Phytoene Synthase Research Articles

The transcription factor CaBBX10 promotes chlorophyll and carotenoid pigment accumulation in Capsicum annuum fruit.

Chlorophylls and carotenoids are two pivotal photosynthetic pigments directly influencing the economic value of pepper (Capsicum annuum L.) fruits. However, the coordinated regulatory mechanisms governing the accumulation of both chlorophylls and carotenoids during pepper fruit development remain elusive. In this study, pepper B-box 10 (CaBBX10), a candidate hub transcription factor, was found to play dual roles in the early development of pepper fruit. CaBBX10 virus-induced gene silencing and overexpression experiments demonstrated that the encoded transcription factor promotes both chlorophyll and carotenoid accumulation in pepper fruit. Further comprehensive analyses showed that CaBBX10 directly binds to the promoter of magnesium chelatase subunit D subunit (CaCHLD) and phytoene synthase 1 (CaPSY1), thereby activating their expression in the chlorophyll and carotenoid biosynthesis pathways, respectively. Additionally, the photomorphogenic factor CaCOP1 was found to physically interact with CaBBX10 and lead to its degradation. Therefore, CaBBX10 may serve as a critical link connecting chlorophyll and carotenoid biosynthesis to light signaling. Altogether, our findings reveal a mechanism for the complex transcriptional regulation that simultaneously promotes chlorophyll and carotenoid accumulation in pepper fruit.

Nostoxanthin Biosynthesis by Sphingomonas Species (COS14-R2): Isolation, Identification, and Optimization of Culture Conditions.

Nostoxanthin, a yellow pigment, belongs to the xanthophyll group of carotenoids found in various species of bacteria and cyanobacteria. Several species of Sphingomonas can produce appropriate carotenoids for survive in various environments. This comprise nostoxanthin, a significant carotenoid. The study isolated the Sphingomonas species strain COS14-R2 from the Cosmos bipinnatus and identified it through the whole-genome sequence. The strain consists of a circular chromosome with a length of 3,677,457 base pairs.s ecThe genome consists of three carotenoid biosynthesis genes, specifically crtB (phytoene synthase), crtI (phytoene desaturase), and crtY (Lycopene beta-cyclase), which are involved in the synthesis of nostoxanthin. The strain has a circular, undulated colony morphology with a deep yellow color. It demonstrates optimal growth in liquid media at 25 to 35°C and exhibits a high tolerance for pH levels between 5 and 11 and requires adequate quantities of carbon and nitrogen. We observed the highest concentration of nostoxanthin was recorded at 35°C, pH of 7.5, glucose concentration of 40g L-1, and a yeast extract concentration of 5g L-1 during dark incubation. The fed-batch fermentation process produced nostoxanthin at a concentration of 217.22 ± 9.60mg L-1, with a selectivity of 72.32% and a productivity of 2.59g/L/h. The dry biomass extract was purified using column chromatography. The LC-MS/MS analysis of the purified fraction indicated that the molecular weight of nostoxanthin is 600.5098m/z. The DPPH assay result of 75.5 ± 0.33% indicates nostoxanthin is highly effective in scavenging free radicals.

Regulation Mechanism of Gibberellic Acid-3 for Astaxanthin Biosynthesis in Heterotrophic Growing Chromochloris zofingiensis.

Chromochloris zofingiensis has been proven as a potential resource for large-scale astaxanthin production, but little information on phytohormones for its growth and astaxanthin accumulation could be obtained. This study explored the impact of gibberellic acid-3 (GA3) on growth and astaxanthin biosynthesis in heterotrophic C. zofingiensis. After 6 days of cultivation with GA3, biomass and astaxanthin yields in 7.5 L fermenters reached 268.5 g·L-1 and 0.34 g·L-1, respectively, which were 6% and 89% higher than those in the control. GA3 changed transcription levels of genes linked to carbon metabolism, lipid metabolism, astaxanthin production, and ABC transporters. Genes related to astaxanthin biosynthesis, such as phytoene synthase (PSY), phytoene desaturase (PDS), beta-carotenoid hydroxylase (CHYb), and beta-carotenoid ketolase (BKT), were up-regulated under GA3 induction. The enhancement of carbon metabolism and lipid metabolism led to elevated consumption of substrates and generation of reducing power, thus facilitating astaxanthin biosynthesis. By using GA3 and arginine together, the astaxanthin yield increased to 0.39 g·L-1, which was 18% higher than that obtained under GA3 induction. It could be concluded that GA3 showed significant effects on astaxanthin biosynthesis. This research proposed novel feasible approaches to enhance astaxanthin production in heterotrophic C. zofingiensis.

Proteomic analysis identified proteins that are differentially expressed in the flavonoid and carotenoid biosynthetic pathways of Camellia Nitidissima flowers

BackgroundCamellia nitidissima Chi is a popular ornamental plant because of its golden flowers, which contain flavonoids and carotenoids. To understand the regulatory mechanism of golden color formation, the metabolites of C. nitidissima petals at five different developmental stages were detected, a proteome map of petals was first constructed via tandem mass tag (TMT) analysis, and the accuracy of the sequencing data was validated via parallel reaction monitoring (PRM).ResultsNineteen color components were detected, and most of these components were carotenoids that gradually accumulated, while some metabolites were flavonoids that were gradually depleted. A total of 97,647 spectra were obtained, and 6,789 quantifiable proteins were identified. Then, 1,319 differentially expressed proteins (DEPs) were found, 55 of which belong to the flavonoid and carotenoid pathways, as revealed by pairwise comparisons of protein expression levels across the five developmental stages. Notably, most DEPs involved in the synthesis of flavonoids, such as phenylalanine ammonium lyase and 4-coumarate-CoA ligase, were downregulated during petal development, whereas DEPs involved in carotenoid synthesis, such as phytoene synthase, 1-deoxy-D-xylulose-5-phosphate synthase, and β-cyclase, tended to be upregulated. Furthermore, protein‒protein interaction (PPI) network analysis revealed that these 55 DEPs formed two distinct PPI networks closely tied to the flavonoid and carotenoid synthesis pathways. Phytoene synthase and chalcone synthase exhibited extensive interactions with numerous other proteins and displayed high connectivity within the PPI networks, suggesting their pivotal biological functions in flavonoid and carotenoid biosynthesis.ConclusionProteomic data on the flavonoid and carotenoid biosynthesis pathways were obtained, and the regulatory roles of the DEPs were analyzed, which provided a theoretical basis for further understanding the golden color formation mechanism of C. nitidissima.

Ectopic expression of the GRAS-type transcriptional regulator NSP2 in Parasponia triggers contrasting effects on symbioses.

Plants strictly control root endosymbioses with nutrient-scavenging arbuscular endomycorrhizal fungi or nodule inducing diazotrophic bacteria. The GRAS-type transcriptional regulator NODULATION SIGNALING PATHWAY 2 (NSP2) is a conserved hub in this process. The NSP2-regulated transcriptional network is instrumental in balancing nutrient homeostasis with symbiotic interactions. NSP2 activity is modulated post-transcriptionally by a specific microRNA. Overriding this control mechanism by ectopic expression of a miRNA-resistant NSP2 transgene enhances the symbiotic permissiveness to arbuscular endomycorrhizal fungi. Such engineered plants may possess enhanced capacities for nutrient uptake. However, the trade-off of this strategy on plant development or other symbiotic interactions, like nodulation, is yet to be fully understood. We used the nodulating Cannabaceae species Parasponia andersonii as an experimental system to study the effect of ectopic NSP2 expression. Parasponia and legumes (Fabaceae) diverged 100 million years ago, providing a unique comparative system to dissect the nodulation trait. Six independent transgenic Parasponia lines were generated that differed in the level of NSP2 expression in the root from 6 to 95-fold higher when compared to the empty vector control plants. Analysis of these plants revealed a positive correlation between mycorrhization and the NSP2 expression level, as well as with the expression of the symbiosis transcription factor CYCLOPS and the rate-limiting enzyme in the carotenoid biosynthetic pathway PHYTOENE SYNTHASE1 (PSY1). Yet ectopic expression of NSP2 affected plant architecture and root nodule organogenesis. This indicates a significant trade-off when leveraging NSP2 over-expression to enhance endomycorrhization.

A low frequency magnetic field regulated the synthesis of carotenoids in Rhodotorula mucilaginosa by influencing iron metabolism

The purpose of this study is to investigate the effect of low frequency magnetic fields (LFMF) on carotenoid synthesis and iron metabolism in Rhodotorula mucilaginosa and to explore potential connections between the two processes. Various LFMF intensities were employed to assess carotenoid production, intracellular iron content, and the expression levels of genes related to carotenoid synthesis and iron metabolism in R. mucilaginosa. The results show that a LFMF could promote carotenoid synthesis, resulting in varying increases in intracellular iron content. Specifically, the expression of the iron metabolism-related gene transferrin receptor (TFR) was upregulated, while the expression of ferroportin 1 (FPN1) was downregulated. Meanwhile, the expressions of the carotenoid synthesis-related genes geranylgeranyl diphosphate synthases (GGPPS) and phytoene synthase (PSY) displayed varying degrees of upregulation at different time points. Further, the TFR and FPN1 gene knockout strains of R. mucilaginosa were subjected to treatment with a 3.5-mT LFMF, the results of which showed that an LFMF is capable of influencing the iron metabolism levels and subsequently regulating carotenoid synthesis in R. mucilaginosa.

Combining systems and synthetic biology for in vivo enzymology

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax, and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

<i>Solanum tuberosum</i> L. phytoene synthase genes (<i>StPSY1, StPSY2, StPSY3</i>) participate in the potato plant’s response to cold stress

The structure and phylogeny of the genes StPSY1, StPSY2 and StPSY3, encoding phytoene synthases from Solanum tuberosum L., were characterized. The expression of these genes in potato seedlings was studied in response to exposure to cold stress in the dark phase of the diurnal cycle as an imitation of night cooling. It was found that all three genes are activated when the temperature decreases, and the greatest response is observed for StPSY1. The response of the StPSY3 gene to cold stress and photoperiod has been demonstrated for the first time. A search was carried out for cis-regulatory elements in the promoter region and 5´-UTR of the StPSY genes and it was shown that the regulation of all three genes is associated with the response to light. The high level of low-temperature activation of the StPSY1 gene may be associated with the presence of cis-elements associated with sensitivity to cold and ABA.

Understanding the role of Bacillus subtilis GEB1 in enhancing guava plant immunity against Meloidogyne enterolobii: A multifaceted approach

Guava root-knot nematode, Meloidogyne enterolobii has emerged as a significant threat to guava cultivation, impacting production worldwide. By integrating the molecular, histopathological and in silico approaches the current study explores the efficacy of Bacillus subtilis GEB1 in enhancing the resistance of guava against M. enterolobii. The gene expression studies reveal that guava plants treated with B. subtilis and challenged with nematodes at 72 h showed 2 fold increase in phytoene synthase, 2.1 fold increase in mitogen-activated protein kinase, 1.54 fold increase in phenylalanine ammonia-lyase and 1.82 fold increase in peroxidase genes when compared to untreated control. The Scanning electron microscope analysis of guava roots treated with B. subtilis revealed the presence of spores in the rhizoplane region which led to deter the invading nematodes. The comparative histological studies with guava roots treated with B. subtilis revealed lesser damage in the cortex, vascular regions with limited hypertrophy and hyperplasia, lesser adult females, and feeding traces with minimum root, stele and cortex diameter and thickness than those of untreated infested roots. Attempts were made to dock the plant functional proteins with B. subtilis peptides to understand their interaction. The results revealed that bacillobactin showed a greater binding affinity of −9.0 kcal/mol with phytoene synthase, plipstatin (−16.0 kcal/mol), and fengycin (−15.9 kcal/mol) with MAPK proteins of guava. Overall, the current study offers insight into the intricate mechanisms of plant-nematode-bacteria interactions and underscores the importance of B. subtilis GEB-1 as a biocontrol agent to combat Meloidogyne enterolobii infection in guava.

Genetic mapping and molecular marker development for white flesh color in tomato.

Fruit color significantly influences the quality of horticultural crops, which affects phytochemical diversity and consumer preferences. Despite its importance, the genetic basis of the white-colored fruit in tomatoes remains poorly understood. In this study, we demonstrate that white-fleshed tomato varieties accumulate fewer carotenoids than yellow-fleshed varieties. We developed various segregating populations by hybridizing red, yellow, and white fruit tomato cultivars. Genetic analysis revealed that the white fruit color trait is controlled by a single gene that dominates both red and yellow fruits. Bulk segregant RNA sequencing provided a preliminary map of a 3.17 Mb region on chromosome 3 associated with the white color trait. Based on kompetitive allele-specific PCR (KASP) markers, we narrowed the candidate gene region to 819 kb. Within this region, we identified a 4906-bp sequence absence variation near Phytoene Synthase 1 (SlPSY1) specific to white-colored tomatoes. Genotyping of the progeny and natural populations using a single nucleotide polymorphism adjacent to this absence of variation confirmed its key role in white fruit formation. Collectively, our findings provide insights into white fruit trait formation in tomatoes, enabling tomato breeders to precisely introduce white fruit traits for commercial exploitation.

Polymorphisms in the Transit Peptide of Phytoene Synthase ZmPSY1 Link to the White Color of Grain Endosperm in Maize Inbred Lines

Polymorphisms in the Transit Peptide of Phytoene Synthase ZmPSY1 Link to the White Color of Grain Endosperm in Maize Inbred Lines

Transcriptome wide analysis of MADS box genes in Crocus sativus and interplay of CstMADS19-CstMADS26 in orchestrating apocarotenoid biosynthesis

Flowers of Crocus sativus L. are immensely important not only for arrangement of floral whorls but more because each floral organ is dominated by a different class of specialized compounds. Dried stigmas of C. sativus flowers form commercial saffron, and are known to accumulate unique apocarotenoids like crocin, picrocrocin and safranal. Inspite of being a high value crop, the molecular mechanism regulating flower development in Crocus remains largely unknown. Moreover, it would be very interesting to explore any co-regulatory mechanism which controls floral architecture and secondary metabolic pathways which exist in specific floral organs. Here we report transcriptome wide identification of MADS box genes in Crocus. A total of 39 full length MADS box genes were identified among which three belonged to type I and 36 to type II class. Phylogeny classified them into 11 sub-clusters. Expression pattern revealed some stigma up-regulated genes among which CstMADS19 encoding an AGAMOUS gene showed high expression. Transient over-expression of CstMADS19 in stigmas of Crocus resulted in increased crocin by enhancing expression of pathway genes. Yeast one hybrid assay demonstrated that CstMADS19 binds to promoters of phytoene synthase and carotenoid cleavage dioxygenase 2 genes. Yeast two hybrid and BiFC assays confirmed interaction of CstMADS19 with CstMADS26 which codes for a SEPALATA gene. Co-overexpression of CstMADS19 and CstMADS26 in Crocus stigmas enhanced crocin content more than was observed when genes were expressed individually. Collectively, these findings indicate that CstMADS19 functions as a positive regulator of stigma based apocarotenoid biosynthesis in Crocus.

Unveiling the regulatory role of DzAGL6-1 in carotenoid biosynthesis during durian (Durio zibethinus) fruit development.

Approximately 119 MADS-box genes have been identified in durian. Moreover, DzAGL6-1 primarily expressed during fruit development, activates theDzPSYpromoter. Transient expression of DzAGL6-1 in tomatoes influences carotenoid production. MADS-box transcription factors play a crucial role in regulating plant biological processes, including fruit ripening and associated events. This study aimed to comprehend the mechanisms involved in durian fruit development and ripening and carotenoid production by conducting a genome-wide analysis of MADS-box proteins in durian (Durio zibethinus L.), an economically important fruit in Southeast Asia. A total of 119 durian MADS-box proteins were identified from the genome of the 'Musang King' cultivar. Based on the phylogenetic analysis, the proteins were classified into types I and II, which exhibited similar conserved motif compositions. Notably, only 16 durian MADS-box genes exhibited fruit-specific expression patterns. Among these genes, DzAGL6-1 was predominantly expressed during fruit development, a stage at which carotenoid biosynthesis is activated. Transient expression of DzAGL6-1 in tomato fruit increased the transcript level of the carotenoid biosynthetic gene phytoene synthase (PSY) and the β-carotene content. Furthermore, DzAGL6-1 activated the promoter activity of DzPSY, as demonstrated by a dual-luciferase assay. These findings provide insights into the role of MADS-box transcription factors in regulating carotenoid biosynthesis during durian fruit development.

Boosting pro-vitamin A content and bioaccessibility in leaves by combining engineered biosynthesis and storage pathways with high-light treatments.

Biofortification of green leafy vegetables with pro-vitamin A carotenoids, such as β-carotene, has remained challenging to date. Here, we combined two strategies to achieve this goal. One of them involves producing β-carotene in the cytosol of leaf cells to avoid the negative impacts on photosynthesis derived from changing the balance of carotenoids and chlorophylls in chloroplasts. The second approach involves the conversion of chloroplasts into non-photosynthetic, carotenoid-overaccumulating chromoplasts in leaves agroinfiltrated or infected with constructs encoding the bacterial phytoene synthase crtB, leaving other non-engineered leaves of the plant to sustain normal growth. A combination of these two strategies, referred to as strategy C (for cytosolic production) and strategy P (for plastid conversion mediated by crtB), resulted in a 5-fold increase in the amount of β-carotene in Nicotiana benthamiana leaves. Following several attempts to further improve β-carotene leaf contents by metabolic engineering, hormone treatments and genetic screenings, it was found that promoting the proliferation of plastoglobules with increased light-intensity treatments not only improved β-carotene accumulation but it also resulted in a much higher bioaccessibility. The combination of strategies C and P together with a more intense light treatment increased the levels of accessible β-carotene 30-fold compared to controls. We further demonstrated that stimulating plastoglobule proliferation with strategy P, but also with a higher-light treatment alone, also improved β-carotene contents and bioaccessibility in edible lettuce (Lactuca sativa) leaves.

A transcriptional cascade involving BBX22 and HY5 finely regulates both plant height and fruit pigmentation in citrus.

Dwarfing is a pivotal agronomic trait affecting both yield and quality. Citrus species exhibit substantial variation in plant height, among which internode length is a core element. However, the molecular mechanism governing internode elongation remains unclear. Here, we unveiled that the transcriptional cascade consisting of B-BOX DOMAIN PROTEIN 22 (BBX22) and ELONGATED HYPOCOTYL 5 (HY5) finely tunes plant height and internode elongation in citrus. Loss-of-function mutations of BBX22 in an early-flowering citrus (Citrus hindsii "SJG") promoted internode elongation and reduced pigment accumulation, whereas ectopic expression of BBX22 in SJG, sweet orange (C. sinensis), pomelo (C. maxima) or heterologous expression of BBX22 in tomato (Solanum lycopersicum) significantly decreased internode length. Furthermore, exogenous application of gibberellin A3 (GA3) rescued the shortened internode and dwarf phenotype caused by BBX22 overexpression. Additional experiments revealed that BBX22 played a dual role in regulation internode elongation and pigmentation in citrus. On the one hand, it directly bound to and activated the expression of HY5, GA metabolism gene (GA2 OXIDASE 8, GA2ox8), carotenoid biosynthesis gene (PHYTOENE SYNTHASE 1, PSY1) and anthocyanin regulatory gene (Ruby1, a MYB DOMAIN PROTEIN). On the other hand, it acted as a cofactor of HY5, enhancing the ability of HY5 to regulate target genes expression. Together, our results reveal the critical role of the transcriptional cascade consisting of BBX22 and HY5 in controlling internode elongation and pigment accumulation in citrus. Unraveling the crosstalk regulatory mechanism between internode elongation and fruit pigmentation provides key genes for breeding of novel types with both dwarf and health-beneficial fortification in citrus.

Carotenoid biosynthesis profiling unveils the variance of flower coloration in Tagetes erecta and enhances fruit pigmentation in tomato

Carotenoids play a pivotal role in plant. Tagetes erecta, commonly called marigold, has increasing nutritional and economic value due to its high level of carotenoids in flower. However, the functional genes in the carotenoid biosynthesis of T. erecta have not been studied. In this work, three T. erecta varieties with flowers of yellow, yellow-orange and orange color, respectively, were examined for carotenoids composition and corresponding expression profiling of biosynthetic genes at four developmental stages. The results indicated that the varieties with higher lutein content, orange-flower ‘Juwang’ and yellow-orange ‘Taishan’, exhibited significant upregulation of genes in the upstream biosynthesis pathway, especially PDS (phytoene desaturase), PSY (phytoene synthase) and ZDS (zeta-carotene desaturase), whereas downstream carotenoid cleavage genes CCD (carotenoid cleavage dioxygenase) were markedly downregulated throughout flower development in the highest lutein containing variety ‘Juwang’. Furthermore, marigold TePDS, TePSYS3 and TeZDS were isolated and transformed into tomato. Overexpression of TePDS or TeZDS resulted in the promotion of fruit ripening and accumulation of carotenoids in the transgenic lines. On the other hand, marigold TePSYS3 showed multiple effects, not only on fruit carotenogenesis but also on pigmentation patterns in vegetative tissues and plant growth. Taken together, the variations in expression profiles of the biosynthetic genes contribute to dynamic change in carotenoid levels and diversity of flower coloration in T. erecta. These functional genes of T. erecta were verified in tomato and provide targets for genetic improvement of fruit carotenoids accumulation.

Light Intensity Enhances the Lutein Production in Chromochloris zofingiensis Mutant LUT-4.

Chromochloris zofingiensis, a unicellular green alga, is a potential source of natural carotenoids. In this study, the mutant LUT-4 was acquired from the chemical mutagenesis pool of C. zofingiensis strain. The biomass yield and lutein content of LUT-4 reached 9.23 g·L-1, and 0.209% of dry weight (DW) on Day 3, which was 49.4%, and 33% higher than that of wild-type (WT), respectively. The biomass yields of LUT-4 under 100, 300, and 500 µmol/m2/s reached 8.4 g·L-1, 7.75 g·L-1, and 6.6 g·L-1, which was 10.4%, 21%, and 29.6% lower compared with the control, respectively. Under mixotrophic conditions, the lutein yields were significantly higher than that obtained in the control. The light intensity of 300 µmol/m2/s was optimal for lutein biosynthesis and the content of lutein reached 0.294% of DW on Day 3, which was 40.7% more than that of the control. When LUT-4 was grown under 300 µmol/m2/s, a significant increase in expression of genes implicated in lutein biosynthesis, including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene epsilon cyclase (LCYe) was observed. The changes in biochemical composition, Ace-CoA, pyruvate, isopentenyl pyrophosphate (IPP), and geranylgeranyl diphosphate (GGPP) contents during lutein biosynthesis were caused by utilization of organic carbon. It was thereby concluded that 300 µmol/m2/s was the optimal culture light intensity for the mutant LUT-4 to synthesize lutein. The results would be helpful for the large-scale production of lutein.

Proteome and metabolome of Caryocar brasiliense camb. fruit and their interaction during development

Considered the symbol fruit of the Brazilian Cerrado, pequi (Caryocar brasiliense Camb.) is an exotic and much-appreciated fruit with an internal mesocarp (edible part) with an eye-catching golden yellow color. In an unprecedented way, this study characterized the proteome throughout pequi development. The most influential and essential transcription factors operating in the regulation of pequi ripening identified were members of the MAD-box family. A group of proteins related to the methionine cycle indicates the high consumption and recycling of methionine. However this consumption does not occur mainly for the biosynthesis of ethylene, a process dependent on methionine consumption. In the bioactive compounds presented, different proteins could be correlated with the presence of these phytochemicals, such as monodehydroascorbate reductase and ascorbate peroxidase in ascorbic acid recycling; pyruvate kinase, fructose bisphosphate aldolase and phytoene synthase with carotenoid biosynthesis; S-adenosylmethionine synthase 1 as a donor of methyl groups in the formation of trigonelline and aspartate aminotransferase as a biomarker of initial regulation of the trigonelline biosynthetic pathway; phenylalanine ammonia lyase, chorismate synthesis and chalcone-flavononone isomerase in the biosynthesis of phenolic compounds. Among the volatile organic compounds identified, the majority compound in pequi was ethyl hexanoate ester, with an area of 50.68 % in the ripe fruit, and in this group of esters that was the most representative, alcohol dehydrogenase, a fundamental enzyme in the synthesis of esters, was identified with an increase of approximately 7.2 times between the first and last stages. Therefore, an extensive group of proteins and some metabolites can serve as biomarkers of ripening in pequi, as most were more expressed in the last stage, which is the ripe fruit suitable for consumption.

Functional Characterization of the First Bona Fide Phytoene Synthase in Red Algae from Pyropia yezoensis.

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.

Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus

Key messageModification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callusMetabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.