoene desaturase through the plastoquinone pool and may play a role in reducing redox-potential-dependent and oxygen-concentration-dependent formation of reactive oxygen species in the chloroplast. Over-expression of both the carotenogenic and PTOX genes confers to the astaxanthin-overproduction mutant more effective photoprotective capability than that of the wild type under photooxidative stress.
Astaxanthin; Haematococcus pluvialis; High light; mRNA expression; Oxidative stress
Since the creation of “Golden Rice”, biofortification of plant-derived foods is a promising strategy for the alleviation of nutritional deficiencies. Potato is the most important staple food for mankind after the cereals rice, wheat and maize, and is extremely poor in provitamin A carotenoids.
We transformed potato with a mini-pathway of bacterial origin, driving the synthesis of beta-carotene (Provitamin A) from geranylgeranyl diphosphate. Three genes, encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene beta-cyclase (CrtY) from Erwinia, under tuber-specific or constitutive promoter control, were used. 86 independent transgenic lines, containing six different promoter/gene combinations, were produced and analyzed. Extensive regulatory effects on the expression of endogenous genes for carotenoid biosynthesis are observed in transgenic lines. Constitutive expression of the CrtY and/or CrtI genes interferes with the establishment of transgenosis and with the accumulation of leaf carotenoids. Expression of all three genes, under tuber-specific promoter control, results in tubers with a deep yellow (“golden”) phenotype without any adverse leaf phenotypes. In these tubers, carotenoids increase approx. 20-fold, to 114 mcg/g dry weight and beta-carotene 3600-fold, to 47 mcg/g dry weight.
This is the highest carotenoid and beta-carotene content reported for biofortified potato as well as for any of the four major staple foods (the next best event being “Golden Rice 2”, with 31 mcg/g dry weight beta-carotene). Assuming a beta-carotene to retinol conversion of 6∶1, this is sufficient to provide 50% of the Recommended Daily Allowance of Vitamin A with 250 gms (fresh weight) of “golden” potatoes.
For photosynthesis, phototrophic organisms necessarily synthesize not only chlorophylls but also carotenoids. Many kinds of carotenoids are found in algae and, recently, taxonomic studies of algae have been developed. In this review, the relationship between the distribution of carotenoids and the phylogeny of oxygenic phototrophs in sea and fresh water, including cyanobacteria, red algae, brown algae and green algae, is summarized. These phototrophs contain division- or class-specific carotenoids, such as fucoxanthin, peridinin and siphonaxanthin. The distribution of α-carotene and its derivatives, such as lutein, loroxanthin and siphonaxanthin, are limited to divisions of Rhodophyta (macrophytic type), Cryptophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta. In addition, carotenogenesis pathways are discussed based on the chemical structures of carotenoids and known characteristics of carotenogenesis enzymes in other organisms; genes and enzymes for carotenogenesis in algae are not yet known. Most carotenoids bind to membrane-bound pigment-protein complexes, such as reaction center, light-harvesting and cytochrome b6f complexes. Water-soluble peridinin-chlorophyll a-protein (PCP) and orange carotenoid protein (OCP) are also established. Some functions of carotenoids in photosynthesis are also briefly summarized.
algal phylogeny; biosynthesis of carotenoids; distribution of carotenoids; function of carotenoids; pigment-protein complex
The orange pigmentation of the fungus Neurospora crassa is due to the accumulation of the xanthophyll neurosporaxanthin and precursor carotenoids. Two key reactions in the synthesis of these pigments, the formation of phytoene from geranylgeranyl pyrophosphate and the introduction of β cycles in desaturated carotenoid products, are catalyzed by two domains of a bifunctional protein, encoded by the gene al-2. We have determined the sequence of nine al-2 mutant alleles and analyzed the carotenoid content in the corresponding strains. One of the mutants is reddish and it is mutated in the cyclase domain of the protein, and the remaining eight mutants are albino and harbor different mutations on the phytoene synthase (PS) domain. Some of the mutations are expected to produce truncated polypeptides. A strain lacking most of the PS domain contained trace amounts of a carotenoid-like pigment, tentatively identified as the squalene desaturation product diapolycopene. In support, trace amounts of this compound were also found in a knock-out mutant for gene al-2, but not in that for gene al-1, coding for the carotene desaturase. The cyclase activity of the AL-2 enzyme from two albino mutants was investigated by heterologous expression in an appropriately engineered E. coli strain. One of the AL-2 enzymes, predictably with only 20% of the PS domain, showed full cyclase activity, suggesting functional independence of both domains. However, the second mutant showed no cyclase activity, indicating that some alterations in the phytoene synthase segment affect the cyclase domain. Expression experiments showed a diminished photoinduction of al-2 transcripts in the al-2 mutants compared to the wild type strain, suggesting a synergic effect between reduced expression and impaired enzymatic activities in the generation of their albino phenotypes.
Plant carotenoids are a family of pigments that participate in light harvesting and are essential for photoprotection against excess light. Furthermore, they act as precursors for the production of apocarotenoid hormones such as abscisic acid and strigolactones. In this review, we summarize the current knowledge on the genes and enzymes of the carotenoid biosynthetic pathway (which is now almost completely elucidated) and on the regulation of carotenoid biosynthesis at both transcriptional and post-transcriptional levels. We also discuss the relevance of Arabidopsis as a model system for the study of carotenogenesis and how metabolic engineering approaches in this plant have taught important lessons for carotenoid biotechnology.
Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogen in planta.
The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene phytoene synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.
Genetic modification; mass spectrometry; multidimensional liquid chromatography; phytoene synthase; proteomics; Solanum lycopersicum
Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat.
We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs.
The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.
Pseudomonas syringae pv. tomato strain DC3000, a pathogen of tomato and Arabidopsis, occurs as an epiphyte. It produces N-acyl homoserine lactones (AHLs) which apparently function as quorum-sensing signals. A Tn5 insertion mutant of DC3000, designated PsrA− (Psr is for Pseudomonas sigma regulator), overexpresses psyR (a LuxR-type regulator of psyI) and psyI (the gene for AHL synthase), and it produces a ca. 8-fold-higher level of AHL than does DC3000. The mutant is impaired in its ability to elicit the hypersensitive reaction and is attenuated in its virulence in tomato. These phenotypes correlate with reduced expression of hrpL, the gene for an alternate sigma factor, as well as several hrp and hop genes during early stages of incubation in a Hrp-inducing medium. PsrA also positively controls rpoS, the gene for an alternate sigma factor known to control various stress responses. By contrast, PsrA negatively regulates rsmA1, an RNA-binding protein gene known to function as negative regulator, and aefR, a tetR-like gene known to control AHL production and epiphytic fitness in P. syringae pv. syringae. Gel mobility shift assays and other lines of evidence demonstrate a direct interaction of PsrA protein with rpoS promoter DNA and aefR operator DNA. In addition, PsrA negatively autoregulates and binds the psrA operator. In an AefR− mutant, the expression of psyR and psyI and AHL production are lower than those in DC3000, the AefR+ parent. In an RpoS− mutant, on the other hand, the levels of AHL and transcripts of psyR and psyI are much higher than those in the RpoS+ parent, DC3000. We present evidence, albeit indirect, that the RpoS effect occurs via psyR. Thus, AefR positively regulates AHL production, whereas RpoS has a strong negative effect. We show that AefR and RpoS do not regulate PsrA and that the PsrA effect on AHL production is exerted via its cumulative, but independent, effects on both AefR and RpoS.
Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin.
Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ɛ-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained.
C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.
Corynebacterium; Carotenoid production; Lycopene production; Phytoene synthase; Phytoene desaturase; Lycopene elongase; Carotenoid epsilon-cyclase; Decaprenoxanthin
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 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
Whole genome duplication is a common evolutionary event in plants. Bread wheat (Triticum aestivum L.) is a good model to investigate the impact of paleo- and neoduplications on the organization and function of modern plant genomes.
We performed an RNA sequencing-based inference of the grain filling gene network in bread wheat and identified a set of 37,695 non-redundant sequence clusters, which is an unprecedented resolution corresponding to an estimated half of the wheat genome unigene repertoire. Using the Brachypodium distachyon genome as a reference for the Triticeae, we classified gene clusters into orthologous, paralogous, and homoeologous relationships. Based on this wheat gene evolutionary classification, older duplicated copies (dating back 50 to 70 million years) exhibit more than 80% gene loss and expression divergence while recent duplicates (dating back 1.5 to 3 million years) show only 54% gene loss and 36 to 49% expression divergence.
We suggest that structural shuffling due to duplicated gene loss is a rapid process, whereas functional shuffling due to neo- and/or subfunctionalization of duplicates is a longer process, and that both shuffling mechanisms drive functional redundancy erosion. We conclude that, as a result of these mechanisms, half the gene duplicates in plants are structurally and functionally altered within 10 million years of evolution, and the diploidization process is completed after 45 to 50 million years following polyploidization.
Phosphomannomutase (PMM) is an essential enzyme in eukaryotes. However, little is known about PMM gene and function in crop plants. Here, we report molecular evolutionary and biochemical analysis of PMM genes in bread wheat and related Triticeae species.
Two sets of homoeologous PMM genes (TaPMM-1 and 2) were found in bread wheat, and two corresponding PMM genes were identified in the diploid progenitors of bread wheat and many other diploid Triticeae species. The duplication event yielding PMM-1 and 2 occurred before the radiation of diploid Triticeae genomes. The PMM gene family in wheat and relatives may evolve largely under purifying selection. Among the six TaPMM genes, the transcript levels of PMM-1 members were comparatively high and their recombinant proteins were all enzymatically active. However, PMM-2 homoeologs exhibited lower transcript levels, two of which were also inactive. TaPMM-A1, B1 and D1 were probably the main active isozymes in bread wheat tissues. The three isozymes differed from their counterparts in barley and Brachypodium distachyon in being more tolerant to elevated test temperatures.
Our work identified the genes encoding PMM isozymes in bread wheat and relatives, uncovered a unique PMM duplication event in diverse Triticeae species, and revealed the main active PMM isozymes in bread wheat tissues. The knowledge obtained here improves the understanding of PMM evolution in eukaryotic organisms, and may facilitate further investigations of PMM function in the temperature adaptability of bread wheat.