Phytoene Synthase Research Articles

Chromoplast plastoglobules recruit the carotenoid biosynthetic pathway and contribute to carotenoid accumulation during tomato fruit maturation.

Tomato (Solanum lycopersicum) fruit maturation is associated with a developmental transition from chloroplasts (in mature green fruit) to chromoplasts (in red fruit). The hallmark red color of ripe tomatoes is due to carotenogenesis and accumulation of the red carotenoid lycopene inside chromoplasts. Plastoglobules (PG) are lipid droplets in plastids that are involved in diverse lipid metabolic pathways. In tomato, information on the possible role of PG in carotogenesis and the PG proteome is largely lacking. Here, we outline the role of PG in carotenogenesis giving particular attention to tomato fruit PG proteomes and metabolomes. The proteome analysis revealed the presence of PG-typical FBNs, ABC1K-like kinases, and metabolic enzymes, and those were decreased in the PG of tomato chromoplasts compared to chloroplasts. Notably, the complete β-carotene biosynthesis pathway was recruited to chromoplast PG, and the enzymes PHYTOENE SYNTHASE 1 (PSY-1), PHYTOENE DESATURASE (PDS), ZETA-CAROTENE DESATURASE (ZDS), and CAROTENOID ISOMERASE (CRTISO) were enriched up to twelvefold compared to chloroplast PG. We profiled the carotenoid and prenyl lipid changes in PG during the chloroplast to chromoplast transition and demonstrated large increases of lycopene and β-carotene in chromoplast PG. The PG proteome and metabolome are subject to extensive remodeling resulting in high accumulation of lycopene during the chloroplast-to-chromoplast transition. Overall, the results indicate that PGs contribute to carotenoid accumulation during tomato fruit maturation and suggest that they do so by functioning as a biosynthetic platform for carotenogenesis.

Phenotypic and genotypic characterization to authenticate and trace a high-quality yellow tomato ecotype through the processing chain

In the last few years considerable interest is growing for high food quality, both for organoleptic and nutritional aspects. As for tomatoes, the yellow fruit varieties are conquering the consumer markets in many Mediterranean countries. Among the great variability of yellow tomatoes, one landrace of “Pomodorino giallo del Vesuvio”, named GiaGiù (E40), differs from the traditional “Pomodorino del Piennolo” for the fruit color and stands out for its quality traits, such as high glutamic acid, pectin content and titratable acidity of fruit, thus increasing its demand for both fresh consumption and cooking purposes. The aim of this work was to phenotypically and genotypically distinguish the GiaGiù landrace through morphological descriptors and molecular markers, in order to provide an effective tool to authenticate this product as fresh and processed tomatoes. Morphological characterization evidenced that the distinctive traits of GiaGiù landrace were the potato leaf morphology and the pyriform shape with a pointed apex of the yellow fruits. The genotypic distinction of E40 was performed by using two Cleaved Amplified Polymorphic Sequence (CAPS) markers designed on a Single Nucleotide Polymorphism (SNP) of the Phytoene synthase 1 (psy1) gene that confers the yellow color to tomato fruit and already known as specific of GiaGiù genotype. Additional CAPS markers were designed on two private mutations of E40 genes derived from data retrieved from a Genotyping-By-Sequencing (GBS) dataset, already available. These findings were confirmed by comparing E40 private mutations with the 360 accessions of the BGI tomato 360 genomes resequencing project. The designed markers allowed to distinguish GiaGiù genotype in all fresh and processed fruit tomato matrices, thus representing a molecular tool able to prevent food fraud and authenticate GiaGiù products.

Soil-to-fruit nitrogen flux mediates the onset of fruit-nitrogen remobilization and color change in citrus

In non-climacteric fruit tree species, color change is primarily controlled by the interplay between environmental conditions, nutritional factors (nitrogen and sugars) and hormones, mainly abscisic acid and gibberellins (GA), through a complex mechanism which is not completely understood. Nitrogen has a strong impact on color change, influenced by environmental changes, either locally or at the whole tree level. We use Citrus trees, as a non-climacteric model species, to understand the long-distance nitrogen signaling mechanism delaying the chloroplast-to-chromoplast transition at the molecular level. It is unknown whether nitrogen regulates the branch point of geranylgeranyl diphosphate (GGPP), the shared precursor for gibberellin, chlorophyll and carotenoid synthesis. We used 15 N to trace the root-to-fruit nitrogen flux in trees grown under soils differing in nitrogen content and temperature, and treated with NH 4 NO 3 and GA. Key genes involved in nitrogen signaling, transport and metabolism, and those from the GGPP branch point were analyzed in the fruit. Results explain how soil temperature modifies 15 N transport to play a key role in signaling citrus color change, and show that fruit-nitrogen remobilization is required for triggering degreening. Nitrogen content in the fruit modulates the onset of glutamate deamination, asparagine synthesis, nitrite assimilation and GA 1 depletion. Expression of the nitrate transporter NRT1.2, glutamate dehydrogenase and asparagine synthetase genes was high right before the start of degreening, together with NH 4 + concentration. Nitrogen delayed carotenoid synthesis (phytoene synthase gene expression) without modifying gibberellin synthesis ( ent -kaurene synthase and oxidase) at the chloroplast level. • N and GA 1 depletion from the fruit is a prerequisite for citrus color change • Cold-soil temperatures reduce 15 N translocation and trigger fruit color change • Low N flux correlates fruit protein hydrolysis, upregulating GDH, AS and NRT1.2 • GA treatment delays protein hydrolysis, NRT1.2 expression and N depletion • The first steps of synthesis of GA in the chloroplast are not modified by N

Differences in commercial quality and carotenoids profile of yellow- and white-fleshed nectarine fruit during low temperature storage and the regulation of carotenoids by sugar

The aim of this study was to assess commercial quality parameters, carotenoids and sugars in two different nectarine cultivars (‘Zijinhong’, yellow flesh, and ‘Ruiguang’, white flesh) to gain insight into their commercial quality differences and the interaction among carotenoid, glucose, sucrose and fructose in nectarines during low temperature storage. In comparison with the white-fleshed cultivar ‘Ruiguang’, higher storability by higher soluble solid content and slower decrease of fruit firmness were exhibited in yellow-fleshed ‘Zijinhong’ nectarine. The increased carotenoids accumulation and inhibition of glucose and fructose degradation occurred in both cultivars, and glucose were positively correlated with total carotenoid contents (0.9825). It was confirmed through an in vitro test that glucose and fructose induced the biosynthesis of carotenoids. In addition, the gene expression of phytoene synthase and carotenoid cleavage dioxygenase were highly correlated with carotenoid content in ‘Zijinhong’ nectarine flesh.

Establishment of Transcriptional Gene Silencing Targeting the Promoter Regions of GFP, PDS, and PSY Genes in Cotton using Virus-Induced Gene Silencing.

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.

The C-terminal region of phytoene synthase is a key element to control carotenoid biosynthesis in the haloarchaeon Haloferax volcanii.

Phytoene synthase (PSY) converts two molecules of geranyl-geranyl diphosphate to phytoene, the key regulatory step in carotenogenesis. However, post-translational mechanisms that control PSY expression are scarcely understood. Carotenoid biosynthesis (mainly bacterioruberin) is a distinctive feature of haloarchaea thriving in hypersaline environments. Carotenogenesis is negatively regulated by the AAA+ LonB protease in the haloarchaeon Haloferax volcanii as it controls PSY degradation. We investigated the relevance of the C-terminal portion of HvPSY as a regulatory element for carotenoid biosynthesis. H. volcanii mutants were constructed to express full-length HvPSY protein (strain HVPSYwt) and truncated HvPSY lacking 10 (HVPSY10), 20 (HVPSY20) or 34 amino acids (HVPSY34) at the C-terminus. Cells of HVPSY20 and HVPSY34 showed hyperpigmentation (bacterioruberin content 3-fold higher than HVPSYwt) which correlated with increased PSY protein abundance (2-fold in HVPSY34) while they contained less psy transcript level compared with HVPSYwt. In vivo degradation assays showed that HvPSY34 was more stable than HvPSYwt. Collectively, these results show that the C-terminal region of HvPSY contains a 'recognition determinant' for proteolysis in H. volcanii. Preliminary evidence suggests that LonB is involved in the recognition mechanism. This study provides the first identification of a regulatory sequence in an archaeal PSY for the post-translational control of carotenogenesis.

Flooding and herbivory: the effect of concurrent stress factors on plant volatile emissions and gene expression in two heirloom tomato varieties

BackgroundIn nature and in cultivated fields, plants encounter multiple stress factors. Nonetheless, our understanding of how plants actively respond to combinatorial stress remains limited. Among the least studied stress combination is that of flooding and herbivory, despite the growing importance of these stressors in the context of climate change. We investigated plant chemistry and gene expression changes in two heirloom tomato varieties: Cherokee Purple (CP) and Striped German (SG) in response to flooding, herbivory by Spodoptera exigua, and their combination.ResultsVolatile organic compounds (VOCs) identified in tomato plants subjected to flooding and/or herbivory included several mono- and sesquiterpenes. Flooding was the main factor altering VOCs emission rates, and impacting plant biomass accumulation, while different varieties had quantitative differences in their VOC emissions. At the gene expression levels, there were 335 differentially expressed genes between the two tomato plant varieties, these included genes encoding for phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA-reductase-like, and phytoene synthase (Psy1). Flooding and variety effects together influenced abscisic acid (ABA) signaling genes with the SG variety showing higher levels of ABA production and ABA-dependent signaling upon flooding. Flooding downregulated genes associated with cytokinin catabolism and general defense response and upregulated genes associated with ethylene biosynthesis, anthocyanin biosynthesis, and gibberellin biosynthesis. Combining flooding and herbivory induced the upregulation of genes including chalcone synthase (CHS), PAL, and genes encoding BAHD acyltransferase and UDP-glucose iridoid glucosyltransferase-like genes in one of the tomato varieties (CP) and a disproportionate number of heat-shock proteins in SG. Only the SG variety had measurable changes in gene expression due to herbivory alone, upregulating zeatin, and O-glucosyltransferase and thioredoxin among others.ConclusionOur results suggest that both heirloom tomato plant varieties differ in their production of secondary metabolites including phenylpropanoids and terpenoids and their regulation and activation of ABA signaling upon stress associated with flooding. Herbivory and flooding together had interacting effects that were evident at the level of plant chemistry (VOCs production), gene expression and biomass markers. Results from our study highlight the complex nature of plant responses to combinatorial stresses and point at specific genes and pathways that are affected by flooding and herbivory combined.

Application of 5-aminolevulinic acid promotes ripening and accumulation of primary and secondary metabolites in postharvest tomato fruit.

5-Aminolevulinic acid (ALA) plays a vital role in promoting plant growth, enhancing stress resistance, and improving fruit yield and quality. In the present study, tomato fruits were harvested at mature green stage and sprayed with 200 mg L-1 ALA on fruit surface. During ripening, the estimation of primary and secondary metabolites, carotenoids, and chlorophyll contents, and the expression levels of key genes involved in their metabolism were carried out. The results showed that ALA significantly promoted carotenoids accumulation by upregulating the gene expression levels of geranylgeranyl diphosphate synthase (GGPPS, encoding geranylgeranyl diphosphate synthase), phytoene synthase 1 (PSY1, encoding phytoene synthase), phytoene desaturase (PDS, encoding phytoene desaturase), and lycopeneβ-cyclase (LCYB, encoding lycopene β-cyclase), whereas chlorophyll content decreased by downregulating the expression levels of Mg-chelatase (CHLH, encoding Mg-chelatase) and protochlorophyllide oxidoreductase (POR, encoding protochlorophyllide oxidoreductase). Besides, the contents of soluble solids, vitamin C, soluble protein, free amino acids, total soluble sugar, organic acid, total phenol, and flavonoid were increased in ALA-treated tomato fruit, but the fruit firmness was decreased. These results indicated that the exogenous ALA could not only promote postharvest tomato fruit ripening but also improve the internal nutritional and flavor quality of tomato fruit.

Transcript expression level analysis of phytoene synthase and phytoene desaturase associated with β-carotene content in selected Kenyan Bitter melon.

Bitter melon (Momordica charantia L.) is a widely cultivated food and medicinal plant native to the world's subtropics and tropics. Constraints affecting cultivation of Bitter melon affect productivity of β-carotene. Knowing the mechanism that controls the transcription of the β-carotene biosynthesis genes in Bitter melon will be of great value in improving the yield of this important metabolite. The expressions of β-carotene biosynthetic genes such as Phytoene Desaturase (PDS) and Phytoene Synthase (PSY) were evaluated in Bitter melon accessions 'GBK027049', 'NS1026', 'Mahy-ventura', '453B' and 'Sibuka532'. Transcript expression level analysis of PSY and PDS, and amount of β-carotene in leaf, stem, and fruit, were determined using quantitative polymerase chain reaction and high-performance liquid chromatography (HPLC). Root transcript expression was used as a negative control for determining relative fold change in other tissues. Expression of PSY in fruit (6 to 27-fold compared to the control) was higher than in the other organs for all accessions. This was also the case of PDS expression (10 to 29-fold compared to the control). Leaves had the highest β-carotene concentration (17.92-45.35µg∙g-1); there was no difference between stems (5.67-12.75µg∙g-1) and fruit (6.18-12.53µg∙g-1). The highest β-carotene content was in accessions 'GBK027049' (12.53-45.35µg∙g-1) and '453B' (6.18-32.09µg∙g-1). The PSY and PDS expressions were positively correlated with amount of β-carotene in leaves, stems, and fruits. Bitter melon leaves, especially those of 'GBK027049' and '453B' accessions, are an alternative to alleviate the β-carotene deficiencies in the world and especially in Africa.

Integrated Metabolomic and Transcriptomic Analyses Reveal the Basis for Carotenoid Biosynthesis in Sweet Potato (Ipomoea batatas (L.) Lam.) Storage Roots.

Carotenoids are important compounds of quality and coloration within sweet potato storage roots, but the mechanisms that govern the accumulation of these carotenoids remain poorly understood. In this study, metabolomic and transcriptomic analyses of carotenoids were performed using young storage roots (S2) and old storage roots (S4) from white-fleshed (variety S19) and yellow-fleshed (variety BS) sweet potato types. S19 storage roots exhibited significantly lower total carotenoid levels relative to BS storage roots, and different numbers of carotenoid types were detected in the BS-S2, BS-S4, S19-S2, and S19-S4 samples. β-cryptoxanthin was identified as a potential key driver of differences in root coloration between the S19 and BS types. Combined transcriptomic and metabolomic analyses revealed significant co-annotation of the carotenoid and abscisic acid (ABA) metabolic pathways, PSY (phytoene synthase), CHYB (β-carotene 3-hydroxylase), ZEP (zeaxanthin epoxidase), NCED3 (9-cis-epoxycarotenoid dioxygenase 3), ABA2 (xanthoxin dehydrogenase), and CYP707A (abscisic acid 8'-hydroxylase) genes were found to be closely associated with carotenoid and ABA content in these sweet potato storage roots. The expression patterns of the transcription factors OFP and FAR1 were associated with the ABA content in these two sweet potato types. Together, these results provide a valuable foundation for understanding the mechanisms governing carotenoid biosynthesis in storage roots, and offer a theoretical basis for sweet potato breeding and management.

Transcriptome and Metabolome Analysis of Color Changes during Fruit Development of Pepper (Capsicum baccatum).

Fruit color is one of the most critical characteristics of pepper. In this study, pepper (Capsicum baccatum L.) fruits with four trans-coloring periods were used as experimental materials to explore the color conversion mechanism of pepper fruit. By transcriptome and metabolome analysis, we identified a total of 307 flavonoid metabolites, 68 carotenoid metabolites, 29 DEGs associated with flavonoid biosynthesis, and 30 DEGs related to carotenoid biosynthesis. Through WGCNA (weighted gene co-expression network analysis) analysis, positively correlated modules with flavonoids and carotenoids were identified, and hub genes associated with flavonoid and carotenoid synthesis and transport were anticipated. We identified Pinobanksin, Naringenin Chalcone, and Naringenin as key metabolites in the flavonoid biosynthetic pathway catalyzed by the key genes chalcone synthase (CHS CQW23_29123, CQW23_29380, CQW23_12748), cinnamic acid 4-hydroxylase (C4H CQW23_16085, CQW23_16084), cytochrome P450 (CYP450 CQW23_19845, CQW23_24900). In addition, phytoene synthase (PSY CQW23_09483), phytoene dehydrogenase (PDS CQW23_11317), zeta-carotene desaturase (ZDS CQW23_19986), lycopene beta cyclase (LYC CQW23_09027), zeaxanthin epoxidase (ZEP CQW23_05387), 9-cis-epoxycarotenoid dioxygenase (NCED CQW23_17736), capsanthin/capsorubin synthase (CCS CQW23_30321) are key genes in the carotenoid biosynthetic pathway, catalyzing the synthesis of key metabolites such as Phytoene, Lycopene, β-carotene and ε-carotene. We also found that transcription factor families such as p450 and NBARC could play important roles in the biosynthesis of flavonoids and carotenoids in pepper fruits. These results provide new insights into the interaction mechanisms of genes and metabolites involved in the biosynthesis of flavonoids and carotenoids in pepper fruit leading to color changes in pepper fruit.

Exogenous Arachidonic Acid Affects Fucoxanthin Biosynthesis and Photoprotection in Phaeodactylum tricornutum.

Fucoxanthin is an oxygenated carotenoid component that has been reported to play important roles in anti-oxidation, anti-obesity and anti-cancer in the human body. Fucoxanthin-chlorophyll protein (FCP) complexes participate in light harvesting and photoprotection in diatom. In order to better understand the change of fucoxanthin content and its role in photoprotection, the growth, fucoxanthin biosynthesis and photosynthetic phenotypes were studied in the diatom Phaeodactylum tricornutum under the treatment of exogenous arachidonic acid (AA). Our results demonstrated that even low concentration of AA at 0.1 mg/L strongly induced fucoxanthin accumulation in algal cells to a maximum of 1.1 mg/g dry weight (DW), which was 36.6% higher than that in the untreated ones. By principal component analysis (PCA), we also identified a close correlation between fucoxanthin accumulation and the expression of genes involved in fucoxanthin biosynthesis, especially phytoene synthase (PSY), suggesting that AA change the metabolism of fucoxanthin by inducing carotenoid metabolic enzymes at the transcriptional level. Furthermore, we found that the exogenous application of AA affected non-photochemical quenching (NPQ) and photoinhibition, which resulted from the changed diadinoxanthin (DD) and diatoxanthin (DT) cycle, and thus played an important role in photoprotection.

Hybrid histidine kinase HisK2301 modulates carotenoid production to counteract cold-induced oxidative stress in Rhodosporidium kratochvilovae YM25235 under low temperature.

Hybrid histidine kinases (HHKs) are major sensor proteins for fungi that contribute to stress tolerance. In the present work, we investigated the roles and mechanisms of the HHK HisK2301 in cold-adapted Rhodosporidium kratochvilovae strain YM25235. The HisK2301 deletion strain was constructed by homologous recombination method and arranged for multiple stress tests. We analysed the content of carotenoid using UV-Vis and HPLC. Relative transcript levels of genes phytoene desaturase (RKCrtI) and phytoene synthase and lycopene cyclase (RKCrtYB) were analysed by RT-qPCR. Intracellular reactive oxygen species (ROS) generation was measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Our results clearly indicated that YM25235 produces γ-carotene, torulene, β-carotene and torularhodin, with the latter two components strongly related to adapt to cold. HisK2301 is crucial for YM25235 adaptation to different types of stress such as cold, salt, osmotic and oxidative stress. Growth at low temperature clearly induced oxidative stress in YM25235, as more ROS accumulated at cold. During cold stress, HisK2301 is suggested to sense cold-induced ROS signals and then promote carotenoid production partially by RKCrtI and RKCrtYB to scavenge excessive ROS production. Such an inducible protective system may confer YM25235 fast response and better adaptation to cold stress. To conclude, our findings give the first insight into the effect of HisK2301 on carotenoid biosynthesis and cold-induced oxidative stress in fungi under low temperature and suggest the potential use of the cold-adapted HHK HisK2301 in industrial production of carotenoid.

Carrot DcALFIN4 and DcALFIN7 Transcription Factors Boost Carotenoid Levels and Participate Differentially in Salt Stress Tolerance When Expressed in Arabidopsis thaliana and Actinidia deliciosa.

ALFIN-like transcription factors (ALs) are involved in several physiological processes such as seed germination, root development and abiotic stress responses in plants. In carrot (Daucus carota), the expression of DcPSY2, a gene encoding phytoene synthase required for carotenoid biosynthesis, is induced after salt and abscisic acid (ABA) treatment. Interestingly, the DcPSY2 promoter contains multiple ALFIN response elements. By in silico analysis, we identified two putative genes with the molecular characteristics of ALs, DcAL4 and DcAL7, in the carrot transcriptome. These genes encode nuclear proteins that transactivate reporter genes and bind to the carrot DcPSY2 promoter in yeast. The expression of both genes is induced in carrot under salt stress, especially DcAL4 which also responds to ABA treatment. Transgenic homozygous T3 Arabidopsis thaliana lines that stably express DcAL4 and DcAL7 show a higher survival rate with respect to control plants after chronic salt stress. Of note is that DcAL4 lines present a better performance in salt treatments, correlating with the expression level of DcAL4, AtPSY and AtDXR and an increase in carotenoid and chlorophyll contents. Likewise, DcAL4 transgenic kiwi (Actinidia deliciosa) lines show increased carotenoid and chlorophyll content and higher survival rate compared to control plants after chronic salt treatment. Therefore, DcAL4 and DcAL7 encode functional transcription factors, while ectopic expression of DcAL4 provides increased tolerance to salinity in Arabidopsis and Kiwi plants.

Overexpression of PSY1 increases fruit skin and flesh carotenoid content and reveals associated transcription factors in apple (Malus × domestica).

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.

Enhancement of violaxanthin accumulation in Nannochloropsis oceanica by overexpressing a carotenoid isomerase gene from Phaeodactylum tricornutum.

Nannochloropsis has been considered as a promising feedstock for the industrial production of violaxanthin. However, a rational breeding strategy for the enhancement of violaxanthin content in this microalga is still vacant, thereby limiting its industrial application. All-trans-lycopene locates in the first branch point of carotenogenesis. The carotenoid isomerase (CRTISO), catalyzing the lycopene formation, is thus regarded as a key enzyme for carotenogenesis. Phaeodactylum tricornutum can accumulate high-level carotenoids under optimal conditions. Therefore, it is feasible to improve violaxanthin level in Nannochloropsis by overexpression of PtCRTISO. Protein targeting analysis of seven PtCRTISO candidates (PtCRTISO1–6 and PtCRTISO-like) demonstrated that PtCRTISO4 was most likely the carotenoid isomerase of P. tricornutum. Moreover, the transcriptional pattern of PtCRTISO4 at different cultivation periods was quite similar to other known carotenogenesis genes. Thus, PtCRTISO4 was transformed into N. oceanica. Compared to the wild type (WT), all three transgenic lines (T1–T3) of N. oceanica exhibited higher levels of total carotenoid and violaxanthin. Notably, T3 exhibited the peak violaxanthin content of 4.48 mg g–1 dry cell weight (DCW), which was 1.68-folds higher than WT. Interestingly, qRT-polymerase chain reaction (PCR) results demonstrated that phytoene synthase (NoPSY) rather than ζ-carotene desaturase (NoZDS) and lycopene β-cyclase (NoLCYB) exhibited the highest upregulation, suggesting that PtCRTISO4 played an additional regulatory role in terms of carotenoid accumulation. Moreover, PtCRTISO4 overexpression increased C18:1n-9 but decreased C16:1n-7, implying that C18:1 may serve as a main feedstock for xanthophyll esterification in Nannochloropsis. Our results will provide valuable information for the violaxanthin production from Nannochloropsis.

Gardenia carotenoid cleavage dioxygenase 4a is an efficient tool for biotechnological production of crocins in green and non-green plant tissues.

Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the colour and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the carotenoid cleavage dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison with CsCCD2L, in citrus callus engineered by Agrobacterium-mediated supertransformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. We demonstrate that co-expression of GjCCD4a with phytoene synthase and β-carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved β-carotene, in planta, and produced crocetin dialdehyde via C30 β-apocarotenoid intermediate. GjCCD4a also cleaved C27 β-apocarotenoids, providing a new route for C17 -dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast-like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis-9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non-green crop tissues/organs.

Metabolic Profiling and Transcriptional Analysis of Carotenoid Accumulation in a Red-Fleshed Mutant of Pummelo (Citrus grandis).

Citrus grandis ‘Tomentosa’, commonly known as ‘Huajuhong’ pummelo (HJH), is used in traditional Chinese medicine and can moisten the lungs, resolve phlegm, and relieve coughs. A spontaneous bud mutant, named R-HJH, had a visually attractive phenotype with red albedo tissue and red juice sacs. In this study, the content and composition of carotenoids were investigated and compared between R-HJH and wild-type HJH using HPLC–MS analysis. The total carotenoids in the albedo tissue and juice sacs of R-HJH were 4.03- and 2.89-fold greater than those in HJH, respectively. The massive accumulation of carotenoids, including lycopene, β-carotene and phytoene, led to the attractive red color of R-HJH. However, the contents of flavones, coumarins and most volatile components (mainly D-limonene and γ-terpinene) were clearly reduced in R-HJH compared with wild-type HJH. To identify the molecular basis of carotenoid accumulation in R-HJH, RNA-Seq transcriptome sequencing was performed. Among 3948 differentially expressed genes (DEGs), the increased upstream synthesis genes (phytoene synthase gene, PSY) and decreased downstream genes (β-carotene hydroxylase gene, CHYB and carotenoid cleavage dioxygenase gene, CCD7) might be the key factors that account for the high level of carotenoids in R-HJH. These results will be beneficial for determining the molecular mechanism of carotenoid accumulation and metabolism in pummelo.

Rice carotenoid biofortification and yield improvement conferred by endosperm-specific overexpression of OsGLK1

Carotenoids, indispensable isoprenoid phytonutrients, are synthesized in plastids and are known to be deficient in rice endosperm. Many studies, involving transgenic manipulations of carotenoid biosynthetic genes, have been performed to obtain carotenoid-enriched rice grains. Nuclear-encoded GOLDEN2-LIKE (GLK) transcription factors play important roles in the regulation of plastid and thylakoid grana development. Here, we show that endosperm-specific overexpression of rice GLK1 gene (OsGLK1) leads to enhanced carotenoid production, increased grain yield, but deteriorated grain quality in rice. Subsequently, we performed the bioengineering of carotenoids biosynthesis in rice endosperm by introducing other three carotenogenic genes, tHMG1, ZmPSY1, and PaCrtI, which encode the enzymes truncated 3-hydroxy-3-methylglutaryl-CoA reductase, phytoene synthase, and phytoene desaturase, respectively. Transgenic overexpression of all four genes (OsGLK1, tHMG1, ZmPSY1, and PaCrtI) driven by rice endosperm-specific promoter GluB-1 established a mini carotenoid biosynthetic pathway in the endosperm and exerted a roughly multiplicative effect on the carotenoid accumulation as compared with the overexpression of only three genes (tHMG1, ZmPSY1, and PaCrtI). In addition, the yield enhancement and quality reduction traits were also present in the transgenic rice overexpressing the selected four genes. Our results revealed that OsGLK1 confers favorable characters in rice endosperm and could help to refine strategies for the carotenoid and other plastid-synthesized micronutrient fortification in bioengineered plants.

Multi-omics analyses of 398 foxtail millet accessions reveal genomic regions associated with domestication, metabolite traits, and anti-inflammatory effects

Foxtail millet (Setaria italica), which was domesticated from the wild species green foxtail (Setaria viridis), is a rich source of phytonutrients for humans. To evaluate how breeding changed the metabolome of foxtail millet grains, we generated and analyzed the datasets encompassing the genomes, transcriptomes, metabolomes, and anti-inflammatory indices from 398 foxtail millet accessions. We identified hundreds of common variants that influence numerous secondary metabolites. We observed tremendous differences in natural variations of the metabolites and their underlying genetic architectures between distinct sub-groups of foxtail millet. Furthermore, we found that the selection of the gene alleles associated with yellow grains led to altered profiles of metabolites such as carotenoids and endogenous phytohormones. Using CRISPR-mediated genome editing we validated the function of PHYTOENE SYNTHASE 1 (PSY1) gene in affecting millet grain color and quality. Interestingly, our in vitro cell inflammation assays showed that 83 metabolites in millet grains have anti-inflammatory effects. Taken together, our multi-omics study illustrates how the breeding history of foxtail millet has shaped its metabolite profile. The datasets we generated in this study also provide important resources for further understanding how millet grain quality is affected by different metabolites, laying the foundations for future millet genetic research and metabolome-assisted improvement.