Expression Of Carotenogenic Genes Research Articles

Grape phytochrome-interacting factor VvPIF1 negatively regulates carotenoid biosynthesis by repressing VvPSY expression

Phytochrome-interacting factors (PIFs) play important roles in light-mediated secondary metabolism; however, the roles of PIFs in grape fruit carotenogenesis remain unclear. Here, by identifying the PIF family genes in grapes, we focused on the role of VvPIF1 in carotenoid metabolism. During grape berry development, VvPIF1 expression was negatively correlated with carotenoid accumulation and the transcription of phytoene synthase 1/2 (VvPSY1/2), which encodes the major flux-controlling enzymes for carotenoid biosynthesis. Light significantly repressed VvPIF1 expression, but induced the expression of carotenogenic genes including VvPSY1/2. VvPIF1 functioned as a nucleus-localized protein and interacted with the light photoreceptor VvphyB. Overexpression of VvPIF1 resulted in the downregulation of the endogenous PIF1 gene, which may unexpectedly induce carotenoid accumulation and PSY expression in tobacco leaves. The transgenic grape leaves and tomato fruits with high VvPIF1 expression produced a significant decrease in carotenoid concentrations, with suppressed transcription of PSY and other carotenogenic genes. Further biochemical assays demonstrated that VvPIF1 bound directly to the promoters of VvPSY1/2 to inhibit their transcription. Collectively, we conclude that VvPIF1 negatively regulates carotenoid biosynthesis by repressing VvPSY expression in grapes. These findings shed light on the role and mode of action of PIFs in the carotenoid regulatory network of grapes.

A carotenoid cleavage dioxygenase 4 from Paulownia tomentosa determines visual and aroma signals in flowers

Paulownia tomentosa is an economically important fast-growing tree, and its flowers and fruits are a rich source of biologically active secondary metabolites. In addition, the flowers of P. tomentosa are distinguished by a strong aroma and are also excellent nectariferous plants. The flowers are pale lilac and characterized by the presence of yellow nectar guides, whose color changes during the development of the flower, representing reliable signals to pollinators while enhancing reproductive success. The chemical analyses of the nectar guides revealed the presence of carotenoids as the pigments responsible for the observed coloration, with β-carotene levels determining the color changes observed after anthesis, with a reduction at anthesis and further increase and accumulation in post anthesis. To understand how β-carotene accumulation was controlled in the nectar guides, the expression of genes related to carotenoid biosynthesis and metabolism was analyzed. Carotenogenic gene expression was not associated with the observed changes in β-carotene during flower development. However, the expression of a gene encoding a carotenoid cleavage dioxygenase, CCD4–4, was co-related with the levels of β-carotene in the nectar guides. In addition, CCD4–4 cleavage β-carotene at C9-C10 and C9'-C10' positions, resulting in the generation of β-ionone, which was detected in flowers at anthesis. The obtained results indicated a developmental stage specific regulation of apocarotenoid formation through β-carotene cleavage, resulting in color changes and volatile production as key traits for plant-pollinator interactions. Data availabilityData will be made available on request.

Citrus β-carotene hydroxylase 2 (BCH2) participates in xanthophyll synthesis by catalyzing the hydroxylation of β-carotene and compensates for BCH1 in citrus carotenoid metabolism.

As an essential horticultural crop, Citrus has carotenoid diversity, which affects its aesthetic and nutritional values. β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits, and non-heme di-iron carotene hydroxylase (BCH) enzymes are mainly responsible for β,β-xanthophyll synthesis. Previous studies have focused on the hydroxylation of BCH1, but the role of its paralogous gene in citrus, BCH2, remains largely unknown. In this study, we revealed the β-hydroxylation activity of citrus BCH2 (CsBCH2) for the first time through the functional complementation assay using Escherichia coli, although CsBCH2 exhibited a lower activity in hydroxylating β-carotene into β-cryptoxanthin than citrus BCH1 (CsBCH1). Our results showed that overexpression of CsBCH2 in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression. This study revealed the distinct expression patterns and functional characteristics of two paralogous genes, CsBCH1 and CsBCH2, and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis. The independent function of CsBCH2 and its cooperative function with CsBCH1 in β-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering.

Red light-transmittance bagging promotes carotenoid accumulation of grapefruit during ripening

Light, a crucial environmental signal, is involved in the regulation of secondary metabolites. To understand the mechanism by which light influences carotenoid metabolism, grapefruits were bagged with four types of light-transmitting bags that altered the transmission of solar light. We show that light-transmitting bagging induced changes in carotenoid metabolism during fruit ripening. Compared with natural light, red light (RL)-transmittance treatment significantly increases the total carotenoid content by 62%. Based on weighted gene co-expression network analysis (WGCNA), ‘blue’ and ‘turquoise’ modules are remarkably associated with carotenoid metabolism under different light treatment (p < 0.05). Transcriptome analysis identifies transcription factors (TFs) bHLH128, NAC2-like/21/72, MYB-like, AGL11/AGL61, ERF023/062, WRKY20, SBPlike-7/13 as being involved in the regulation of carotenoid metabolism in response to RL. Under RL treatment, these TFs regulate the accumulation of carotenoids by directly modulating the expression of carotenogenic genes, including GGPPS2, PDS, Z-ISO, ZDS2/7, CRTISO3, CYP97A, CHYB, ZEP2, CCD1-2. Based on these results, a network of the regulation of carotenoid metabolism by light in citrus fruits is preliminarily proposed. These results show that RL treatments have great potential to improve coloration and nutritional quality of citrus fruits.

Development and carotenoid synthesis in dark-grown carrot taproots require PHYTOCHROME RAPIDLY REGULATED1.

Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange carrot (Daucus carota) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes, such as DcPSY1 and DcPSY2, reducing carotenoid accumulation. By means of RNA sequencing, in a previous analysis, we observed that carrot PHYTOCHROME RAPIDLY REGULATED1 (DcPAR1) is more highly expressed in the underground grown taproot compared with those grown in light. PAR1 is a transcriptional cofactor with a negative role in shade avoidance syndrome regulation in Arabidopsis (Arabidopsis thaliana) through the dimerization with PHYTOCHROME-INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here, we show that overexpressing AtPAR1 in carrot increases carotenoid production in taproots grown underground as well as DcPSY1 expression. The high expression of AtPAR1 and DcPAR1 led us to hypothesize a functional role of DcPAR1 that was verified through in vivo binding to AtPIF7 and overexpression in Arabidopsis, where AtPSY expression and carotenoid accumulation increased together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoid levels and in relative expression of key carotenogenic genes as well as impaired taproot development. These results suggest that DcPAR1 is a key factor for secondary root development and carotenoid synthesis in carrot taproot grown underground.

Analysis of the light regulatory mechanism in carotenoid production in Rhodosporidium toruloides NBRC 10032.

Light stimulates carotenoid production in an oleaginous yeast Rhodosporidium toruloides NBRC 10032 by promoting carotenoid biosynthesis genes. These genes undergo two-step transcriptional activation. The potential light regulator, Cryptochrome DASH (CRY1), has been suggested to contribute to this mechanism. In this study, based on KU70 (a component of nonhomologous end joining (NHEJ)) disrupting background, CRY1 disruptant was constructed to clarify CRY1 function. From analysis of CRY1 disruptant, it was suggested that CRY1 has the activation role of the carotenogenic gene expression. To obtain further insights into the light response, mutants varying carotenoid production were generated. Through analysis of mutants, the existence of the control two-step gene activation was proposed. In addition, our data analysis showed the strong possibility that R. toruloides NBRC 10032 is a homo-diploid strain.

Lycopene β-cyclase plays a critical role in carotenoid biosynthesis during persimmon fruit development and postharvest ripening

The aim of the current study was to characterize the functional role of DkLCYb in carotenoid biosynthesis in persimmon fruit. The accumulation of carotenoids and the expression of carotenogenic genes were investigated during persimmon fruit development and postharvest ripening. DkLCYb was cloned and its functional role in carotenoid biosynthesis was characterized. The results showed that carotenoids constantly accumulated during persimmon fruit development and postharvest ripening. More carotenoids accumulated during postharvest storage than during fruit development. The carotenoid composition of unsaponified extracts of the persimmon fruit flesh showed that the major carotenoids were lycopene and β-carotene in ‘Huoguan’ and ‘Heishi’, respectively. Consistent with this result, the expression of six carotenogenic genes increased to their highest levels during postharvest storage. The selected carotenoid biosynthesis genes showed an increased expression pattern during fruit development and in the early period of postharvest storage. A significant decrease in carotenogenic genes was found after stage 6 (DkPSY, DkLCYb, DkZDS and DkBCH) or stage 5 (DkLCYe,). Both DkLCYe and DkBCH showed a steady increase from stage 1 to stage 5; however, dramatically decreased expression of DkLCYe and sharply increased expression of DkBCH were found at stage 6. Correlation analysis showed that the expression of DkPSY, DkPDS, DkZDS, DkLCYb and DkBCH was significantly correlated with the accumulation of one or more carotenoid compounds analysed in this study. However, a negative correlation was found between the expression of DkLCYe and the accumulation of lycopene, β-cryptoxanthin and zeaxanthin. Specifically, expression of DkLCYb showed the highest correlation (p < 0.01) with β-carotene (r = 0.74) accumulation. We cloned DkLCYb and analysed its functions in vitro and in vivo. DkLCYb contained typical domains that were highly conserved and essential for lcyb protein activity. The cyclization activity of the encoded protein was demonstrated by functional analysis in E. coli BL21. Transient overexpression of DkLCYb significantly enhanced the accumulation of β-carotene in persimmon fruit. These results indicate that DkLCYb is a genuine lycopene cyclase gene and it plays an essential role in β, β-carotenoid (β-carotene and β-cryptoxanthin) biosynthesis in persimmon fruit.

A fruit ripening-associated transcription factor CsMADS5 positively regulates carotenoid biosynthesis in citrus

Carotenoids in citrus contribute to the quality of the fruit, but the mechanism of its transcriptional regulation is fairly unknown. Here, we characterized a citrus FRUITFULL sub-clade MADS gene, CsMADS5, that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid concentrations. The expression of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β-cyclase 1 (LCYb1) was increased in the peels of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY, PDS, and LCYb1 by directly binding to their promoters. We concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy of carotenoid biosynthesis during fruit ripening.

A Citrus Phosphate Starvation Response Factor CsPHL3 Negatively Regulates Carotenoid Metabolism.

Carotenoids provide precursors for the biosynthesis of strigolactones, which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway, but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was upregulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene β-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents and elevated expression of LCYb genes but downregulation of other key carotenogenic genes, including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis.

Metabolic engineering of Saccharomyces cerevisiae for production of β-carotene from hydrophobic substrates.

ABSTRACTβ-Carotene is a yellow–orange–red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for β-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding β-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest β-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Engineering Saccharomyces cerevisiae for the Overproduction of β-Ionone and Its Precursor β-Carotene.

β-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment β-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current β-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of β-carotene and its subsequent conversion to β-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on β-carotene accumulation, and later, on β-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For β-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest β-carotene-producing strains, reaching up to 33 mg/L of β-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing β-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

Functional Validation of Phytoene Synthase and Lycopene ε-Cyclase Genes for High Lycopene Content in Autumn Olive Fruit (Elaeagnus umbellata).

Lycopene is the most potent antioxidant among all carotenoids and is beneficial to human health. A ripe fruit of autumn olive (Elaeagnus umbellata Thunb.) accumulates a high level of lycopene, which is 5-20 times higher than that in an ordinary tomato fruit. During fruit ripening of autumn olive, only phytoene synthase (EutPSY) expression pattern shows a tight positive correlation with the increased lycopene content observed at four ripening stages, while the lycopene ε-cyclase (EutLCYe) transcript could not be detected throughout fruit ripening. Here, we investigated whether the two genes are important targets for engineering lycopene biosynthesis. The full-length cDNAs of EutPSY and EutLCYe were first isolated. Fruit-specific overexpression of EutPSY in tomato fruits resulted in elevated contents of lycopene and β-carotene through feedforward regulation of carotenogenic genes, i.e., downregulation of SlLCYe and upregulation of SlLCYb and SlCYCB. These fruits were decreased in ethylene production throughout ripening. Transcript levels of genes for system-2 ethylene synthesis (SlACS2, SlACS4, SlACO1, and SlACO3), perception (SlNR/ETR3 and SlETR4), and response (SlE4 and SlE8) were also inhibited in EutPSY-overexpressing fruits. Repressing ethylene synthesis and signaling transduction delayed fruit climacteric ripening of transgenic tomato plants. Additionally, RNAi suppression of SlLCYe enhanced β-carotene but not lycopene accumulation through altered expression of carotenogenic genes in transgenic tomato fruits by both feedforward and feedback regulatory mechanisms. Ethylene production in SlLCYe-RNAi fruits decreased, thereby delaying fruit ripening. Collectively, these results confirmed that transcriptional regulation of EutPSY and EutLCYe plays a crucial role and a part in massive lycopene accumulation in autumn olive fruits, respectively. EutPSY overexpression enhanced lycopene accumulation in tomato fruits independently of the ethylene pathway but did not influence the size and weight of tomato fruits. EutPSY can be used as an effective strategy capable of elevating the lycopene content in fruits for improving quality.

The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds

BackgroundDeoxyxylulose 5-phosphate synthase (DXS) and deoxyxylulose 5-phosphate reductoisomerase (DXR) are the enzymes that catalyze the first two enzyme steps of the methylerythritol 4-phosphate (MEP) pathway to supply the isoprene building-blocks of carotenoids. Plant DXR and DXS enzymes have been reported to function differently depending on the plant species. In this study, the differential roles of rice DXS and DXR genes in carotenoid metabolism were investigated.ResultsThe accumulation of carotenoids in rice seeds co-expressing OsDXS2 and stPAC was largely enhanced by 3.4-fold relative to the stPAC seeds and 315.3-fold relative to non-transgenic (NT) seeds, while the overexpression of each OsDXS2 or OsDXR caused no positive effect on the accumulation of either carotenoids or chlorophylls in leaves and seeds, suggesting that OsDXS2 functions as a rate-limiting enzyme supplying IPP/DMAPPs to seed carotenoid metabolism, but OsDXR doesn’t in either leaves or seeds. The expressions of OsDXS1, OsPSY1, OsPSY2, and OsBCH2 genes were upregulated regardless of the reductions of chlorophylls and carotenoids in leaves; however, there was no significant change in the expression of most carotenogenic genes, even though there was a 315.3-fold increase in the amount of carotenoid in rice seeds. These non-proportional expression patterns in leaves and seeds suggest that those metabolic changes of carotenoids were associated with overexpression of the OsDXS2, OsDXR and stPAC transgenes, and the capacities of the intermediate biosynthetic enzymes might be much more important for those metabolic alterations than the transcript levels of intermediate biosynthetic genes are. Taken together, we propose a ‘Three Faucets and Cisterns Model’ about the relationship among the rate-limiting enzymes OsDXSs, OsPSYs, and OsBCHs as a “Faucet”, the biosynthetic capacity of intermediate metabolites as a “Cistern”, and the carotenoid accumulations as the content of “Cistern”.ConclusionOur study suggests that OsDXS2 plays an important role as a rate-limiting enzyme supplying IPP/DMAPPs to the seed-carotenoid accumulation, and rice seed carotenoid metabolism could be largely enhanced without any significant transcriptional alteration of carotenogenic genes. Finally, the “Three Faucets and Cisterns model” presents the extenuating circumstance to elucidate rice seed carotenoid metabolism.

Characterization of Carotenoid Accumulation and Carotenogenic Gene Expression During Fruit Ripening in Red Colored Pulp of ‘Siam Red Ruby’ Pumelo (&lt;i&gt;Citrus grandis&lt;/i&gt;) Cultivated in Thailand

The ‘Siam Red Ruby’ (Citrus grandis) is a new lycopene-accumulating pumelo variety, which is grown only in the Pakpanang area of the Nakhon Si Thammarat province of Thailand. In this study, we harvested ‘Siam Red Ruby’ fruit at five ripening stages, and the changes in the accumulation of carotenoid and carotenogenic gene expression were investigated in the pulp. During the ripening process, the contents of phytoene, ζ-Carotene, lycopene, β-carotene, β-cryptoxanthin, and all-trans-violaxanthin increased gradually, while the contents of α-carotene and lutein decreased in the pulp of ‘Siam Red Ruby’. In the mature fruit of ‘Siam Red Ruby’, a high amount of lycopene was accumulated in the pulp, accounting for more than 93.26% of the total carotenoid content. The massive accumulation of lycopene led to an attractive red color of the ‘Siam Red Ruby’ pulp. In addition, gene expression results showed that the increases in the expression of upstream genes (CitPSY, CitPDS, CitZDS, CitZISO, and CitCRTISO), and decreases in the expression of genes downstream (CitLCYb1, CitLCYb2, CitLCYe, CitHYb, and CitHYe) was the main mechanism of lycopene accumulation in the pulp of ‘Siam Red Ruby’ pumelo.

Carotenoid composition and expression of carotenogenic genes in the peel and pulp of commercial mango fruit cultivars

Fruits of two mango cultivars (‘Tainong’ and ‘Jinhuang’) with commercial maturity were analyzed for their firmness, TSS (Total soluble solids), color index and soluble sugar content. Compared to ‘Jinhuang’, ‘Tainong’ was texture soften and had higher soluble sugar content and the color judged visually the maturity. To study the regulation mechanism of carotenoid metabolism, carotenoid component and the expression levels of 12 carotenogenic genes in peel and pulp of two mango fruit cultivars with commercial maturity were analsyed. Total carotenoid content in the peel and pulp of ‘Tainong’ was 116.78 ug/g FW and 77.48 ug/g FW, which were 1.39 times and 1.63 times in the peel and pulp of ‘Jinhuang’, respectively. Among the carotenoid components, the main carotenoids in the peel and pulp were α-carotene and β-Carotene, and a small number of carotenoids were lycopene, β-cryptoxanthin, zeaxanthin and lutein. qRT-PCR analysis showed that the accumulation of carotenoids in the peel and pulp of different mango cultivars was controlled by the differential expression of carotenogenic genes. Our results provide new clues for the molecular mechanism of carotenoids metabolism in postharvest ripening of different mango cultivars.

Effect of NaCl stress and supplemental CaCl2 on carotenoid accumulation in germinated yellow maize kernels

Germinated edible seeds and sprouts are becoming increasingly common in the human diet because they are rich in bioactive compounds and antioxidants and are highly nutritious. In this study, the effects of NaCl stress and supplemental CaCl2 on carotenoid accumulation, antioxidant capacity and expression of key enzymes in yellow maize kernels were investigated. The results showed that the lutein and zeaxanthin contents increased with NaCl treatment, and further increased with supplemental CaCl2. Additionally, germinated yellow maize kernels showed increased antioxidant capacity in response to NaCl and CaCl2. The transcript levels of carotenogenic genes ZmPSY and ZmCYP97C were upregulated and the expression levels of ZmLCYB and ZmBCH1 were downregulated under NaCl stress. The expression of all key carotenogenic genes was upregulated by CaCl2 supplementation. These results suggested that NaCl and CaCl2 contribute to carotenoid accumulation via increased expression of related carotenogenic genes and increased antioxidant capacity in germinated yellow maize kernels.

A new approach to promote astaxanthin accumulation via Na2WO4 in Haematococcus pluvialis

The optimum concentration of Na2WO4 was explored in relation to the cell density and astaxanthin content in Haematococcus pluvialis. Then, the cellular morphology, nitrate reductase (NR) activity, soluble sugar and protein contents, and chlorophyll fluorescence were measured, and the transcriptional expression of carotenogenic genes was determined by quantitative real-time PCR. The results showed that 3.0 mmol/L of Na2WO4 was the optimum concentration to induce astaxanthin accumulation, with a maximum content of 49.41±0.13 pg/cell reached on the tenth day. The NR activity decreased significantly and continually after Na2WO4 treatment. The soluble sugar content increased gradually during the experimental period and was eventually significantly higher than that in the control. The soluble protein content increased rapidly, reached a maximum in day 0.5 and day 1 and then decreased. The effective photochemical efficiency of PSII ( Fv'/Fm') and light saturation ( Ek) first decreased and then tended to stabilize, and NADP+-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was correlated with photosynthesis. The transcriptional expression of ipi, psy and bkt was significantly increased compared with that in the control after application of Na2WO4, and the relative expression of ipi reached the highest level on the fifth day, with a 98.03±1.92-fold increase. Our results describe a new approach to promote the effective accumulation of astaxanthin in H. pluvialis by NR inhibitor Na2WO4.

Production of astaxanthin at moderate temperature in Xanthophyllomyces dendrorhous using a two-step process.

In order to achieve continuous industrial production of astaxanthin in Xanthophyllomyces dendrorhous, a moderate temperature (25-37°C) fermentation process was needed. In this study, a two-step process with a 20°C pre-culture for 18 h and a 30°C culture for 30 h was performed to achieve the astaxanthin yields of 116.42μgg-1 dry cell weight, which was lower than that in the normal process (20°C, 96h). However, cell yield (YX/S) and product yield (YP/S) showed no significant differences between the two processes, suggesting that moderate temperature did not affect the productivity of astaxanthin. The transcriptional levels of genes involved in astaxanthin synthesis were compared in different culture times and a negative correlation between temperature and expression of carotenogenic genes was found. This work provided a potential method for continuous production of astaxanthin using X. dendrorhous at moderate temperature throughout the year.

Transcript profiling of carotenoid/apocarotenoid biosynthesis genes during corm development of saffron (Crocus sativus L.).

The dried stigmas of saffron constitute the world's costliest spice. Saffron has many therapeutic applications due to the presence of apocarotenoids. The latter are synthesized at different stages of development, and the biosynthetic pathway involves several genes encoding different enzymes. In order to understand the differential expression of various genes of the pathway, eight distinct developmental stages (S1-early to S8-late) were identified. The corms were assorted into three groups (I, II, and III) based on corm weight. Expression profiles of 12 carotenoid/apocarotenoid genes were studied. The expression of all genes was minimum/least in groups I and II corms during bud development. Lowest expression of carotenogenic genes (CsPSY, CsPDS, CsZDS, CsCRTISO, CsLYC-β1, CsLYC-ε, CsBCH2, and CsNCED) was observed during early stages (S1-S3) of corm growth (dormant period). In group III corms, increased expression of apocarotenoid genes (CsZCO, CsCCD2, CsUGT, and CsALDH) was observed during S4 to S8 stages (reproductive period, floral differentiation). Besides, expression profiles of genes in apical and axillary buds were also examined. Of all the genes studied, apocarotenoid biosynthesis genes (CsBCH2, CsZCO, CsCCD2, CsALDH, and CsUGT) were found to be upregulated in apical bud than in the axillary bud. The results indicated that interaction of phytohormones and sugars, mother corm reserves and the influence of internal and external factors may be contributing to the growth of saffron corm/bud. The study has laid a foundation for further research on the molecular mechanisms underlying bud dormancy/growth in saffron.

The Citrus Transcription Factor CsMADS6 Modulates Carotenoid Metabolism by Directly Regulating Carotenogenic Genes.

Although remarkable progress has been made toward understanding carotenoid biosynthesis, the mechanisms that regulate the transcription of carotenogenic genes remain poorly understood. Lycopene β-cyclases (LCYb) are critical enzymes located at the branch point of the carotenoid biosynthetic pathway. Here, we used the promoter sequence of LCYb1 as bait in a yeast one-hybrid screen for promoter-binding proteins from sweet orange (Citrus sinensis). This screen identified a MADS transcription factor, CsMADS6, that was coordinately expressed with fruit development and coloration. Acting as a nucleus-localized transcriptional activator, CsMADS6 directly bound the promoter of LCYb1 and activated its expression. Overexpression of CsMADS6 in citrus calli increased carotenoid contents and induced the expression of LCYb1 and other carotenogenic genes, including phytoene synthase (PSY), phytoene desaturase (PDS), and carotenoid cleavage dioxygenase1 (CCD1). CsMADS6 up-regulated the expression of PSY, PDS, and CCD1 by directly binding to their promoters, which suggested the multitargeted regulation of carotenoid metabolism by CsMADS6. In addition, the ectopic expression of CsMADS6 in tomato (Solanum lycopersicum) affected carotenoid contents and the expression of carotenogenic genes. The sepals of CsMADS6-overexpressing tomato lines exhibited dramatic changes in carotenoid profiles, accompanied by changes in plastid ultrastructure. Global transcriptome analysis of transgenic sepals revealed that CsMADS6 regulates a series of pathways that promote increases in flux through the carotenoid pathway. Overall, these findings establish that CsMADS6 directly regulates LCYb1 and other carotenogenic genes to coordinately and positively modulate carotenoid metabolism in plants, which may provide strategies to improve the nutritional quality of crops.