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We found that ApGGPPS1, ApGGPPS2, and ApGGPPS3 can convert IPP and DMAPP to GGPP. ApGGPPS2 is probably involved in andrographolide biosynthesis. ApGGPPS3 may be responsible for the synthesis of the cytosolic GGPP. Andrographis paniculata is a traditional herb for the treatment of sore throat, flu, upper respiratory tract infections and other disorders. In A. paniculata, GGPP is not only the precursor of andrographolide and its primary bioactive compounds, but also the precursor of chlorophylls, carotenoids, gibberellins, and abscisic acid, which are the biomolecules of photosynthesis, growth regulation and other physiological and ecological processes. In this study, four cDNAs (named ApGGPPS1, ApGGPPS2, ApGGPPS3 and ApGGPPS4) encoding geranylgeranyl pyrophosphate synthases from A. paniculata were putatively isolated. Bioinformatic and phylogenetic analyses suggested that these ApGGPPS are highly similar to the geranylgeranyl pyrophosphate synthases in other plants. Prokaryotic expression showed that ApGGPPS1, ApGGPPS2 and ApGGPPS3 could convert IPP and DMAPP to GGPP, although ApGGPPS4 lacks a similar function. The expression of ApGGPPS2 was similar as ApCPS2 under MeJA treatment, ApCPS2 involved in the biosynthesis pathway of andrographolide (Shen et al., Biotechnol Lett 38:131–137, 2016a), has been proven through Virus-induced Gene Siliencing (VIGS) (Shen et al., Acta Bot Boreal 36:17–22, 2016b), and the subcellular localization of ApGGPPS2 was shown to localize in the plastid, suggested that ApGGPPS2 could be the key synthase in the biosynthesis pathway of andrographolide. In addition, ApGGPPS3 was shown to localize in the cytoplasm, suggested that ApGGPPS3 may be responsible for the synthesis of cytosolic GGPP, which may participate in the synthesis of cytosolic oligoprenols as side chains to produce ubiquinone, dolichols or other isoprenoids, in the synthesis of polyisoprenoids, and in protein prenylation.

Carotenoids play important roles in many biological processes, such as light harvesting, photoprotection and visual attraction in plants. However, the regulation of carotenoid biosynthesis is still not fully understood. Here, we demonstrate that SlBBX20, a B-box (BBX) zinc-finger transcription factor, is a positive regulator of carotenoid accumulation in tomato (Solanum lycopersicum). Overexpression of SlBBX20 leads to dark green fruits and leaves and higher levels of carotenoids relative to the wild-type. Interactions between SlBBX20 and DE-ETIOLATED 1 (SlDET1) lead to the ubiquitination and 26S proteasome-mediated degradation of SlBBX20. Moreover, deficiencies in the components of the CUL4-DDB1-DET1 complex enhanced the stability of the SlBBX20 protein. Thus, we conclude that SlBBX20 is a substrate of the CUL4-DDB1-DET1 E3 ligase. SlBBX20 can activate the expression of PHYTOENE SYNTHASE 1, encoding a key enzyme in carotenoid biosynthesis, by directly binding to a G-box motif in its promoter, which results in the elevated levels of carotenoids in SlBBX20 overexpression lines. We identified a key regulator of carotenoid biosynthesis and demonstrated that the stability of SlBBX20 is regulated by ubiquitination. These findings provide us a new target for the genetic improvement of the nutritional quality of tomato fruit.

Under environmental stresses, Haematococcus pluvialis accumulates large amounts of carotenoids. Scale of carotenoid biosynthesis depends on availability of geranylgeranyl pyrophosphate (GGPP) precursor, which is supplied by GGPP synthase (GGPPS) through sequential 1'-4 condensation of three isopentenyl pyrophosphates (IPPs) into dimethylallyl pyrophosphate (DMAPP). Using IPP and DMAPP as substrates, a tri-functional HpGGPPS was identified in this study to promiscuously synthesize allylic prenyl pyrophosphates (PPPs), e.g. C10 geranyl pyrophosphate (GPP), C15 farnesyl pyrophosphate (FPP), and C20 GGPP. Intriguingly, HpGGPPS can utilize GPP or FPP as a single substrate to synthesize GGPP by hydrolyzing the allylic PPP substrate into C5 IPP. Transcription of HpGGPPS and key carotenogenesis genes, morphological transformation, and carotenoid biosynthesis were differentially induced by environmental stresses, while HpGGPPS's products were low in vivo, implying that most of PPP flux had been shunted into carotenoid biosynthesis. Hydrolyzing allylic PPP intermediates into C5 building blocks by promiscuous HpGGPPS may be a fail safe for carotenoid accumulation against environmental stress.

The membrane protease LonB is an essential protein in the archaeon Haloferax volcanii and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a H. volcanii LonB mutant under reduced and physiological protease levels. LC-MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis. Fil: Cerletti, Micaela. Universidad Nacional de Mar del Plata; Argentina Fil: Paggi, Roberto Alejandro. Universidad Nacional de Mar del Plata; Argentina Fil: Troetschel, Christian. Ruhr Universität Bochum; Alemania Fil: Ferrari, María Celeste. Universidad Nacional de Mar del Plata; Argentina Fil: Guevara, Carina Ramallo. Ruhr Universität Bochum; Alemania Fil: Albaum, Stefan. Universitat Bielefeld; Alemania Fil: Poetsch, Ansgar. Plant Biochemistry, Ruhr University Bochum; Alemania Fil: de Castro, Rosana Esther. Universidad Nacional de Mar del Plata; Argentina

Chemical imaging of plant-bacteria co-cultures makes it possible to characterize bacterial populations and behaviors and their interactions with proximal organisms, under conditions closest to the environment in the rhizosphere. Here Raman micro-spectroscopy and confocal Raman imaging are used as minimally invasive probes to study the rhizosphere bacterial isolate, Pantoea sp. YR343, and its co-culture with model plant Arabidopsis thaliana by combining enhanced Raman spectroscopies with electron microscopy and principal component analysis (PCA). The presence of carotenoid pigments in the wild type Pantoea sp. YR343 was characterized using resonance Raman scattering, which was also used to confirm successful disruption of the crtB gene in an engineered carotenoid mutant strain. Other components of the Pantoea sp. YR343 cells were imaged in the presence of resonantly enhanced pigments using a combination of surface enhanced Raman imaging and PCA. Pantoea sp. YR343 cells decorated with Ag colloid synthesized ex situ gave spectra dominated by carotenoid scattering, whereas colloids synthesized in situ produced spectral signatures characteristic of flavins in the cell membrane. Scanning electron microscopy (SEM) of whole cells and transmission electron microscopy (TEM) images of thinly sliced cross-sections were used to assess structural integrity of the coated cells and to establish the origin of spectral signatures based on the position of Ag nanoparticles in the cells. Raman imaging was also used to characterize senescent green Arabidopsis thaliana plant roots inoculated with Pantoea sp. YR343, and PCA was used to distinguish spectral contributions from plant and bacterial cells, thereby establishing the potential of Raman imaging to visualize the distribution of rhizobacteria on plant roots.

A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34 kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg(2+) binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in the carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control carotenoid biosynthesis and accumulation in plants. However, little is known about the mechanisms underlying their posttranscriptional regulation. Here we report that PSY and OR family proteins [Arabidopsis thaliana OR (AtOR) and AtOR-like] physically interacted with each other in plastids. We found that alteration of OR expression in Arabidopsis exerted minimal effect on PSY transcript abundance. However, overexpression of AtOR significantly increased the amount of enzymatically active PSY, whereas an ator ator-like double mutant exhibited a dramatically reduced PSY level. The results indicate that the OR proteins serve as the major posttranscriptional regulators of PSY. The ator or ator-like single mutant had little effect on PSY protein levels, which involves a compensatory mechanism and suggests partial functional redundancy. In addition, modification of PSY expression resulted in altered AtOR protein levels, corroborating a mutual regulation of PSY and OR. Carotenoid content showed a correlated change with OR-mediated PSY level, demonstrating the function of OR in controlling carotenoid biosynthesis by regulating PSY. Our findings reveal a novel mechanism by which carotenoid biosynthesis is controlled via posttranscriptional regulation of PSY in plants.

Provitamin A (PVA) bio-fortification of crops offers a sustainable strategy to prevent the prevalence of vitamin A deficiency (VAD), one of the world’s major public health problems. The present work aimed to enhance PVA accumulation in cottonseed, the main by-product in the production of cotton fibers and the third largest source of edible plant oil in the world. On the basis of comprehensive identification of carotenoid synthase genes and their expression levels in various cotton tissues, we selected phytoene synthase as the target for manipulating carotenoid biosynthesis in the developing cottonseeds. After functional verification in transgenic tobacco, a cotton phytoene synthase gene (GhPSY2D) driven by a seed-specific promoter was transformed into cotton. The transgenic cottonseeds showed golden appearance and contained over 6-fold higher carotenoid contents in the extracted oil than the non-transgenic control. Thin layer chromatograph analysis indicated that the main PVA carotenoid β-carotene was predominant in the transgenic cottonseeds, but undetectable in the wild-type control. By simultaneously providing economically valuable fibers and edible oils, the transgenic cottons bio-fortified with β-carotene in seeds may be a new powerful tool against VAD in low-income regions.

The dried stigmas of Crocus sativus constitute the saffron, which is considered to be the costliest spice of the world. Saffron is valuable for its constituents, which are mainly apocarotenoids. In order to enhance the production of apocarotenoids, it is imperative to understand the regulation of apocarotenoid biosynthetic pathway. In C. sativus, although the pathway has been elucidated, the information regarding the regulation of the pathwaygenes is scanty. During the present investigation, the characterization of promoters regulating the expression of two important genes i.e. CsPSY and CsUGT was performed. We successfully cloned the promoters of both the genes, which were functionally characterized in Crocus sativus and Nicotiana tabaccum. In silico analysis of the promoters demonstrated the presence of several important cis regulatory elements responding tolight, hormonesand interaction with transcription factors (TFs). Further analysis suggested the regulation of CsPSY promoter by Abscisic acid (ABA) and that of CsUGT by Gibberellic acid (GA). In addition, we also observed ABA and GA mediated modulation in the expression of significant TFs and CsPSY and CsUGT transcripts. Overall, the study addresses issues related to regulation of key genes of apocarotenoid pathway in C.sativus.

Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants., This work was supported by Agriculture and Food Research Initiative competitive award no. 2016-67013-24612 from the USDA National Institute of Food and Agriculture and by the HarvestPlus research consortium (2014H6320.FRE).

Bisphosphonates are widely used for the treatment of bone disorders. These drugs also inhibit the growth of a variety of protozoan parasites, such as Toxoplasma gondii, the etiologic agent of toxoplasmosis. The target of the most potent bisphosphonates is the isoprenoid biosynthesis pathway enzyme farnesyl diphosphate synthase (FPPS). Based on our previous work on the inhibitory effect of sulfur-containing linear bisphosphonates against T. gondii, we investigated the potential synergistic interaction between one of these derivatives, 1-[(n-heptylthio)ethyl]-1,1-bisphosphonate (C7S), and statins, which are potent inhibitors of the host 3-hydroxy-3-methyl glutaryl-coenzyme A reductase (3-HMG-CoA reductase). C7S showed high activity against the T. gondii bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS), which catalyzes the formation of FPP and GGPP (50% inhibitory concentration [IC50] = 31 ± 0.01 nM [mean ± standard deviation]), and modest effect against the human FPPS (IC50 = 1.3 ± 0.5 μM). We tested combinations of C7S with statins against the in vitro replication of T. gondii. We also treated mice infected with a lethal dose of T. gondii with similar combinations. We found strong synergistic activities when using low doses of C7S, which were stronger in vivo than when tested in vitro. We also investigated the synergism of several commercially available bisphosphonates with statins both in vitro and in vivo. Our results provide evidence that it is possible to develop drug combinations that act synergistically by inhibiting host and parasite enzymes in vitro and in vivo. Fil: Li, Zhu-Hong. University of Georgia; Estados Unidos Fil: Li, Catherine. University of Georgia; Estados Unidos Fil: Szajnman, Sergio Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Rodriguez, Juan Bautista. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Moreno, Silvia N. J.. University of Georgia; Estados Unidos

Background Photosynthetic organisms utilize carotenoids for photoprotection as well as light harvesting. Our previous study revealed that high-intensity light increases the expression of the gene for phytoene synthase (EgcrtB) in Euglena gracilis (a unicellular phytoflagellate), the encoded enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway. To examine carotenoid synthesis of E. gracilis in response to light stress, we analyzed carotenoid species and content in cells grown under various light intensities. In addition, we investigated the effect of suppressing EgcrtB with RNA interference (RNAi) on growth and carotenoid content. Results After cultivation for 7 days under continuous light at 920 μmol m−2 s−1, β-carotene, diadinoxanthin (Ddx), and diatoxanthin (Dtx) content in cells was significantly increased compared with standard light intensity (55 μmol m−2 s−1). The high-intensity light (920 μmol m−2 s−1) increased the pool size of diadinoxanthin cycle pigments (i.e., Ddx + Dtx) by 1.2-fold and the Dtx/Ddx ratio from 0.05 (control) to 0.09. In contrast, the higher-intensity light treatment caused a 58% decrease in chlorophyll (a + b) content and diminished the number of thylakoid membranes in chloroplasts by approximately half compared with control cells, suggesting that the high-intensity light-induced accumulation of carotenoids is associated with an increase in both the number and size of lipid globules in chloroplasts and the cytoplasm. Transient suppression of EgcrtB in this alga by RNAi resulted in significant decreases in cell number, chlorophyll, and total major carotenoid content by 82, 82 and 86%, respectively, relative to non-electroporated cells. Furthermore, suppression of EgcrtB decreased the number of chloroplasts and thylakoid membranes and increased the Dtx/Ddx ratio by 1.6-fold under continuous illumination even at the standard light intensity, indicating that blocking carotenoid synthesis increased the susceptibility of cells to light stress. Conclusions Our results indicate that suppression of EgcrtB causes a significant decrease in carotenoid and chlorophyll content in E. gracilis accompanied by changes in intracellular structures, suggesting that Dtx (de-epoxidized form of diadinoxanthin cycle pigments) contributes to photoprotection of this alga during the long-term acclimation to light-induced stress. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1066-7) contains supplementary material, which is available to authorized users.

Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity.

Genetic modulation of the carotenogenesis in I. germanica `Fire Bride' by ectopic expression of a crtB gene causes several flower parts to develop novel orange and pink colors. Flower color in tall bearded irises (Iris germanica L.) is determined by two distinct biochemical pathways; the carotenoid pathway, which imparts yellow, orange and pink hues and the anthocyanin pathway, which produces blue, violet and maroon flowers. Red-flowered I. germanica do not exist in nature and conventional breeding methods have thus far failed to produce them. With a goal of developing iris cultivars with red flowers, we transformed a pink iris I. germanica, `Fire Bride', with a bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under the control of the promoter region of a gene for capsanthin-capsorubin synthase from Lilium lancifolium (Llccs). This approach aimed to increase the flux of metabolites into the carotenoid biosynthetic pathway and lead to elevated levels of lycopene and darker pink or red flowers. Iris callus tissue ectopically expressing the crtB gene exhibited a color change from yellow to pink-orange and red, due to accumulation of lycopene. Transgenic iris plants, regenerated from the crtB-transgenic calli, showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink), while the standards and falls showed no significant differences in color when compared to control plants. HPLC and UHPLC analysis confirmed that the color changes were primarily due to the accumulation of lycopene. In this study, we showed that ectopic expression of a crtB can be used to successfully alter the color of certain flower parts in I. germanica `Fire Bride' and produce new flower traits. Cooley's Gardens Inc., Silverton, OR {[}ARF3711]; Ministry of Education, Science and Technological Development, Republic of Serbia {[}TR31019]

Carotenoids are important accessory pigments in plants that are essential for photosynthesis. Phytoene synthase (PSY), a rate-controlling enzyme in the carotenoid biosynthesis pathway, has been widely characterized in rice, maize, and sorghum, but at present there are no reports describing this enzyme in cotton. In this study, GhPSY was identified as a candidate gene for the red plant phenotype via a combined strategy using: (1) molecular marker data for loci closely linked to R1; (2) the whole-genome scaffold sequence from Gossypium raimondii; (3) gene expression patterns in cotton accessions expressing the red plant and green plant phenotypes; and (4) the significant correlation between a single nucleotide polymorphisms (SNP) in GhPSY and leaf phenotypes of progeny in the (Sub16 × T586) F2 segregating population. GhPSY was relatively highly expressed in leaves, and the protein was localized to the plastid where it appeared to be mostly attached to the surface of thylakoid membranes. GhPSY mRNA was expressed at a significantly higher level in T586 and SL1-7-1 red plants than TM-1 and Hai7124 green plants. SNP analysis in the GhPSY locus showed co-segregation with the red and green plant phenotypes in the (Sub16 × T586) F2 segregating population. A phylogenetic analysis showed that GhPSY belongs to the PSY2 subfamily, which is related to photosynthesis in photosynthetic tissues. Using a reverse genetics approach based on Tobacco rattle virus-induced gene silencing, we showed that the knockdown of GhPSY caused a highly uniform bleaching of the red color in newly-emerged leaves in both T586 and SL1-7-1 plants with a red plant phenotype. These findings indicate that GhPSY is important for engineering the carotenoid metabolic pathway in pigment production.

Carotenoid content is the primary determinant of fruit color that affects nutritional value and appearance in tomato. Phytoene synthase (PSY) is the key regulatory enzyme in the carotenoid biosynthesis pathway. Absent function of PSY1 in tomato fruit results in yellow flesh phenotype. We, here, report that two different transcripts, a wild-type (Psy1) and a chimeric mRNA (Psy1/Unknown), exist in a yellow-fruited tomato accession PI 114490. Psy1/Unknown is generated by joining exons from two different genes, Psy1 and an unknown gene, transcribed using both complementary DNA strands. The Psy1 shows low expression in the fruit of PI 114490, while the expression of Psy1/Unknown in the fruit of PI 114490 shows the same pattern as Psy1 in red fruit. The PSY1/Unknown has a lower function than PSY1 in a bacterial expression system. Coincidence of one single-nucleotide polymorphism (SNP) in the fourth intron and one simple sequence repeat (SSR) with 19 AT repeats in the downstream sequence of Psy1 gene with Psy1/Unknown in a set of yellow-fruited tomato lines indicates that Psy1/Unknown might be caused by the SNP and/or SSR. One possible explanation of these observations is trans-splicing. Severely reduced Psy1 transcript caused by Psy1/Unknown results in low accumulation of carotenoid and yellow flesh in PI 114490.

Lycopene is an effective antioxidant proposed as a possible treatment for some cancers and other degenerative human conditions. This study aims at generation of a yeast strain (Saccharomyces cerevisiae) of efficient productivity of lycopene by overexpressing synthetic genes derived from crtE, crtB and crtI genes of Erwinia uredovora. These synthetic genes were constructed in accordance with the preferred codon usage in S. cerevisiae but with no changes in amino acid sequences of the gene products. S. cerevisiae cells were transformed with these synthetic crt genes, whose expression was regulated by the ADH2 promoter, which is de-repressed upon glucose depletion. The RT-PCR and Western blotting analyses indicated that the synthetic crt genes were efficiently transcribed and translated in crt-transformed S. cerevisiae cells. The highest level of lycopene in one of the transformed lines was 3.3mglycopene/g dry cell weight, which is higher than the previously reported levels of lycopene in other microorganisms transformed with the three genes. These results suggest the excellence of using the synthetic crt genes and the ADH2 promoter in generation of recombinant S. cerevisiae that produces a high level of lycopene. The level of ergosterol was reversely correlated to that of lycopene in crt-transformed S. cerevisiae cells, suggesting that two pathways for lycopene and ergosterol syntheses compete for the use of farnesyl diphosphate.

Corynebacterium glutamicum, a yellow-pigmented soil bacterium that synthesizes the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides, has been engineered for the production of various carotenoids. CrtE was assumed to be the major geranylgeranyl pyrophosphate (GGPP) synthase in carotenogenesis; however, deletion of crtE did not abrogate carotenoid synthesis. In silico analysis of the repertoire of prenyltransferases encoded by the C. glutamicum genome revealed two candidate GGPPS genes (idsA and ispB). The absence of pigmentation of an idsA deletion mutant and complementation experiments with a double deletion mutant lacking both idsA and crtE showed that IdsA is the major GGPPS of C. glutamicum and that crtE overexpression compensated for the lack of IdsA, whereas plasmid-borne overexpression of ispB did not. Purified His-tagged CrtE was active as a homodimer, whereas the active form of IdsA was homotetrameric. Both enzymes catalyzed prenyl transfer with isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesylphosphate (FPP) as substrates. IdsA showed the highest catalytic efficiency with dimethylallyl pyrophosphate and IPP, whereas the catalytic efficiency of CrtE was highest with geranyl pyrophosphate and IPP. Finally, application of prenyltransferase overexpression revealed that combined overexpression of idsA and the IPP isomerase gene idi in the absence of crtE led to the highest decaprenoxanthin titer reported to date.

Chromoplasts are special organelles that possess superior ability to synthesize and store massive amounts of carotenoids. They are responsible for the distinctive colors found in fruits, flowers, and roots. Chromoplasts exhibit various morphologies and are derived from either pre-existing chloroplasts or other non-photosynthetic plastids such as proplastids, leucoplasts or amyloplasts. While little is known about the molecular mechanisms underlying chromoplast biogenesis, research progress along with proteomics study of chromoplast proteomes signifies various processes and factors important for chromoplast differentiation and development. Chromoplasts act as a metabolic sink that enables great biosynthesis and high storage capacity of carotenoids. The formation of chromoplasts enhances carotenoid metabolic sink strength and controls carotenoid accumulation in plants. The objective of this review is to provide an integrated view on our understanding of chromoplast biogenesis and carotenoid accumulation in plants.

Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.

Three 1-deoxy-D-xylulose-5-phosphate synthases (DXS) and three phytoene synthases (PSY) were identified in citrus, from Affymetrix GeneChip Citrus Genome Array, GenBank and public orange genome databases. Tissue-specific expression analysis of these genes was carried out on fruit peel and flesh, flower and leaf of Satsuma mandarin (Citrus unshiu Marc.) in order to determine their roles in carotenoid accumulation in different tissues. Expression of CitDXS1 and CitPSY1 was highest in all test tissues, while that of CitDXS2 and CitPSY2 was lower, and that of CitDXS3 and CitPSY3 undetectable. The transcript profiles of CitDXS1 and CitPSY1 paralleled carotenoid accumulation in flesh of Satsuma mandarin and orange (Citrus sinensis Osbeck) during fruit development, and CitPSY1 expression was also associated with carotenoid accumulation in peel, while the CitDXS1 transcript level was only weakly correlated with carotenoid accumulation in peel. Similar results were obtained following correlation analysis between expression of CitDXS1 and CitPSY1 and carotenoid accumulation in peel and flesh of 16 citrus cultivars. These findings identify CitPSY1 and CitDXS1 as the main gene members controlling carotenoid biosynthesis in citrus fruit. Furthermore, chromoplasts were extracted from flesh tissue of these citrus, and chromoplasts of different shape (spindle or globular), different size, and color depth were observed in different cultivars, indicating chromoplast abundance, number per gram tissue, size and color depth were closely correlated with carotenoid content in most cultivars. The relationship between carotenoid biosynthesis and chromoplast development was discussed.

Carotenoids are conjugated isoprenoid molecules with many important physiological functions in organisms, including roles in photosynthesis, oxidative stress reduction, vision, diapause, photoperiodism, and immunity. Until recently, it was believed that only plants, microorganisms, and fungi were capable of synthesizing carotenoids and that animals acquired them from their diet, but recent studies have demonstrated that two arthropods (pea aphid and spider mite) possess a pair of genes homologous to those required for the first step of carotenoid biosynthesis. Absent in all other known animal genomes, these genes appear to have been acquired by aphids and spider mites in one or several lateral gene transfer events from a fungal donor. We report the third case of fungal carotenoid biosynthesis gene homologs in an arthropod: flies from the family Cecidomyiidae, commonly known as gall midges. Using phylogenetic analyses we show that it is unlikely that lycopene cyclase/phytoene synthase and phytoene desaturase homologs were transferred singly to an ancient arthropod ancestor; instead we propose that genes were transferred independently from related fungal donors after divergence of the major arthropod lineages. We also examine variation in intron placement and copy number of the carotenoid genes that may underlie function in the midges. This trans-kingdom transfer of carotenoid genes may represent a key innovation, underlying the evolution of phytophagy and plant-galling in gall midges and facilitating their extensive diversification across plant lineages.

Summary Four phytoene synthase genes and several variants were characterized, and their evolution and function in differential carotenoid accumulation in leaf, peel, and flesh of white- and red-fleshed loquats were established., Differences in carotenoid accumulation between tissues and cultivars is common in plants. White-fleshed loquat cultivars had low levels of carotenoids in the flesh, but accumulated carotenoids in peel when ripe, and the leaves accumulated similar carotenoids to those in the red-fleshed loquat cultivars. The catalytic activity and expression patterns of four phytoene synthase (PSY) genes, EjPSY1, EjPSY2A, EjPSY2B, and EjPSY3, were analysed to understand their roles in different loquat (Eriobotrya japonica Lindl.) types. EjPSY1 was responsible for carotenoid synthesis in the fruit peel but not the flesh, whereas EjPSY2A was responsible for carotenoid accumulation in flesh of ripening fruit. A mutant EjPSY2A d, with the same tissue specificity and expression level as EjPSY2A, but lacking the C-terminal region and corresponding catalytic activity, was discovered in white-fleshed varieties, explaining the lack of carotenoids in the white flesh. The catalytic role of EjPSY2B was most significant in leaves. The tissue-specific expression of EjPSY1 and EjPSY2B explained well how peel and leaf tissues can still accumulate carotenoids in white-fleshed cultivars, which have lost the functional EjPSY2A. EjPSY3 mRNA abundance was ~1000-fold less than that of other PSY mRNAs in all tissues examined. In addition, neither the normal sized transcript nor two alternatively spliced forms, EjPSY3α in LYQ and EjPSY3β in BS cultivars, encoded functional enzymes, and it is concluded that EjPSY3 plays no role in carotenoid accumulation. In addition, it was noted that recruitment of PSY genes for expression in specific tissues of different plants has occurred independently of gene structure and evolutionary origin.

Cassava (Manihot esculenta (L.) Crantz) is a starchy root crop cultivated in the tropics for fresh consumption and commercial processing. Dry matter content and micronutrient density, particularly of provitamin A – traits that are negatively correlated – are among the primary selection objectives in cassava breeding. This study aimed at identifying genetic markers associated with these traits and uncovering the potential underlying cause of their negative correlation – whether linkage and/or pleiotropy. A genome-wide association mapping using 672 clones genotyped at 72,279 SNP loci was carried out. Root yellowness was used indirectly to assess variation in carotenoid content. Two major loci for root yellowness was identified on chromosome 1 at positions 24.1 and 30.5 Mbp. A single locus for dry matter content that co-located with the 24.1 Mbp peak for carotenoid content was identified. Haplotypes at these loci explained a large proportion of the phenotypic variability. Evidence of mega-base-scale linkage disequilibrium around the major loci of the two traits and detection of the major dry matter locus in independent analysis for the white- and yellow-root subpopulations suggests that physical linkage rather that pleiotropy is more likely to be the cause of the negative correlation between the target traits. Moreover, candidate genes for carotenoid (phytoene synthase) and starch biosynthesis (UDP-glucose pyrophosphorylase and sucrose synthase) occurred in the vicinity of the identified locus at 24.1 Mbp. These findings elucidate on the genetic architecture of carotenoids and dry matter in cassava and provides an opportunity to accelerate genetic improvement of these traits.CORE IDEASCassava, a starchy root crop, is a major source of dietary calories in the tropics.Most varieties consumed are poor in micronutrients, including pro-vitamin A.These two traits are governed by few major loci on chromosome one.Genetic linkage, rather than pleiotropy, is the most likely cause of their negative correlation.

Nearly half of the world’s population obtains its daily calories from rice grains, which lack or have insufficient levels of essential micronutrients. The deficiency of micronutrients vital for normal growth is a global health problem, and iron, zinc and vitamin A deficiencies are the most prevalent ones. We developed rice lines expressing Arabidopsis NICOTIANAMINE SYNTHASE 1 (AtNAS1), bean FERRITIN (PvFERRITIN), bacterial CAROTENE DESATURASE (CRTI) and maize PHYTOENE SYNTHASE (ZmPSY) in a single genetic locus in order to increase iron, zinc and β-carotene content in the rice endosperm. NAS catalyzes the synthesis of nicotianamine (NA), which is a precursor of deoxymugeneic acid (DMA) iron and zinc chelators, and also chelate iron and zinc for long distance transport. FERRITIN provides efficient storage of up to 4500 iron ions. PSY catalyzes the conversion of GGDP to phytoene, and CRTI performs the function of desaturases required for the synthesis of β-carotene from phytoene. All transgenic rice lines have significantly increased β-carotene, iron, and zinc content in the polished rice grains. Our results establish a proof-of-concept for multi-nutrient enrichment of rice grains from a single genetic locus, thus offering a sustainable and effective approach to address different micronutrient deficiencies at once., Scientific Reports, 7, ISSN:2045-2322

In the course of screening for an inhibitor of farnesyl transferase (FTase), we identified two compounds, N-benzyl-aclacinomycin A (ACM) and N-allyl-ACM, which are new derivatives of ACM. N-benzyl-ACM and N-allyl-ACM inhibited FTase activity with IC50 values of 0.86 and 2.93 μM, respectively. Not only ACM but also C-10 epimers of each ACM derivative failed to inhibit FTase. The inhibition of FTase by N-benzyl-ACM and N-allyl-ACM seems to be specific, because these two compounds did not inhibit geranylgeranyltransferase or geranylgeranyl pyrophosphate (GGPP) synthase up to 100 μM. In cultured A431 cells, N-benzyl-ACM and N-allyl-ACM also blocked both the membrane localization of H-Ras and activation of the H-Ras-dependent PI3K/Akt pathway. In addition, they inhibited epidermal growth factor (EGF)-induced migration of A431 cells. Thus, N-benzyl-ACM and N-allyl-ACM inhibited EGF-induced migration of A431 cells by inhibiting the farnesylation of H-Ras and subsequent H-Ras-dependent activation of the PI3K/Akt pathway.

Selection for yellow- and white-grain types has been central to postdomestication improvement of maize. While genetic control of carotenoid biosynthesis in endosperm is attributed primarily to the

Plastoglobules (PGs) are thylakoid membrane microdomains within plastids that are known as specialized locations of carotenogenesis. Three rice phytoene synthase proteins (OsPSYs) involved in carotenoid biosynthesis have been identified. Here, the N-terminal 80-amino-acid portion of OsPSY2 (PTp) was demonstrated to be a chloroplast-targeting peptide by displaying cytosolic localization of OsPSY2(Delta PTp): mCherry in rice protoplast, in contrast to chloroplast localization of OsPSY2: mCherry in a punctate pattern. The peptide sequence of a PTp was predicted to harbor two transmembrane domains eligible for a putative PG-targeting signal. To assess and enhance the PG-targeting ability of PTp, the original PTp DNA sequence (PTp) was modified to a synthetic DNA sequence (stPTp), which had 84.4% similarity to the original sequence. The motivation of this modification was to reduce the GC ratio from 75% to 65% and to disentangle the hairpin loop structures of PTp. These two DNA sequences were fused to the sequence of the synthetic green fluorescent protein (sGFP) and drove GFP expression with different efficiencies. In particular, the RNA and protein levels of stPTp-sGFP were slightly improved to 1.4-fold and 1.3-fold more than those of sGFP, respectively. The green fluorescent signals of their mature proteins were all observed as speckle-like patterns with slightly blurred stromal signals in chloroplasts. These discrete green speckles of PTp-sGFP and stPTp-sGFP corresponded exactly to the red fluorescent signal displayed by OsPSY2: mCherry in both etiolated and greening protoplasts and it is presumed to correspond to distinct PGs. In conclusion, we identified PTp as a transit peptide sequence facilitating preferential translocation of foreign proteins to PGs, and developed an improved PTp sequence, a stPTp, which is expected to be very useful for applications in plant biotechnologies requiring precise micro-compartmental localization in plastids.

Background Phytoene synthase 1 (PSY1) is the most important regulatory enzyme in carotenoid biosynthesis, whereas its function is hardly known in common wheat. The aims of the present study were to investigate Psy1 function and genetic regulation using reverse genetics approaches. Results Transcript levels of Psy1 in RNAi transgenic lines were decreased by 54–76 % and yellow pigment content (YPC) was reduced by 26–35 % compared with controls, confirming the impact of Psy1 on carotenoid accumulation. A series of candidate genes involved in secondary metabolic pathways and core metabolic processes responded to Psy1 down-regulation. The aspartate rich domain (DXXXD) was important for PSY1 function, and conserved nucleotides adjacent to the domain influenced YPC by regulating gene expression, enzyme activity or alternative splicing. Compensatory responses analysis indicated that three Psy1 homoeologs may be coordinately regulated under normal conditions, but separately regulated under stress. The period 14 days post anthesis (DPA) was found to be a key regulation node during grain development. Conclusion The findings define key aspects of flour color regulation in wheat and facilitate the genetic improvement of wheat quality targeting color/nutritional specifications required for specific end products. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0916-z) contains supplementary material, which is available to authorized users.

Phytoene synthase (PSY) regulates the first committed step of the carotenoid biosynthetic pathway in plants. The present work reports identification and characterization of the three PSY genes (TaPSY1, TaPSY2 and TaPSY3) in wheat (Triticum aestivum L.). The TaPSY1, TaPSY2, and TaPSY3 genes consisted of three homoeologs on the long arm of group 7 chromosome (7L), short arm of group 5 chromosome (5S), and long arm of group 5 chromosome (5L), respectively in each subgenomes (A, B, and D) with a similarity range from 89% to 97%. The protein sequence analysis demonstrated that TaPSY1 and TaPSY3 retain most of conserved motifs for enzyme activity. Phylogenetic analysis of all TaPSY revealed an evolutionary relationship among PSY proteins of various monocot species. TaPSY derived from A and D subgenomes shared proximity to the PSY of Triticum urartu and Aegilops tauschii, respectively. The differential expression of TaPSY1, TaPSY2, and TaPSY3 in the various tissues, seed development stages, and stress treatments suggested their role in plant development, and stress condition. TaPSY3 showed higher expression in all tissues, followed by TaPSY1. The presence of multiple stress responsive cis-regulatory elements in promoter region of TaPSY3 correlated with the higher expression during drought and heat stresses has suggested their role in these conditions. The expression pattern of TaPSY3 was correlated with the accumulation of β-carotene in the seed developmental stages. Bacterial complementation assay has validated the functional activity of each TaPSY protein. Hence, TaPSY can be explored in developing genetically improved wheat crop.

Geranylgeranyl pyrophosphate synthase enzyme is one of the key enzymes in the synthesis pathway of diterpenoid. Nine Lamiaceae genus GGPS synthase in Genebank was analyzed in this article. GGPS synthase the nucleic acid sequences and amino acid sequences, physicochemical properties, the signal peptide, leader peptides, transmembrane topological structure, hydrophobic, hydrophilic, subcellular localization, secondary structure, function domain, tertiary structure and evolutional relationship were predicted by using bioinformatics methods.Phylogenetic tree was constructed for the geranylgeranyl pyrophosphate synthase enzyme protein family. The results showed that GGPS amino acid sequence of the physical and chemical properties were basically identical, mainly hydrophilic protein, there existed chloroplast transit peptide, and no signal peptide and membrane structure domain, which mainly located in the chloroplast, the minor part located in mitochondria. The main secondary structures of the proteins are alpha helix and random coil. All these proteins have catalytic residues, aspartate-rich region, active site lid residues, substrate-Mg2+ binding site. The results provide theoretical reference for study on both the enzymatic characteristics of GGPS and the biosynthesis pathway of diterpenoid.

A conventional breeding program was established to transfer the bacterial phytoene synthase transgene-crtB-from a transgenic, white-rooted cassava to yellow-rooted cassava plants carrying the endogenous phytoene synthase alleles named psy2-y

Background β-carotene, the most active provitamin A molecule produced by plants, plays important roles in human nutrition and health. β-carotene does not usually accumulate in the endosperm (i.e. flour) of mature wheat grains, which is a major food source of calories for humans. Therefore, enriching β-carotene accumulation in wheat grain endosperm will enable a sustainable dietary supplementation of provitamin A. Several metabolic genes affecting β-carotene accumulation have already been isolated from wheat, including phytoene synthase 1 (PSY1), lycopene ε-cyclase (LCYe) and carotenoid β-ring hydroxylase1/2 (HYD1/2). Results In this work, we cloned and biochemically characterized two carotenoid cleavage dioxygenases (CCDs), CCD1 and CCD4, from wheat. While CCD1 homoeologs cleaved β-apo-8′-carotenal, β-carotene, lutein and zeaxanthin into apocarotenoid products, CCD4 homoeologs were inactive towards these substrates in in vitro assays. When analyzed by real-time qPCR, PSY1, LCYe, HYD1/2 and CCD1/4 homoeologs showed distinct expression patterns in vegetative tissues and sections of developing tetraploid and hexaploid wheat grains, suggesting that carotenoid metabolic genes and homoeologs are differentially regulated at the transcriptional level in wheat. Conclusions The CCD1/4 enzyme activity and the spatial-temporal gene expression data provide critical insights into the specific carotenoid metabolic gene homoeologs that control β-carotene accumulation in wheat grain endosperm, thus establishing the knowledge base for generation of wheat varieties with enhanced β-carotene in the endosperm through breeding and genome editing approaches. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0848-7) contains supplementary material, which is available to authorized users.

Tomato ( Solanum lycopersicum ) fruit accumulate the red carotenoid pigment lycopene. The recessive mutation yellow-flesh (locus r ) in tomato eliminates fruit carotenoids by disrupting the activity of the fruit-specific phytoene synthase (PSY1), the first committed step in the carotenoid biosynthesis pathway. Fruits of the recessive mutation tangerine ( t ) appear orange due to accumulation of 7,9,7′,9′-tetra- cis -lycopene (prolycopene) as a result of a mutation in the carotenoid cis – trans isomerase. It was established 60 y ago that tangerine is epistatic to yellow-flesh . This uncharacteristic epistasis interaction defies a paradigm in biochemical genetics arguing that mutations that disrupt enzymes acting early in a biosynthetic pathway are epistatic to other mutations that block downstream steps in the same pathway. To explain this conundrum, we have investigated the interaction between tangerine and yellow-flesh at the molecular level. Results presented here indicate that allele r 2997 of yellow-flesh eliminates transcription of PSY1 in fruits. In a genetic background of tangerine , transcription of PSY1 is partially restored to a level sufficient for producing phytoene and downstream carotenoids. Our results revealed the molecular mechanism underlying the epistasis of t over r and suggest the involvement of cis -carotenoid metabolites in a feedback regulation of PSY1 gene expression.

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.

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to β-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene β-cyclase. Intermediates of the pathway beyond β-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3'-HO group results in the formation of fucoxanthin.

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.

The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSY homologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSY genes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.

The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [(3)H]farnesyl diphosphate and [(3)H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [(3)H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 d (enzyme assays) to up to 3 months (autoradiography of [(3)H]-labeled proteins).

Carotenoids play important roles in photosynthesis where they are involved in light-harvesting, in photo-protection and in the assembly and structural stability of light-harvesting and reaction centre complexes. In order to examine the effects of carotenoids on the oligomeric state of the reaction centre–light-harvesting 1 -PufX (RC–LH1–PufX) core complex of Rhodobacter sphaeroides two carotenoid-less mutants, TC70 and R-26, were studied. Detergent fractionation showed that in the absence of carotenoids LH2 complexes do not assemble, as expected, but also that core complexes are predominantly found as monomers, although levels of the PufX polypeptide appeared to be unaffected. Analysis of R-26 membranes by electron microscopy and atomic force microscopy reveals arrays of hexagonally packed monomeric RC–LH1–PufX complexes. Transfer of the crtB gene encoding phytoene synthase to TC70 and R-26 restores the normal synthesis of carotenoids demonstrating that the R-26 mutant of Rba. sphaeroides harbours a mutation in crtB , among its other defects. The transconjugant TC70 and R-26 strains containing crtB had regained their ability to assemble wild-type levels of dimeric RC–LH1–PufX core complexes and normal energy transfer pathways were restored, demonstrating that carotenoids are essential for the normal assembly and function of both the LH2 and RC–LH1–PufX complexes in this bacterial photosystem.

The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon (ΔpyrF) as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding ΔpyrF host. After positive selection of the single-crossover integration recombinants (pop-in) on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants (pop-out) with 5-fluoroorotic acid (5-FOA), the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.

Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.

Phytoene synthase (PSY) and phytoene desaturase (PDS), which catalyze the first and second steps of the carotenoid biosynthetic pathway, respectively, are key enzymes for the accumulation of carotenoids in many plants. We isolated 2 partial cDNAs encoding PSY (AsPSY-1 and AsPSY-2) and a partial cDNA encoding PDS (AsPDS) from Allium sativum. They shared high sequence identity and conserved motifs with other orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of AsPSY1, AsPSY2, and AsPDS in the bulbils, scapes, leaves, stems, bulbs, and roots of garlic. High-performance liquid chromatography demonstrated that carotenoids were not biosynthesized in the underground organs (roots and bulbs), but were very abundant in the photosynthetic organs (leaves) of A. sativum. A significantly higher amount of β-carotene (73.44 μg·g(-1)) was detected in the leaves of A. sativum than in the other organs.

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. © 2011 The Author(s)., This study was supported by a financial grant from Ministerio Español de Educación y Ciencia (AGL 2007-65303-CO2), and Junta de Andalucía, group BIO-299. The authors are grateful to Prof. F. Cunningham for providing pAC-85b plasmid. We also would like to thank Marco Mejías and Carlos Parejo for their excellent technical assistance. Finally, we would like to thank to Dr. Irina Obraztsova for her help in the design of some primers.

Flour colour measured as a Commission Internationale de l'Eclairage (CIE) b* value is an important wheat quality attribute for a range of end-products, with genes and enzymes of the xanthophyll biosynthesis pathway providing potential sources of trait variation. In particular, the phytoene synthase 1 (Psy1) gene has been associated with quantitative trait loci (QTL) for flour b* colour variation. Several Psy1 alleles on chromosome 7A (Psy-A1) have been described, along with proposed mechanisms for influencing flour b* colour. This study sought to identify evolutionary relationships among known Psy-A1 alleles, to establish which Psy-A1 alleles are present in selected Australian wheat genotypes and establish their role in controlling variation for flour b* colour via QTL analysis. Phylogenetic analyses showed seven of eight known Psy-A1 alleles clustered with sequences from T. urartu, indicating the majority of alleles in Australian germplasm share a common evolutionary lineage. In this regard, Psy-A1a, Psy-A1c, Psy-A1e and Psy-A1p were common in Australian genotypes with flour b* colour ranging from white to yellow. In contrast Psy-A1s was found to be related to A. speltoides, indicating a possible A-B genome translocation during wheat polyploidisation. A new allele Psy-A1t (similar to Psy-A1s) was discovered in genotypes with yellow flour, with QTL analyses indicating Psy-A1t strongly influences flour b* colour in Australian germplasm. QTL LOD value maxima did not coincide with Psy-A1 gene locus in two of three populations and, therefore, Psy-A1a and Psy-A1p may not be involved in flour colour. Instead two other QTL were identified, one proximal and one distal to Psy-A1 in Australian wheat lines. Comparison of Psy-A1t and Psy-A1p predicted protein sequences suggests differences in putative sites for post-translational modification may influence enzyme activity and subsequent xanthophyll accumulation in the wheat endosperm. Psy-A1a and Psy-A1p were not involved in flour b* colour variation, indicating other genes control variation on chromosome 7A in some wheat genotypes.

Phytoene synthase (PSY) catalyses the first step in the production of carotenoids, which has been described as a key regulatory step in the carotenoids biosynthetic pathway. PSY gene from Dunaliella salina was constitutively expressed in Chlamydomonas reinhardtii under the control of the RBCS2 and HSP70A promoters and targeted to the chloroplast by the RBCS2 transit peptide. DsPSY overexpression resulted in a stable increase in the corresponding PSY transcript level and in the content of carotenoids such as violaxanthin, lutein, and β-carotene, reaching between 125 and 260% the levels in control untransformed cells.