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The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga Pyropia yezoensis—a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned PyPSY should encode a phytoene synthase; this was empirically confirmed by pigment complementation in E. coli. This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis. [ABSTRACT FROM AUTHOR]

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4 , psy-90 , psy-130 , and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme–substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or β-carotene accumulation in ccr2 , yet they reduced specific acyclic linear cis -carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis -carotene abundance can be fine-tuned through holoenzyme–metabolon interactions to control plastid development. [ABSTRACT FROM AUTHOR]

Dunaliella salina, a unicellular marine microalga, has emerged as a promising host for genetic manipulation to enhance the production of commercially valuable products, including carotenoids, xanthophylls, vitamins, fatty acids, and enzymes. Carotenoids, in particular, are in high demand in the food, medical, and cosmetic industries. In this study, the focus was on manipulating the phytoene synthase (PSY) gene in Dunaliella salina, which is a key enzyme in the carotenoid biosynthesis pathway. Both homologous (PSY gene from Dunaliella salina) and heterologous (PSY gene from Haematococcus pluvialis) expression of PSY were performed. The PSY genes were cloned into the plant expression vector pMDC45 and then introduced into Dunaliella salina through nuclear transformation using electroporation. Positive clones resistant to hygromycin were identified, and transgenic lines carrying the transformed PSY genes were screened using PCR. Six transgenic lines of PSY-transformed Dunaliella salina were selected for further analysis. Real-time PCR was used to measure the expression levels of the PSY gene and other genes involved in the carotenoid pathway, such as phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), lycopene β-cyclase (LCYb), and β-carotene hydroxylase (CHYb). Compared to the wild-type strain, the homologous expression line (DS-2) showed an up to 2.9-fold increase in PSY gene expression. Moreover, the expression of other carotenoid-related genes also increased in both the homologous and heterologous transgenic lines. The enhanced expression of the PSY gene resulted in a significant increase in carotenoid production. The DS-2 transgenic line exhibited a 5.4-fold increase in β-carotene content, while the HP-4 transgenic line showed a 2.4-fold increase. Additionally, the DS-4 and HP-3 transgenic lines had a 7.6-fold increase in lutein content. Furthermore, the modified D. salina, with increased carotenoid production, was used as a feed for Artemia (brine shrimp). The results indicated that Artemia fed with genetically manipulated D. salina showed improved growth and survival rates. These Artemia also exhibited increased uptake of carotenoids, which are beneficial for their reproductive capacity. This study demonstrates the potential of manipulating the PSY gene in Dunaliella salina to enhance carotenoid production. It also highlights the application of genetically modified D. salina as a valuable feed source to improve the reproductive capacity and overall health of aquatic organisms, such as Artemia. [ABSTRACT FROM AUTHOR]

Dunaliella salina is a rich source of carotenoids. Carotenoid production is induced under specific conditions, i.e., high light intensity, high salt concentration, nutrient limitation, and suboptimal temperatures in this microalga. The control of environmental factors is vital for high productivity of carotenoids. In this paper, the effect of different ethanol concentrations in combination with nitrogen deficiency was investigated to induce carotenoid production in D. salina CCAP 19/18. Also, some biochemical and molecular parameters were investigated in response to ethanol in the cells. It was shown that ethanol at 0.5% concentration increased cell number but, at 5% concentration, reduced cell viability compared to the control. The highest carotenoid production was achieved at 3% ethanol concentration, which was 1.46 fold higher than the nitrogen deficiency condition. Investigation of the 3 carotenoid biosynthesis genes revealed that their expression levels increased at 3% ethanol concentration, and the phytoene synthase gene was the most upregulated one. Lipid peroxidation increased at both 3% and 5% ethanol concentrations. At 3% concentration, the activity of catalase and superoxide dismutase increased, but no significant changes were seen at 5% ethanol concentration. Peroxidase activity reduced at both 3% and 5% concentrations. Moreover, proline and reducing sugar content increased at 3% concentration while decreased at 5% ethanol concertation. The results showed that at 3% ethanol concentration, higher carotenoid productivity was associated with an increase in other intracellular responses (molecular and biochemical). Ethanol as a controllable element may be beneficial to increase carotenoid production even under inappropriate environmental conditions in D. salina. [ABSTRACT FROM AUTHOR]

The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga Pyropia yezoensis—a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned PyPSY should encode a phytoene synthase; this was empirically confirmed by pigment complementation in E. coli. This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis.

Summary: Carotenoids are photoprotectant pigments and precursors of hormones such as strigolactones (SL). Carotenoids are produced in plastids from geranylgeranyl diphosphate (GGPP), which is diverted to the carotenoid pathway by phytoene synthase (PSY). In tomato (Solanum lycopersicum), three genes encode plastid‐targeted GGPP synthases (SlG1 to SlG3) and three genes encode PSY isoforms (PSY1 to PSY3).Here, we investigated the function of SlG1 by generating loss‐of‐function lines and combining their metabolic and physiological phenotyping with gene co‐expression and co‐immunoprecipitation analyses.Leaves and fruits of slg1 lines showed a wild‐type phenotype in terms of carotenoid accumulation, photosynthesis, and development under normal growth conditions. In response to bacterial infection, however, slg1 leaves produced lower levels of defensive GGPP‐derived diterpenoids. In roots, SlG1 was co‐expressed with PSY3 and other genes involved in SL production, and slg1 lines grown under phosphate starvation exuded less SLs. However, slg1 plants did not display the branched shoot phenotype observed in other SL‐defective mutants. At the protein level, SlG1 physically interacted with the root‐specific PSY3 isoform but not with PSY1 and PSY2.Our results confirm specific roles for SlG1 in producing GGPP for defensive diterpenoids in leaves and carotenoid‐derived SLs (in combination with PSY3) in roots. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) by deploying viral-based vectors such as tobacco rattle virus (TRV) is a homology-based gene silencing technique in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) to validate the function of particular genes. The study presented here showed the induction of DNA methylation in the promoter regions of three phenotypic marker genes in different cotton accessions, including two endogenous genes such as phytoene desaturase (PDS) and phytoene synthase (PSY), and an exogenous gene, such as green fluorescent protein (GFP). First, DNA methylation was established in transgenic GFP cotton where methylation persisted up to S3 generation. Afterward, the promoter of PSY was targeted following the same conditions. Significant silencing of PSY was observed and methylation of the promoter was found up to S2 generation in red leaf cotton as detected in GFP cotton. Silencing of PDS resulted in a photobleaching phenotype; interestingly, the strength of this phenotype was diverse within the plants and was not observed in the next generation. Bisulfite sequencing results showed methylation percentage of the cytosine residues was high at CG and CHG sites of the targeted promoter sequences in the silenced plants. The findings of this paper suggest that TRV-based vector system can be used to monitor DNA methylation for both exogenous and endogenous gene levels in cotton and offer a very useful tool for plant epigenetic modification. [ABSTRACT FROM AUTHOR]

MADS transcription factors are involved in the regulation of fruit development and carotenoid metabolism in plants. However, whether and how carotenoid accumulation is regulated by algal MADS are largely unknown. In this study, we first used functional complementation to confirm the functional activity of phytoene synthase from the lutein‐rich Dunaliella sp. FACHB‐847 (DbPSY), the key rate‐limiting enzyme in the carotenoid biosynthesis. Promoters of DbPSY and DbLcyB (lycopene β‐cyclase) possessed multiple cis‐acting elements such as light‐, UV‐B‐, dehydration‐, anaerobic‐, and salt‐responsive elements, W‐box, and C‐A‐rich‐G‐box (MADS‐box). Meanwhile, we isolated one nucleus‐localized MADS transcription factor (DbMADS), belonging to type I MADS gene. Three carotenogenic genes, DbPSY, DbLcyB, and DbBCH (β‐carotene hydroxylase) genes were upregulated at later stages, which was well correlated with the carotenoid accumulation. In contrast, DbMADS gene was highly expressed at lag phase with low carotenoid accumulation. Yeast one‐hybrid assay and dual‐luciferase reporter assay demonstrated that DbMADS could directly bind to the promoters of two carotenogenic genes, DbPSY and DbLcyB, and repress their transcriptions. This study suggested that DbMADS may act as a negative regulator of carotenoid biosynthesis by repressing DbPSY and DbLcyB at the lag phase, which provide new insights into the regulatory mechanisms of carotenoid metabolism in Dunaliella. [ABSTRACT FROM AUTHOR]

Background: Carotenoids, which are secondary metabolites derived from isoprenoids, play a crucial role in photo-protection and photosynthesis, and act as precursors for abscisic acid, a hormone that plays a significant role in plant abiotic stress responses. The biosynthesis of carotenoids in higher plants initiates with the production of phytoene from two geranylgeranyl pyrophosphate molecules. Phytoene synthase (PSY), an essential catalytic enzyme in the process, regulates this crucial step in the pathway. In Daucus carota L. (carrot), two PSY genes (DcPSY1 and DcPSY2) have been identified but only DcPSY2 expression is induced by ABA. Here we show that the ectopic expression of DcPSY2 in Nicotiana tabacum L. (tobacco) produces in L3 and L6 a significant increase in total carotenoids and chlorophyll a, and a significant increment in phytoene in the T1L6 line. Tobacco transgenic T1L3 and T1L6 lines subjected to chronic NaCl stress showed an increase of between 2 and 3- and 6-fold in survival rate relative to control lines, which correlates directly with an increase in the expression of endogenous carotenogenic and abiotic-related genes, and with ABA levels. Conclusions: These results provide evidence of the functionality of DcPSY2 in conferring salt stress tolerance in transgenic tobacco T1L3 and T1L6 lines. [ABSTRACT FROM AUTHOR]

Watermelon (Citrullus lanatus) is an economically important cucurbit crop. Its pulp is rich in antioxidant carotenoids, which confer a variety of flesh colors. ClPsy1 (Phytoene Synthase) is the rate-limiting enzyme for carotenoid synthesis; however, the promoter activity of ClPsy1 is still unknown. In the present study, promoter sequences were isolated from four watermelon accessions: Cream of Saskatchewan pale yellow (COS), canary yellow flesh (PI 635597), golden flesh (PI 192938), and red flesh (LSW-177), all of which express ClPsy1 at extremely high levels. Sequence alignment and cis-element analysis disclosed six SNPs between the four lines all in COS, two of which (at the 598th and 1257th positions) caused MYC and MYB cis-element binding sequence variations, respectively. To confirm ClPsy1 gene promoter activity, full-length and deletion fragments of the promoter were constructed and connected to a β-D-glucosidase (GUS) vector and transferred into tomato fruits. GUS staining was performed to analyze the key segment of the promoter. The activity of the PI 192938 ClPsy1 full-length promoter exceeded that of COS. The deletion fragment from −1521 bp to −1043 bp exhibited strong promoter activity, and contained a MYB transcription factor-binding site mutation. We combined RNA-seq with qRT-PCR to analyze the gene expression pattern between the MYB transcription factor Cla97C10G196920 and ClPsy1 gene and found that Cla97C10G196920 (ClMYB21) showed the same expression trend with ClPsy1, which positively regulates carotenoid synthesis and metabolism. [ABSTRACT FROM AUTHOR]

Bread wheat has traditionally been selected for whitish derived flours. As a consequence, the current varieties carry carotenogenic alleles associated with low grain carotenoid. In contrast, high grain yellow pigment content (YPC) has been a major target in durum wheat programs since yellow colour is an important aesthetic factor for pasta production. Phytoene synthase 1 (Psy1) genes have an important role in the determination of the carotenoid content in wheat. In this work, we have transferred the genes Psy1-A1 and Psy1-B1 from durum to bread wheat by inter-specific hybridization in order to evaluate the combined effect of these genes for the improvement of grain carotenoid content, as well as the development of carotenoid-enriched bread wheat lines. Inter-specific breeding coupled with a MAS approach based on Psy1-A1 and Psy1-B1 alleles has allowed the development of bread wheat pre-breeding lines with enhanced grain carotenoid content (16–23% mean). These biofortified lines have the potential to become new varieties or to be used as recurrent parents in bread wheat breeding programs. [ABSTRACT FROM AUTHOR]

Thraustochytrids (phylum: Labyrinthulomycota) are nonphotosynthetic marine protists. Some thraustochytrids have crtIBY , a trifunctional fusion gene encoding a protein capable of β-carotene biosynthesis from geranylgeranyl pyrophosphate. Here we show that crtIBY is essential in, and encodes the sole pathway for, carotenoid biosynthesis in the thraustochytrid Aurantiochytrium limacinum ATCC MYA-1381. We explore the evolutionary origins of CrtIBY and discover that the closest related protein domains are present in a small but diverse group of other heterotrophic protists, including the apusomonad Thecamonas trahens and the dinoflagellates Oxyrrhis marina and Noctiluca scintillans. Each organism within this cluster also contains one or more β-carotene 15-15′ oxygenase genes (blh and rpe65), suggesting that the acquisition of β-carotene biosynthesis genes may have been related to the production of retinal. Our findings support a novel origin of eukaryotic (apo)carotenoid biosynthesis by horizontal gene transfer from Actinobacteria, Bacteroidetes, and/or Archaea. This reveals a remarkable case of parallel evolution of eukaryotic (apo)carotenogenesis in divergent protistan lineages by repeated gene transfers. [ABSTRACT FROM AUTHOR]

The unique color and type characteristics of watermelon fruits are regulated by many molecular mechanisms. However, it still needs to be combined with more abundant genetic data to fine-tune the positioning. We assembled genomes of two Korean inbred watermelon lines (cv. 242-1 and 159-1) with unique color and fruit-type characteristics and identified 23,921 and 24,451 protein-coding genes in the two genomes, respectively. To obtain more precise results for further study, we resequenced one individual of each parental line and an F2 population composed of 87 individuals. This identified 1,539 single-nucleotide polymorphisms (SNPs) and 80 InDel markers that provided a high-density genetic linkage map with a total length of 3,036.9 cM. Quantitative trait locus mapping identified 15 QTLs for watermelon fruit quality-related traits, including b-carotene and lycopene content in fruit flesh, fruit shape index, skin thickness, flesh color, and rind color. By investigating the mapping intervals, we identified 33 candidate genes containing variants in the coding sequence. Among them, Cla97C01G008760 was annotated as a phytoene synthase with a singlenucleotide variant (A!G) in the first exon at 9,539,129 bp of chromosome 1 that resulted in the conversion of a lysine to glutamic acid, indicating that this gene might regulate flesh color changes at the protein level. These findings not only prove the importance of a phytoene synthase gene in pigmentation but also explain an important reason for the color change of watermelon flesh. [ABSTRACT FROM AUTHOR]

ABSTRACT Phytoene synthase (PSY) is a key enzyme in carotenoid metabolism and often regulated by orange protein. However, few studies have focused on the functional differentiation of the two PSYs and their regulation by protein interaction in the β-carotene-accumulating Dunaliella salina CCAP 19/18. In this study, we confirmed that DsPSY1 from D. salina possessed high PSY catalytic activity, whereas DsPSY2 almost had no activity. Two amino acid residues at positions 144 and 285 responsible for substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Moreover, orange protein from D. salina (DsOR) could interact with DsPSY1/2. DbPSY from Dunaliella sp. FACHB-847 also had high PSY activity, but DbOR could not interact with DbPSY, which might be one reason why it could not highly accumulate β-carotene. Overexpression of DsOR, especially the mutant DsORHis, could significantly improve the single-cell carotenoid content and change cell morphology (with larger cell size, bigger plastoglobuli, and fragmented starch granules) of D. salina. Overall, DsPSY1 played a dominant role in carotenoid biosynthesis in D. salina, and DsOR promoted carotenoid accumulation, especially β-carotene via interacting with DsPSY1/2 and regulating the plastid development. Our study provides a new clue for the regulatory mechanism of carotenoid metabolism in Dunaliella. IMPORTANCE Phytoene synthase (PSY) as the key rate-limiting enzyme in carotenoid metabolism can be regulated by various regulators and factors. We found that DsPSY1 played a dominant role in carotenogenesis in the β-carotene-accumulating Dunaliella salina, and two amino acid residues critical in the substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Orange protein from D. salina (DsOR) can promote carotenoid accumulation via interacting with DsPSY1/2 and regulating the plastid development, which provides new insights into the molecular mechanism of massive accumulation of β-carotene in D. salina.

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis and photoprotectors for plants to adapt to high light stress. Here, we report a small subunit (SSU) of geranylgeranyl diphosphate synthase (GGPPS) in Nicotiana tabacum, NtSSU II, which takes part in the regulation carotenoid biosynthesis by forming multiple enzymatic components with NtGGPPS1 and downstream phytoene synthase (NtPSY1). NtSSU II transcript is widely distributed in various tissues and stimulated by low light and high light treatments. The confocal image revealed that NtSSU II was localized in the chloroplast. Bimolecular fluorescence complementation (BiFC) indicated that NtSSU II and NtGGPPS1 formed heterodimers, which were able to interact with phytoene synthase (NtPSY1) to channel GGPP into the carotenoid production. CRISPR/Cas9-induced ntssu II mutant exhibited decreased leaf area and biomass, along with a decline in carotenoid and chlorophyll accumulation. Moreover, the genes involved in carotenoid biosynthesis were also downregulated in transgenic plants of ntssu II mutant. Taken together, the newly identified NtSSU II could form multiple enzymatic components with NtGGPPS1 and NtPSY1 to regulate carotenoid biosynthesis in N. tabacum, in addition to the co-expression of genes in carotenoids biosynthetic pathways. [ABSTRACT FROM AUTHOR]

Colored wheat has piqued the interest of breeders and consumers alike. The chromosomal segment from 7E of Thinopyrum ponticum, which carries a leaf rust resistant gene, Lr19, has been rarely employed in wheat breeding operations due to its association with the Y gene, which gives a yellow tint to the flour. By prioritizing nutritional content over color preferences, consumer acceptance has undergone a paradigm change. Through marker-assisted backcross breeding, we introduced an alien segment harboring the Y (PsyE1) gene into a high yielding commercial bread wheat (HD 2967) background to generate rust resistant carotenoid biofortified bread wheat. Agro-morphological characterization was also performed on a subset of developed 70 lines having enhanced grain carotene content. In the introgression lines, carotenoid profiling using HPLC analysis demonstrated a considerable increase in β-carotene levels (up to 12 ppm). Thus, the developed germplasm caters the threat to nutritional security and can be utilized to produce carotenoid fortified wheat. [ABSTRACT FROM AUTHOR]

Orange protein (OR) is known to interact with phytoene synthase (PSY) that commits the first step in carotenoid biosynthesis, and functions as a major post-transcriptional regulator on PSY. We here tried to reveal enzymatic characteristics of OR, that is, protein disulfide reductase (PDR) activity of the Arabidopsis thaliana OR protein (AtOR) was analyzed using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The AtOR part containing only the zinc (Zn)-finger motif was found to show PDR activity, with an apparent Km of 12,632 nM, Kcat of 11.85 min−1, and KcatKm−1 of 15.6 × 103 M−1sec−1. To evaluate the significance of the N-terminal region of AtOR, we examined the kinetic parameters of a fusion protein composed of the N-terminal region and the Zn-finger motif from AtOR. Consequently, the fusion protein had lower values for Km (2,074 nM) and Kcat (3.18 min−1) and higher catalytic efficiency (25.9 × 103 M−1sec−1) than that of only the Zn-finger motif part, suggesting that the N-terminal region of AtOR should be important for substrate affinity and catalytic efficiency of PDR activity. Complementation experiments with E. coli further demonstrated that AtOR containing the N-terminal region and the Zn-finger motif increases phytoene synthase activity of AtPSY especially under reduced circumstances retaining a NADPH- and H+-regeneration system.

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis.

Background: Carotenoids, which are secondary metabolites derived from isoprenoids, play a crucial role in photo-protection and photosynthesis, and act as precursors for abscisic acid, a hormone that plays a significant role in plant abiotic stress responses. The biosynthesis of carotenoids in higher plants initiates with the production of phytoene from two geranylgeranyl pyrophosphate molecules. Phytoene synthase (PSY), an essential catalytic enzyme in the process, regulates this crucial step in the pathway. In Daucus carota L. (carrot), two PSY genes (DcPSY1 and DcPSY2) have been identified but only DcPSY2 expression is induced by ABA. Here we show that the ectopic expression of DcPSY2 in Nicotiana tabacum L. (tobacco) produces in L3 and L6 a significant increase in total carotenoids and chlorophyll a, and a significant increment in phytoene in the T1L6 line. Tobacco transgenic T1L3 and T1L6 lines subjected to chronic NaCl stress showed an increase of between 2 and 3- and 6-fold in survival rate relative to control lines, which correlates directly with an increase in the expression of endogenous carotenogenic and abiotic-related genes, and with ABA levels. Conclusions: These results provide evidence of the functionality of DcPSY2 in conferring salt stress tolerance in transgenic tobacco T1L3 and T1L6 lines.

Phytoene synthase (PSY) plays an essential role in carotenoid biosynthesis. In this study, three ClPSY genes were identified through the watermelon genome, and their full-length cDNA sequences were cloned. The deduced proteins of the three ClPSY genes were ranged from 355 to 421 amino acid residues. Phylogenetic analysis suggested that the ClPSYs are highly conserved with bottle gourd compared to other cucurbit crops PSY proteins. Variation in ClPSY1 expression in watermelon with different flesh colors was observed; ClPSY1 was most highly expressed in fruit flesh and associated with the flesh color formation. ClPSY1 expression was much lower in the white-fleshed variety than the colored fruits. Gene expression analysis of ClPSY genes in root, stem, leaf, flower, ovary and flesh of watermelon plants showed that the levels of ClPSY2 transcripts found in leaves was higher than other tissues; ClPSY3 was dominantly expressed in roots. Functional complementation assays of the three ClPSY genes suggested that all of them could encode functional enzymes to synthesize the phytoene from Geranylgeranyl Pyrophosphate (GGPP). Some of the homologous genes clustered together in the phylogenetic tree and located in the synteny chromosome region seemed to have similar expression profiles among different cucurbit crops. The findings provide a foundation for watermelon flesh color breeding with regard to carotenoid synthesis and also provide an insight for the further research of watermelon flesh color formation. [ABSTRACT FROM AUTHOR]

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis as well as photoprotectors for plants to adapt to high light stress. Phytoene synthase (PSY) is the entry enzyme and also the major rate-limiting enzyme in the carotenoid pathway. Here, we report a dehydration-responsive element-binding protein (DREB) transcription factor member in Nicotiana tabacum K326, NtDREB-1BL1, which regulates carotenoids biosynthesis by binding to the NtPSY promoter. The NtDREB-1BL1 transcript was widely distributed in leaves by Real-time PCR. Confocal image revealed that NtDREB-1BL1 was localized in the nucleus. The chromatin immunoprecipitation (ChIP) with the qPCR technique indicated that NtDREB-1BL1 could anchor the promoter region of NtPSY. Overexpression (NtDREB-1BL1 OE) and RNA interference (NtDREB-1BL1 RNAi) of NtDREB-1BL1 were performed to evaluate its biological function in N. tabacum. Both carotenoid and chlorophyll contents increased in transgenic plants of NtDREB-1BL1 OE compared with wild-type (WT) plants, with the augment of the genes involved in carotenoid biosynthesis. In contrast, the contents of carotenoid and chlorophyll significantly decreased in transgenic plants of NtDREB-1BL1 RNAi compared to WT, along with the decline in the expression of genes related to carotenoid biosynthesis. Moreover, transgenic plants of NtDREB-1BL1 OE exhibited enhanced tolerance under drought stress, with the weakened tolerance of drought stress in transgenic plants of NtDREB-1BL1 RNAi. In conclusion, our results illustrated the new role of transcription factor NtDREB-1BL1 in improving carotenoid biosynthesis through regulating NtPSY expression. [ABSTRACT FROM AUTHOR]

Abstract Background Carotenoid biosynthesis is essential for the generation of photosynthetic pigments, phytohormone production, and flower color development. The light harvesting like 3 (LIL3) protein, which belongs to the light-harvesting complex protein family in photosystems, interacts with geranylgeranyl reductase (GGR) and protochlorophyllide oxidoreductase (POR) both of which are known to regulate terpenoid and chlorophyll biosynthesis, respectively, in both rice and Arabidopsis. Results In our study, a CRISPR-Cas9 generated 4-bp deletion mutant oslil3 showed aberrant chloroplast development, growth defects, low fertility rates and reduced pigment contents. A comparative transcriptomic analysis of oslil3 suggested that differentially expressed genes (DEGs) involved in photosynthesis, cell wall modification, primary and secondary metabolism are differentially regulated in the mutant. Protein-protein interaction assays indicated that LIL3 interacts with phytoene synthase (PSY) and in addition the gene expression of PSY genes are regulated by LIL3. Subcellular localization of LIL3 and PSY suggested that both are thylakoid membrane anchored proteins in the chloroplast. We suggest that LIL3 directly interacts with PSY to regulate carotenoid biosynthesis. Conclusion This study reveals a new role of LIL3 in regulating pigment biosynthesis through interaction with the rate limiting enzyme PSY in carotenoid biosynthesis in rice presenting it as a putative target for genetic manipulation of pigment biosynthesis pathways in crop plants.

Watermelon (Citrullus lanatus) is an economically important cucurbit crop. Its pulp is rich in antioxidant carotenoids, which confer a variety of flesh colors. ClPsy1 (Phytoene Synthase) is the rate-limiting enzyme for carotenoid synthesis; however, the promoter activity of ClPsy1 is still unknown. In the present study, promoter sequences were isolated from four watermelon accessions: Cream of Saskatchewan pale yellow (COS), canary yellow flesh (PI 635597), golden flesh (PI 192938), and red flesh (LSW-177), all of which express ClPsy1 at extremely high levels. Sequence alignment and cis-element analysis disclosed six SNPs between the four lines all in COS, two of which (at the 598th and 1257th positions) caused MYC and MYB cis-element binding sequence variations, respectively. To confirm ClPsy1 gene promoter activity, full-length and deletion fragments of the promoter were constructed and connected to a β-D-glucosidase (GUS) vector and transferred into tomato fruits. GUS staining was performed to analyze the key segment of the promoter. The activity of the PI 192938 ClPsy1 full-length promoter exceeded that of COS. The deletion fragment from −1521 bp to −1043 bp exhibited strong promoter activity, and contained a MYB transcription factor-binding site mutation. We combined RNA-seq with qRT-PCR to analyze the gene expression pattern between the MYB transcription factor Cla97C10G196920 and ClPsy1 gene and found that Cla97C10G196920 (ClMYB21) showed the same expression trend with ClPsy1, which positively regulates carotenoid synthesis and metabolism.

Bread wheat has traditionally been selected for whitish derived flours. As a consequence, the current varieties carry carotenogenic alleles associated with low grain carotenoid. In contrast, high grain yellow pigment content (YPC) has been a major target in durum wheat programs since yellow colour is an important aesthetic factor for pasta production. Phytoene synthase 1 (Psy1) genes have an important role in the determination of the carotenoid content in wheat. In this work, we have transferred the genes Psy1-A1 and Psy1-B1 from durum to bread wheat by inter-specific hybridization in order to evaluate the combined effect of these genes for the improvement of grain carotenoid content, as well as the development of carotenoid-enriched bread wheat lines. Inter-specific breeding coupled with a MAS approach based on Psy1-A1 and Psy1-B1 alleles has allowed the development of bread wheat pre-breeding lines with enhanced grain carotenoid content (16–23% mean). These biofortified lines have the potential to become new varieties or to be used as recurrent parents in bread wheat breeding programs.

The accumulation of the red carotenoid pigment lycopene in tomato (Solanum lycopersicum) fruit is achieved by increased carotenoid synthesis during ripening. The first committed step that determines the flux in the carotenoid pathway is the synthesis of phytoene catalyzed by phytoene synthase (PSY). Tomato has three PSY genes that are differentially expressed. PSY1 is exclusively expressed in fruits, while PSY2 mostly functions in green tissues. It has been established that PSY1 is mostly responsible for phytoene synthesis in fruits. Although PSY2 is found in the chromoplasts, it is inactive because loss-of-function mutations in PSY1 in the locus yellow flesh (r) eliminate carotenoid biosynthesis in the fruit. Here we demonstrate that specific perturbations of carotenoid biosynthesis downstream to phytoene prior and during the transition from chloroplast to chromoplast cause the recovery of phytoene synthesis in yellow flesh (r) fruits without significant transcriptional changes of PSY1 and PSY2. The recovery of carotenoid biosynthesis was abolished when the expression of PSY2 was silenced, indicating that the perturbations of carotenoid biosynthesis reactivated the chloroplast-specific PSY2 in fruit chromoplasts. Furthermore, it is demonstrated that PSY2 can function in fruit chromoplasts under certain conditions, possibly due to alterations in the plastidial sub-organelle organization that affect its association with the carotenoid biosynthesis metabolon. This finding provides a plausible molecular explanation to the epistasis of the mutation tangerine in the gene carotenoid isomerase over yellow flesh. [ABSTRACT FROM AUTHOR]

Abstract Background Carotenoids play important roles in photosynthesis, hormone signaling, and secondary metabolism. Phytoene synthase (PSY) catalyzes the first step of the carotenoid biosynthetic pathway. In this study, we aimed to characterize the PSY genes in tobacco and analyze their function. Results In this study, we identified three groups of PSY genes, namely PSY1, PSY2, and PSY3, in four Nicotiana species; phylogenetic analysis indicated that these genes shared a high similarity with those in tomato but not with those in monocots such as rice and maize. The expression levels of PSY1 and PSY2 were observed to be highest in leaves compared to other tissues, and they could be elevated by treatment with certain phytohormones and exposure to strong light. No PSY3 expression was detected under these conditions. We constructed virus-induced PSY1 and PSY2 silencing in tobacco and found that the newly emerged leaves in these plants were characterized by severe bleaching and markedly decreased carotenoid and chlorophyll content. Thylakoid membrane protein complex levels in the gene-silenced plants were also less than those in the control plants. The chlorophyll fluorescence parameters such as Fv/Fm, ΦPSII, qP, and NPQ, which reflect photosynthetic system activities, of the gene-silenced plants were also significantly decreased. We further performed RNA-Seq and metabonomics analysis between gene-silenced tobacco and control plants. RNA-Seq results showed that abiotic stress, isoprenoid compounds, and amino acid catabolic processes were upregulated, whereas the biosynthesis of cell wall components was downregulated. Metabolic analysis results were consistent with the RNA-Seq. We also found the downstream genes in carotenoid biosynthesis pathways were upregulated, and putative transcription factors that regulate carotenoid biosynthesis were identified. Conclusions Our results suggest that PSY can regulate carotenoid contents not only by controlling the first biosynthesis step but also by exerting effects on the expression of downstream genes, which would thereby affect photosynthetic activity. Meanwhile, PSY may affect other processes such as amino acid catabolism and cell wall organization. The information we report here may aid further research on PSY genes and carotenoid biosynthesis.

The accumulation of the red carotenoid pigment lycopene in tomato (Solanum lycopersicum) fruit is achieved by increased carotenoid synthesis during ripening. The first committed step that determines the flux in the carotenoid pathway is the synthesis of phytoene catalyzed by phytoene synthase (PSY). Tomato has three PSY genes that are differentially expressed. PSY1 is exclusively expressed in fruits, while PSY2 mostly functions in green tissues. It has been established that PSY1 is mostly responsible for phytoene synthesis in fruits. Although PSY2 is found in the chromoplasts, it is inactive because loss-of-function mutations in PSY1 in the locus yellow flesh (r) eliminate carotenoid biosynthesis in the fruit. Here we demonstrate that specific perturbations of carotenoid biosynthesis downstream to phytoene prior and during the transition from chloroplast to chromoplast cause the recovery of phytoene synthesis in yellow flesh (r) fruits without significant transcriptional changes of PSY1 and PSY2. The recovery of carotenoid biosynthesis was abolished when the expression of PSY2 was silenced, indicating that the perturbations of carotenoid biosynthesis reactivated the chloroplast-specific PSY2 in fruit chromoplasts. Furthermore, it is demonstrated that PSY2 can function in fruit chromoplasts under certain conditions, possibly due to alterations in the plastidial sub-organelle organization that affect its association with the carotenoid biosynthesis metabolon. This finding provides a plausible molecular explanation to the epistasis of the mutation tangerine in the gene carotenoid isomerase over yellow flesh.

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis and photoprotectors for plants to adapt to high light stress. Here, we report a small subunit (SSU) of geranylgeranyl diphosphate synthase (GGPPS) in Nicotiana tabacum, NtSSU II, which takes part in the regulation carotenoid biosynthesis by forming multiple enzymatic components with NtGGPPS1 and downstream phytoene synthase (NtPSY1). NtSSU II transcript is widely distributed in various tissues and stimulated by low light and high light treatments. The confocal image revealed that NtSSU II was localized in the chloroplast. Bimolecular fluorescence complementation (BiFC) indicated that NtSSU II and NtGGPPS1 formed heterodimers, which were able to interact with phytoene synthase (NtPSY1) to channel GGPP into the carotenoid production. CRISPR/Cas9-induced ntssu II mutant exhibited decreased leaf area and biomass, along with a decline in carotenoid and chlorophyll accumulation. Moreover, the genes involved in carotenoid biosynthesis were also downregulated in transgenic plants of ntssu II mutant. Taken together, the newly identified NtSSU II could form multiple enzymatic components with NtGGPPS1 and NtPSY1 to regulate carotenoid biosynthesis in N. tabacum, in addition to the co-expression of genes in carotenoids biosynthetic pathways.

The purpose of the study is to determine the variability and stability of traits related to the amount of yellow pigments in the grain, semolina and the finished pasta product. In addition the polymorphism in one of the phytoene synthase gene on chromosome 7A related to carotenoids synthesis was detected. In the investigation were included 10 durum wheat genotypes - cultivars and breeding lines of different origin - Bulgaria - Field Crops Institute - Chirpan and Dobroudja Agricultural Institute - General Toshevo, Europe, CYMIT-Mexico. All genotypes were grown in field conditions in competitive variety trials in three replications in harvesting years 2014/2016. The results obtained were processed statistically via basic statistics (TIBCO statistica 13.3.0 software package). Great variability was found between genotypes on the study traits. The breeding lines: M-431 (10.71 ppm in grain, 10.42 ppm in semolina and 8.96 ppm in pasta) and TD-97 (9.83 ppm in grain, 9.58 ppm in semolina and 8.52 ppm in pasta) and the Bulgarian standard cultivar Predel (9.25 ppm in grain, 8.87 ppm in semolina and 7.27 ppm in pasta) are characterized by the highest yellow pigment content in grain, semolina and pasta. Both lines are also distinguished by the lowest total pigment losses (2.6%) in milling grain to semolina and in processing to pasta product (11 - 11.4 %), while line TD-97 has the lowest total pigment losses (13.6%). Furthermore the genotypes characterized by the highest yellow pigment content (M-431 and TD-97) have an acceptable stability and can be used in the hybridization program to improve the color potential of Bulgarian durum wheat varieties. The Psy-A1 codominant marker, based on a 37 bp insertion at the 5' end of intron 2 of the PsyA1 gene amplifies two fragments. The Psy-A1a allele with a 194 bp fragment in length was observed in all 7 studied cultivars and lines of durum wheat. The 231-bp Psy-A1b allele is found in 3 genotypes. The obtained results could not be associated with phenotypic differences in the yellow pigment content in the durum wheat genotypes studied. [ABSTRACT FROM AUTHOR]

Background: Carotenoid biosynthesis is essential for the generation of photosynthetic pigments, phytohormone production, and flower color development. The light harvesting like 3 (LIL3) protein, which belongs to the light-harvesting complex protein family in photosystems, interacts with geranylgeranyl reductase (GGR) and protochlorophyllide oxidoreductase (POR) both of which are known to regulate terpenoid and chlorophyll biosynthesis, respectively, in both rice and Arabidopsis. Results: In our study, a CRISPR-Cas9 generated 4-bp deletion mutant oslil3 showed aberrant chloroplast development, growth defects, low fertility rates and reduced pigment contents. A comparative transcriptomic analysis of oslil3 suggested that differentially expressed genes (DEGs) involved in photosynthesis, cell wall modification, primary and secondary metabolism are differentially regulated in the mutant. Protein-protein interaction assays indicated that LIL3 interacts with phytoene synthase (PSY) and in addition the gene expression of PSY genes are regulated by LIL3. Subcellular localization of LIL3 and PSY suggested that both are thylakoid membrane anchored proteins in the chloroplast. We suggest that LIL3 directly interacts with PSY to regulate carotenoid biosynthesis. Conclusion: This study reveals a new role of LIL3 in regulating pigment biosynthesis through interaction with the rate limiting enzyme PSY in carotenoid biosynthesis in rice presenting it as a putative target for genetic manipulation of pigment biosynthesis pathways in crop plants. [ABSTRACT FROM AUTHOR]

The carotenoids available in food are vital dietary micronutrients for human health. Plants synthesize and accumulate different carotenoids in plastids in a tissue-specific manner. The level of β-carotene (provitamin A) and other nutritionally important carotenoids is substantially low in the green tissues such as leaves compared to the fruits and roots. In photosynthetic tissues, chloroplasts can accumulate a moderate level of carotenoids, mainly to facilitate photosynthesis and environmental stress tolerance. However, chromoplasts from the storage tissues such as tomato fruit and carrot root can synthesize and accumulate carotenoids to a substantially higher level. A synthetic biology approach that utilizes a transient expression of bacterial phytoene synthase (crtB) gene in the photosynthetic leaves can induce the transition of chloroplasts into chromoplasts. The plastid-localized heterologous expression of crtB in leaves can induce the overaccumulation of phytoene, triggering the chloroplast-to-chromoplast transition; therefore, enhancing the biosynthesis and accumulation of carotenoids, including provitamin A. The transition of chloroplasts into chromoplasts, however, altered the photosynthetic thylakoids, consequently reducing the photosynthetic efficiency and plant growth. An efficient metabolic engineering strategy is desirable to enhance the production of targeted carotenoids in leaves without perturbing the photosynthetic efficiency and plant growth. Collectively, a synthetic biology strategy that triggers the transformation of chloroplasts into chromoplasts in photosynthetic tissues unfolds new avenues for carotenoid biofortification in the leafy food and vegetable crops, which can increase the dietary intake of carotenoids, therefore, combating the crisis of vitamin A deficiency. [ABSTRACT FROM AUTHOR]

Background: Carotenoid biosynthesis is essential for the generation of photosynthetic pigments, phytohormone production, and flower color development. The light harvesting like 3 (LIL3) protein, which belongs to the light-harvesting complex protein family in photosystems, interacts with geranylgeranyl reductase (GGR) and protochlorophyllide oxidoreductase (POR) both of which are known to regulate terpenoid and chlorophyll biosynthesis, respectively, in both rice and Arabidopsis. Results: In our study, a CRISPR-Cas9 generated 4-bp deletion mutant oslil3 showed aberrant chloroplast development, growth defects, low fertility rates and reduced pigment contents. A comparative transcriptomic analysis of oslil3 suggested that differentially expressed genes (DEGs) involved in photosynthesis, cell wall modification, primary and secondary metabolism are differentially regulated in the mutant. Protein-protein interaction assays indicated that LIL3 interacts with phytoene synthase (PSY) and in addition the gene expression of PSY genes are regulated by LIL3. Subcellular localization of LIL3 and PSY suggested that both are thylakoid membrane anchored proteins in the chloroplast. We suggest that LIL3 directly interacts with PSY to regulate carotenoid biosynthesis. Conclusion: This study reveals a new role of LIL3 in regulating pigment biosynthesis through interaction with the rate limiting enzyme PSY in carotenoid biosynthesis in rice presenting it as a putative target for genetic manipulation of pigment biosynthesis pathways in crop plants. [ABSTRACT FROM AUTHOR]

In the present study, the PSY1 and PSY2 homologous genes were identified in five accessions of three pepper species Capsicum annuum, Capsicum chinense, and Capsicum frutescens, differing in fruit pigmentation pattern. Within the group of studied accessions, the variability of PSY1 and PSY2 genomic and cDNA sequences was determined. The PSY1 and PSY2 sequences were 82% similar and differed in the N- and C-terminal NAGLRYSD and KLTSSSL indels, as well as in the conserved motifs characteristic of the PSY2 and PSY1 homologs. The expression of PSY1 and PSY2 was analyzed in leaves, sepals, petals, and ovaries, as well as in the peel and pulp of the fruits at the three stages of ripening in all five analyzed accessions. The maximum level of PSY1 expression was shown in the petals and in the pericarp of mature fruits of C. annuum and C. frutescens accessions. In the C. chinense cv. Pimenta da Neyde, PSY1 was expressed only in leaves. PSY2 transcripts were found in all analyzed organs of all pepper accessions; the maximum level was in the leaves, and the minimum level was in the fruit pericarp. The obtained data suggest that the PSY1 and PSY2 homologs of pepper species retained conserved key functions in the carotenoid synthesis in fruits (PSY1) and photosynthetic tissues (PSY2). [ABSTRACT FROM AUTHOR]

Background: Carotenoids play important roles in photosynthesis, hormone signaling, and secondary metabolism. Phytoene synthase (PSY) catalyzes the first step of the carotenoid biosynthetic pathway. In this study, we aimed to characterize the PSY genes in tobacco and analyze their function. Results: In this study, we identified three groups of PSY genes, namely PSY1, PSY2, and PSY3, in four Nicotiana species; phylogenetic analysis indicated that these genes shared a high similarity with those in tomato but not with those in monocots such as rice and maize. The expression levels of PSY1 and PSY2 were observed to be highest in leaves compared to other tissues, and they could be elevated by treatment with certain phytohormones and exposure to strong light. No PSY3 expression was detected under these conditions. We constructed virus-induced PSY1 and PSY2 silencing in tobacco and found that the newly emerged leaves in these plants were characterized by severe bleaching and markedly decreased carotenoid and chlorophyll content. Thylakoid membrane protein complex levels in the gene-silenced plants were also less than those in the control plants. The chlorophyll fluorescence parameters such as Fv/Fm, ΦPSII, qP, and NPQ, which reflect photosynthetic system activities, of the gene-silenced plants were also significantly decreased. We further performed RNA-Seq and metabonomics analysis between gene-silenced tobacco and control plants. RNA-Seq results showed that abiotic stress, isoprenoid compounds, and amino acid catabolic processes were upregulated, whereas the biosynthesis of cell wall components was downregulated. Metabolic analysis results were consistent with the RNA-Seq. We also found the downstream genes in carotenoid biosynthesis pathways were upregulated, and putative transcription factors that regulate carotenoid biosynthesis were identified. Conclusions: Our results suggest that PSY can regulate carotenoid contents not only by controlling the first biosynthesis step but also by exerting effects on the expression of downstream genes, which would thereby affect photosynthetic activity. Meanwhile, PSY may affect other processes such as amino acid catabolism and cell wall organization. The information we report here may aid further research on PSY genes and carotenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Phytoene synthase (PSY) plays a key role in the biosynthesis and accumulation of carotenoids as the first rate-limiting enzyme in the plant carotenoid pathway. In the present study, the ‘Granny Smith’ apple PSY gene, MdPSY, was cloned using in silico cloning. The length of the MdPSY was 1500 bp, which included a complete open reading frame of 1194 bp, encoding a predicted protein of 397 amino acids. Sequence analysis revealed that the ‘Granny Smith’ apple PSY protein shared high homology with MdPSY2 (99%), MdPSY1 (93.9%), FaPSY (71.8%), CuPSY (70.7%) and NtPSY (69.7%). Phylogenetic analysis showed that the MdPSY protein was closely related to the PSY protein of Rosaceae. An expression profile analysis of different tissues indicated that MdPSY expression in the roots was higher than that in other tissues. MdPSY expression was tested throughout the fruit development period. The results showed that MdPSY plays a role in carotenoid biosynthesis in ‘Granny Smith’ apple. In addition, bagging treatment inhibits PSY gene expression, which in turn affects the synthesis and accumulation of carotenoids.

Abstract 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.

Crocus sativus stigmas are the main source of crocins, which are glucosylated apocarotenoids derived from zeaxanthin cleavage that give saffron its red color. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis in plants. Four PSY genes encoding functional enzymes were isolated from saffron. All the proteins were localized in plastids, but the expression patterns of each gene, CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3, in different saffron tissues and during the development of the stigma showed different tissue specialization. The CsPSY2 transcript was primarily detected in the stigmas where it activates and stimulates the accumulation of crocins, while its expression was very low in other tissues. In contrast, CsPSY1a and CsPSY1b were mainly expressed in the leaves, but only CsPSY1b showed stress-light regulation. Interestingly, CsPSY1b showed differential expression of two alternative splice variants, which differ in the intron retention at their 5′ UTRs, resulting in a reduction in their expression levels. In addition, the CsPSY1a and CsPSY1b transcripts, together with the CsPSY3 transcript, were induced in roots under different stress conditions. The CsPSY3 expression was high in the root tip, and its expression was associated with mycorrhizal colonization and strigolactone production. CsPSY3 formed a separate branch to the stress-specific Poaceae homologs but was closely related to the dicot PSY3 enzymes.

Lycopene attracts increasing interests in the pharmaceutical, food, and cosmetic industries due to its anti-oxidative and anti-cancer properties. Compared with other lycopene production methods, such as chemical synthesis or direct extraction from plants, the biosynthesis approach using microbes is more economical and sustainable. In this work, we engineered Haloferax mediterranei, a halophilic archaeon, as a new lycopene producer. H. mediterranei has the de novo synthetic pathway for lycopene but cannot accumulate this compound. To address this issue, we reinforced the lycopene synthesis pathway, blocked its flux to other carotenoids and disrupted its competitive pathways. The reaction from geranylgeranyl-PP to phytoene catalyzed by phytoene synthase (CrtB) was identified as the rate-limiting step in H. mediterranei. Insertion of a strong promoter PphaR immediately upstream of the crtB gene, or overexpression of the heterologous CrtB and phytoene desaturase (CrtI) led to a higher yield of lycopene. In addition, blocking bacterioruberin biosynthesis increased the purity and yield of lycopene. Knock-out of the key genes, responsible for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis, diverted more carbon flux into lycopene synthesis, and thus further enhanced lycopene production. The metabolic engineered H. mediterranei strain produced lycopene at 119.25 ± 0.55 mg per gram of dry cell weight in shake flask fermentation. The obtained yield was superior compared to the lycopene production observed in most of the engineered Escherichia coli or yeast even when they were cultivated in pilot scale bioreactors. Collectively, this work offers insights into the mechanism involved in carotenoid biosynthesis in haloarchaea and demonstrates the potential of using haloarchaea for the production of lycopene or other carotenoids.

ABSTRACT Phytoene synthase (PSY) is the rate-limiting enzyme for carotenoid biosynthesis. To date, several studies focused on PSY genes in the context of abiotic stress responses. In this study, two phytoene synthase encoding genes, IbPSY1 and IbPSY2, were identified from a published transcriptome and bioinformatic analysis showed that they shared conserved domains with phytoene synthases from other plants. The IbPSY1 gene was cloned and carefully characterized. Digital gene expression profiling (DGE) showed that the highest transcription level of IbPSY1 was in young leaves, and the lowest level was in stems. In vivo expression levels of IbPSY1 under abiotic stress were observed to be highest in stems at day 11. Over-expression of IbPSY1 in Escherichia coli and yeast cells endowed the cells with better growth under salt and drought stress than the control cells. This study demonstrated that IbPSY1 not only played an important role in vivo, but also in E. coli and yeast to improve tolerance to salinity and drought stress. Thus, IbPSY1 may be aid in the development of transgenic plants with enhanced stress tolerance.

Carotenoids are isoprenoid pigments synthesized in plants, algae, and photosynthetic bacteria and fungus. Their role is essential in light capture, photoprotection, pollinator attraction, and phytohormone production. Furthermore, they can regulate plant development when they are processed as small signaling molecules. Due to their importance for human health, as promoters of the immune system and antioxidant activity, carotenoids have been used in the pharmaceutical, food, and nutraceutical industries. Regulation of carotenoid synthesis and accumulation has been extensively studied. Excellent work has been done unraveling the mode of action of phytoene synthase (PSY), a rate-limiting enzyme of carotenoid biosynthesis pathway, in model species and staple crops. Lately, interest has been turned to Orange protein and its interaction with PSY during carotenoid biosynthesis. Discovered as a dominant mutation in Brassica oleracea , Orange protein regulates carotenoid accumulation by posttranscriptionally regulating PSY, promoting the formation of carotenoid-sequestering structures, and also preventing carotenoid degradation. Furthermore, Orange protein contributes to homeostasis regulation, improving plant tolerance to abiotic stress. In this mini review, the focus is made on recent evidence that elucidates Orange protein mode of action and expression in different plant species. Additionally, strategies are proposed to modify Orange gene by utilization of genome editing techniques. A better understanding of carotenoid biosynthesis and accumulation will lead to a positive impact on the development of healthy food for a growing population. [ABSTRACT FROM AUTHOR]

Petroleum is one of the critical environmental pollutants. Salicornia can grow in petroleum-contaminated soil. Therefore, the potential of two Iranian Salicornia species, S. persica Akhani and S. iranica Akhani, for phytoremediation of soils contaminated with 0.2% or 2% petroleum was evaluated over short (1 and 10 h) and long (100 days) periods of time. In addition, some key factors including the expression analysis of phytoene synthase, physiological and morphological factors were studied. Both species reduced the petroleum in 0.2% and 2% petroleum-contaminated soils to 40% and 60% of the initial amount, respectively. The expression of PSY increased twice more than the control 10 h after 0.2% petroleum stress and the carotenoid content increased twice more than the control. Chlorophyll a and total chlorophyll decreased three times less than the control in both contamination levels, while chlorophyll b decreased three times less than the control only in 2% contamination. The proline content peaked 10 h after 2% stress as it was 10 times more than the control. Promoter analysis of PSY showed the existence of responsive cis-acting elements to abscisic acid suggesting the key role of this gene in abiotic stresses. [ABSTRACT FROM AUTHOR]

Summary: The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1,PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways. [ABSTRACT FROM AUTHOR]

Summary: Diatoms are eukaryotic, unicellular algae that are responsible for c. 20% of the Earth's primary production. Their dominance and success in contemporary oceans have prompted investigations on their distinctive metabolism and physiology. One metabolic pathway that remains largely unexplored in diatoms is isoprenoid biosynthesis, which is responsible for the production of numerous molecules with unique features.We selected the diatom species Haslea ostrearia because of its characteristic isoprenoid content and carried out a comprehensive transcriptomic analysis and functional characterization of the genes identified.We functionally characterized one farnesyl diphosphate synthase, two geranylgeranyl diphosphate synthases, one short‐chain polyprenyl synthase, one bifunctional isopentenyl diphosphate isomerase – squalene synthase, and one phytoene synthase. We inferred the phylogenetic origin of these genes and used a combination of functional analysis and subcellular localization predictions to propose their physiological roles.Our results provide insight into isoprenoid biosynthesis in H. ostrearia and propose a model of the central steps of the pathway. This model will facilitate the study of metabolic pathways of important isoprenoids in diatoms, including carotenoids, sterols and highly branched isoprenoids. [ABSTRACT FROM AUTHOR]

Watermelon mosaic virus (WMV), a plus-strand RNA virus that belongs to the family Potyviridae, is one of the most damaging viruses that infect cucurbit crops. However, isolates of this species frequently induce mild symptoms, which makes difficult to manage the disease in the field and breeding programs. A new generation of marker genes has been recently developed to visually track plant virus infection. Virus-mediated expression of the Pantoea ananatis phytoene synthase (crtB) induces accumulation in infected tissues of pigmented carotenoids that can be readily detected by the naked eye. Here we investigated whether this visual marker may facilitate visual diagnosis of WMV infection in cucurbits. First, we cloned a mild WMV isolate (WMV-Vera) and built a recombinant clone that expresses crtB (WMV-crtB). Next, we inoculated a series of cucurbit cultivars frequently used in breeding programs. We observed that WMV-mediated expression of crtB helps to visually score infection in two susceptible and highly appreciated melon cultivars, such as Piñonet Piel de Sapo and Vedrantais, but not in susceptible Cucurbita spp. cultivars. An increase in yellow pigmentation was experimentally confirmed in susceptible melon cultivars by measuring the Hue angle using a solid colorimeter. Carotenoid analyses in infected tissues explained why the visual crtB marker performs better in susceptible melon than in Cucurbita spp. cultivars. These results support that the visual marker crtB may facilitate visual diagnosis of WMV infection in melon breeding programs. [ABSTRACT FROM AUTHOR]

Crocus sativus stigmas are the main source of crocins, which are glucosylated apocarotenoids derived from zeaxanthin cleavage that give saffron its red color. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis in plants. Four PSY genes encoding functional enzymes were isolated from saffron. All the proteins were localized in plastids, but the expression patterns of each gene, CsPSY1a , CsPSY1b , CsPSY2 , and CsPSY3 , in different saffron tissues and during the development of the stigma showed different tissue specialization. The CsPSY2 transcript was primarily detected in the stigmas where it activates and stimulates the accumulation of crocins, while its expression was very low in other tissues. In contrast, CsPSY1a and CsPSY1b were mainly expressed in the leaves, but only CsPSY1b showed stress-light regulation. Interestingly, CsPSY1b showed differential expression of two alternative splice variants, which differ in the intron retention at their 5′ UTRs, resulting in a reduction in their expression levels. In addition, the CsPSY1a and CsPSY1b transcripts, together with the CsPSY3 transcript, were induced in roots under different stress conditions. The CsPSY3 expression was high in the root tip, and its expression was associated with mycorrhizal colonization and strigolactone production. CsPSY3 formed a separate branch to the stress-specific Poaceae homologs but was closely related to the dicot PSY3 enzymes. [ABSTRACT FROM AUTHOR]

Carotenoids are tetraterpenes (40-carbon isoprenoids) derived from the five-carbon isoprene units, isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). These molecules are the major determinants of fruit pigmentation in many plant species, including tomato. The IDP isomerase (IDI) catalyzes the isomerization of IDP to DMADP. Phytoene synthase (PSY) catalyzes the dimerization of two diterpenes from geranylgeranyl pyrophosphate to phytoene, which is the rate-limiting step in carotenoid biosynthesis. Mutations in the tomato PSY1, yellow flesh, are widely used for breeding yellow tomatoes. We report a series of allelic variations in yellow flesh, and in the newly identified apricot, which has yellow fruit as a result of a mutation in IDI1. In HPLC analysis, both the cultivars showed yellow ripe fruits that lacked carotenoids. However, unlike wild type and yellow flesh, apricot mutants had reduced carotenoid levels in flowers. The allelic variations in yellow flesh were determined to have a deletion in the promoter region and a nonsense mutation in the PSY1 gene, whereas apricot contained a 'T' insertion that resulted in premature termination codon in the IDI1 gene. DNA markers to identify allelic variations in two yellow flesh and three apricot mutant lines were developed. These markers and germplasms would be useful for breeding yellow tomatoes. [ABSTRACT FROM AUTHOR]