Encoding Phytoene Synthase Research Articles

The transcription factor ClWRKY61 interacts with ClLEA55 to enhance salt tolerance in watermelon

Abstract High salinity can severely inhibit the growth and development of watermelon (Citrullus lanatus L.). WRKY proteins are believed to mediate the adaptation of plants to abiotic stresses. Here, we identified the ClWRKY61 gene, which positively regulates the tolerance of watermelon to salt stress. Knockout of the ClWRKY61 reduced salt tolerance, while overexpression of the ClWRKY61 enhanced salt tolerance in watermelon according to phenotypic and physiological analyses. Yeast two-hybrid assays revealed that ClWRKY61 interacts with the ClLEA55 protein, and this interaction was further confirmed by luciferase complementation imaging, transient bimolecular fluorescence complementation, and GST pull-down assays. Knockout of the ClLEA55 resulted in lower salt tolerance compared to the wild-type plants. RNA-seq analysis indicated 421 up-regulated and 133 down-regulated genes in the ClWRKY61 knockout line under salt stress, containing 293 differentially expressed genes with W-box in their promoters. After salt treatment of watermelon seedlings, qRT-PCR assays evidenced that the expression of genes encoding phytoene synthase, MYB transcription factor, sucrose synthase, alpha/beta-hydrolases superfamily protein, glutathione reductase, and sugar transporter were significantly increased; while the expression of genes encoding LEA protein, WRKY transcription factor, ERF transcription factor, alpha-glucan water dikinase, and calcium-dependent protein kinase were significantly decreased in ClWRKY61 knockout lines. These results provide an opportunity to mediate the regulation of salt stress in watermelon with WRKY proteins.

Abscisic acid activates transcription factor module MdABI5-MdMYBS1 during carotenoid-derived apple fruit coloration.

Carotenoids are major pigments contributing to fruit coloration. We previously reported that the apple (Malus domestica Borkh.) mutant fruits of "Beni Shogun" and "Yanfu 3" show a marked difference in fruit coloration. However, the regulatory mechanism underlying this phenomenon remains unclear. In this study, we determined that carotenoid is the main factor influencing fruit flesh color. We identified an R1-type MYB transcription factor (TF), MdMYBS1, which was found to be highly associated with carotenoids and abscisic acid (ABA) contents of apple fruits. Overexpression of MdMYBS1 promoted, and silencing of MdMYBS1 repressed, β-branch carotenoids synthesis and ABA accumulation. MdMYBS1 regulates carotenoid biosynthesis by directly activating the major carotenoid biosynthetic genes encoding phytoene synthase (MdPSY2-1) and lycopene β-cyclase (MdLCYb). 9-cis-epoxycarotenoid dioxygenase 1 (MdNCED1) contributes to ABA biosynthesis, and MdMYBS1 enhances endogenous ABA accumulation by activating the MdNCED1 promoter. In addition, the basic leucine zipper domain TF ABSCISIC ACID-INSENSITIVE5 (MdABI5) was identified as an upstream activator of MdMYBS1, which promotes carotenoid and ABA accumulation. Furthermore, ABA promotes carotenoid biosynthesis and enhances MdMYBS1 and MdABI5 promoter activities. Our findings demonstrate that the MdABI5-MdMYBS1 cascade activated by ABA regulates carotenoid-derived fruit coloration and ABA accumulation in apple, providing avenues in breeding and planting for improvement of fruit coloration and quality.

Identification of carotenoids and candidate genes shaping high pigment chili pepper variety

Pepper (Capsicum spp.) is a major vegetable grown and consumed worldwide. Fruit color is an important exterior quality of fresh and processed peppers. The typical red color of pepper fruit is mainly determined by the content of capsanthin and capsorubin. The biosynthetic pathway of capsanthin and capsorubin has been largely clarified, but the regulators of this pathway remain to be investigated. To decipher the regulatory mechanism of high pigment accumulation in mature red pepper (C. annuum) fruit, carotenoid and transcriptome profiling were performed on two red chili peppers with distinct levels of capsanthin and capsorubin. The total carotenoids of high-pigment genotype (GB23) increased by 168 times during fruit ripening, especially carotenoid compounds such as zeaxanthin, β-cryptoxanthin and capsanthin. Moreover, the difference in the transcript level of genes encoding phytoene synthase 1 (PSY1), β-carotene hydroxylase 1 (BCH1), and capsanthin-capsorubin synthase (CCS) was consistent with the difference in carotenoid accumulation between GB23 and GB42 (low-pigment genotype). Further investigation revealed several transcription factors exhibiting similar expression patterns and correlated with capsanthin accumulation. Utilizing weighted gene co-expression network analysis, we identified eight transcription factors, namely bHLH13, Zinc finger protein ZFP3, MADS-RIN, NAC68, PIF3, and WRKY6, as potential regulators of PSY1, BCH1, or CCS genes. Yeast one-hybrid and luciferase reporter assays demonstrated that ZFP3 activates PSY1 and CCS while WRKY6 activates CCS through promoter binding. This comprehensive study sheds light on the regulatory mechanism underlying capsanthin accumulation in chili pepper, offering new insights into this important trait.

Development of a Gene-Based Marker Set for Orange-Colored Watermelon Flesh with a High β-Carotene Content.

The fruit flesh of watermelons differs depending on the distinct carotenoid composition. Orange-colored flesh relates to the accumulation of β-carotene, which is beneficial to human health. Canary-yellow-fleshed OTO-DAH and orange-β-fleshed (orange-fleshed with high β-carotene) NB-DAH near-isogenic lines (NILs) were used to determine the genetic mechanism attributed to orange watermelon flesh. For genetic mapping, an F2 population was developed by crossing the two NILs. The segregation ratio of flesh color in the F2 population indicated that the orange-β flesh of the NB-DAH NIL was controlled by a single incompletely dominant gene. Through a comparative analysis of the whole-genome sequences of the parent lines and NILs, a major introgression region unique to the NB-DAH NIL was detected on Chr. 1; this was considered a candidate region for harboring genes that distinguish orange from canary-yellow and red flesh. Among the 13 genes involved in the carotenoid metabolic pathway in watermelons, only ClPSY1 (ClCG01G008470), which encodes phytoene synthase 1, was located within the introgression region. The genotyping of F2 plants using a cleaved amplified polymorphic sequence marker developed from a non-synonymous SNP in ClPSY1 revealed its relationship with orange-β flesh. The insights gained in this study can be applied to marker-assisted breeding for this desirable trait.

Chromoplast differentiation: a central role for plastoglobule lipid droplets comes into focus.

This article is a Commentary on Morelli et al. (2023), 237: 1696–1710.

Jasmonate activates a CsMPK6-CsMYC2 module that regulates the expression of β-citraurin biosynthetic genes and fruit coloration in orange (Citrus sinensis).

Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid β-citraurin is responsible for the peel color in "Newhall" orange (Citrus sinensis). Although jasmonates are known to regulate the biosynthesis and accumulation of carotenoids, their effects on β-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and β-citraurin production in "Newhall" orange. A MeJA treatment induced the expression of CsMYC2, which encodes a transcription factor that serves as a master regulator of jasmonate responses. CsMYC2 bound the promoter of the gene that encodes carotenoid cleavage dioxygenase 4b (CsCCD4b), the key gene for β-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene β-cyclase (CsLCYb), and β-carotene hydroxylase (CsBCH) and induced their expression. In addition, CsMYC2 promoted CsMPK6 expression. Notably, we found that CsMPK6 interacted with CsMYC2 and that this interaction decreased the stability and DNA-binding activity of CsMYC2. Thus, we conclude that negative feedback regulation attenuates JA signaling during the jasmonate-induced coloration of citrus fruit. Together, our findings indicate that jasmonates induce β-citraurin biosynthesis in citrus by activating a CsMPK6-CsMYC2 cascade, thereby affecting fruit coloration.

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.

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.

Nucleotide variation in the phytoene synthase (ClPsy1) gene contributes to golden flesh in watermelon (Citrullus lanatus L.).

A gene controlling golden flesh trait in watermelon was discovered and fine mapped to a 39.08 Kb region on chromosome 1 through a forward genetic strategy, and Cla97C01G008760 (annotated as phytoene synthase protein, ClPsy1 ) was recognized as the most likely candidate gene. Vitamin A deficiency is a worldwide public nutrition problem, and β-carotene is the precursor for vitamin A synthesis. Watermelon with golden flesh (gf, which occurs due to an accumulated abundance of β-carotene) is an important germplasm resource. In this study, a genetic analysis of segregated gf gene populations indicated that gf was controlled by a single recessive gene. BSA-seq (Bulked segregation analysis) and an initial linkage analysis placed the gf locus in a 290-Kb region on watermelon chromosome 1. Further fine mapping in a large population including over 1000 F2 plants narrowed this region to 39.08Kb harboring two genes, Cla97C01G008760 and Cla97C01G008770, which encode phytoene synthase (ClPsy1) and GATA zinc finger domain-containing protein, respectively. Gene sequence alignment and expression analysis between parental lines revealed Cla97C01G008760 as the best possible candidate gene for the gf trait. Nonsynonymous SNP mutations in the first exon of ClPsy1 between parental lines co-segregated with the gf trait only among individuals in the genetic population and were not related to flesh color in natural watermelon panels. Promoter sequence analysis of 26 watermelon accessions revealed two SNPs in the cis-acting element sequences corresponding to MYB and MYC2 transcription factors. RNA-seq data and qRT-PCR verification showed that two MYBs exhibited expression trends similar to that of ClPsy1 in the parental lines and may regulate the ClPsy1 expression. Further research findings indicate that the gf trait is determined not only by ClPsy1 but also by ClLCYB, ClCRTISO and ClNCED7, which play important roles in watermelon β-carotene accumulation.

Metabolic engineering of Deinococcus radiodurans for pinene production from glycerol

BackgroundThe objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries.ResultsTo enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli.ConclusionsIn this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules.

Variability and Phylogeny of the Pepper Phytoene Synthase Paralogs PSY1 and PSY2 in Species of Various Capsicum Complexes.

Genes homologous to PSY1 and PSY2 that encode phytoene synthase isoforms in Capsicum species C. baccatum, C. chinense, C. frutescens, C. tovarii, C. eximium, and C. chacoense were identified. High conservatism of functionally significant sites of phytoene synthases of the analyzed accessions was revealed. It was found that only PSY1-based clustering of pepper species corresponds to the traditional Capsicum phylogeny; C. eximium was a part of the Purple corolla complex, and C. chacoense was equidistant from Annuum and Baccatum clades. The absence of significant differences between PSY1 and PSY2 of yellow-fruited C. chinense and red-fruited pepper accessions was shown. The yellow color of C. chinense fruit may be the result of both decreased PSY1 expression and increased PSY2 transcription. Thus, it was demonstrated that the acquired fruit pigmentation retains strict phylogenetic limitations, which, however, can be overcome using artificial selection for the activity of phytoene synthase PSY1.

Improving Fucoxanthin Production in Mixotrophic Culture of Marine Diatom Phaeodactylum tricornutum by LED Light Shift and Nitrogen Supplementation.

Fucoxanthin (Fx), a kind of primary carotenoids in brown seaweeds and diatoms, has attractive efficacy in human’s healthcare including loss weight, the prevention of diabetes and Alzheimer’s disease. Marine diatom Phaeodactylum tricornutum is now realized as a promising producer for commercial Fx production due to its higher content of Fx than brown seaweeds with easily artificial cultivation and Fx extraction. In the present study, to improve Fx production in P. tricornutum, the mixotrophic cultures were applied to optimize initial cell density, light intensity, light regime and nitrogen supplementation. The results showed that the higher initial cell density (1 × 107 cells mL–1) and lower light intensity (20 μmol m–2 s–1) were favorable for biomass production and Fx accumulation. The maximal Fx content [16.28 mg g–1 dry weight (DW)] could be achieved under blue light (BL), but the highest biomass concentration (5.53 g L–1) could be attained under red: blue light (R: B, 6:1) in the batch culture. A novel two-phase culture approach was developed to increase the biomass concentration to the highest value (6.52 g L–1) with the maximal productivity of Fx (8.22 mg L–1 d–1) through light shift from R:B ratio (6:1) in phase 1 to R:B ratio (5:1) by enhancing BL and tryptone addition in phase 2. The content and intracellular amount of Fx were also increased 8% and 12% in phase 2 compared to phase 1. The expression levels analysis revealed that genes encoding phytoene synthase (PSY), zeaxanthin epoxidase (ZEP), and fucoxanthin-chlorophyll-protein b (FCPb) were upregulated significantly, with downregulation of the gene encoding violaxanthin de-epoxidase (VDE), leading to the improvement of Fx in phase 2. The present study demonstrated the two-phase culture strategy could promote Fx productivity through enhancing biomass production and increasing Fx content, indicating that strengthening BL coupled with adding tryptone were effective to facilitate Fx production by mixotrophic cultivation of marine diatom P. tricornutum.

Candidate Gene Analysis Reveals That the Fruit Color Locus C1 Corresponds to PRR2 in Pepper (Capsicum frutescens).

The diverse fruit colors of peppers (Capsicum spp.) are due to variations in carotenoid composition and content. Mature fruit color in peppers is regulated by three independent loci, C1, C2, and Y. C2 and Y encode phytoene synthase (PSY1) and capsanthin-capsorubin synthase (CCS), respectively; however, the identity of the C1 gene has been unknown. With the aim of identifying C1, we analyzed two pepper accessions with different fruit colors: Capsicum frutescens AC08-045 and AC08-201, whose fruits are light yellow and white, respectively. Ultra-performance liquid chromatography showed that the total carotenoid content was six times higher in AC08-045 than in AC08-201 fruits, with similar composition of main carotenoids and slight difference in minor components. These results suggest that a genetic factor in AC08-201 may down-regulate overall carotenoid biosynthesis. Analyses of candidate genes related to carotenoid biosynthesis and plastid abundance revealed that both accessions carry non-functional alleles of CCS, golden2-like transcription factor (GLK2), and PSY1. However, a nonsense mutation (C2571T) in PRR2, a homolog of Arabidopsis pseudo response regulator2-like (APRR2), was present in only AC08-201. In a population derived from a cross between AC08-045 and AC08-201, a SNP marker based on the nonsense mutation co-segregated fully with fruit color, implying that the mutation in PRR2 may cause the white color of AC08-201 fruits. Transmission electron microscopy (TEM) of AC08-201 fruit pericarp also showed less developed granum structure in chloroplast and smaller plastoglobule in chromoplast compared to those of AC08-045. Virus-induced gene silencing (VIGS) of PRR2 significantly reduced carotenoid accumulation in Capsicum annuum ‘Micropep Yellow’, which carries non-functional mutations in both PSY1 and CCS. Furthermore, sequence analysis of PSY1, CCS, and PRR2 in other white pepper accessions of C. annuum and Capsicum chinense showed that they commonly have non-functional alleles in PSY1, CCS, and PRR2. Thus, our data demonstrate that the fruit color locus C1 in Capsicum spp. corresponds to the gene PRR2.

Comparative transcriptome profiling of different tissues from beta-carotene-enhanced transgenic soybean and its non-transgenic counterpart

Beta-carotene-enhanced transgenic soybeans, harboring genes encoding phytoene synthase and carotene desaturase under the control of a seed-specific promoter, were developed to alleviate vitamin A deficiency in populations, the diet of which was deficient in this vitamin. However, metabolic engineering of carotenoid biosynthetic pathways often has unintended effects, leading to major metabolic changes in plants that harbor endogenous beta-carotene biosynthesis pathways. In the present study, we performed transcriptome profiling analysis using RNA-seq to investigate the changes in the transcriptome and some unintended pleiotropic effects on the leaves, stems, roots, and seeds of beta-carotene-enhanced transgenic soybean lines, and compared them to those of their non-transgenic counterpart donor variety Kwangan. We observed that transgenic soybeans showed significant changes in secondary metabolic biosynthesis in leaves and down-regulated galactose metabolism in roots. Differentially expressed genes in the transgenic group, which were significantly up-regulated, included those encoding glycine-aspartic acid-serine-leucine-motif esterase/lipase, known as cutin synthase and cutinase. These results suggested enhanced beta-carotene biosynthesis may affect related enzymes to carbohydrate metabolism and fatty acid metabolism. Hence, we speculated that upregulation of cutin polymerization resulted in thickened seed coat and delayed seed germination of transgenic soybeans. Furthermore, downregulation of raffinose family oligosaccharide biosynthesis may cause redundancy of myo-inositol, a substrate of phytin formation. This could lead to phytic globoids accumulation in transgenic soybean seeds. The present imformation would be important for transgenic plant development via carotenoid metabolic engineering, with focus on beta-carotene over-production.

Metabolic Engineering of Deinococcus radiodurans for the Production of Phytoene.

A metabolically-engineered Deinococcus radiodurans R1 strain capable of producing phytoene, a colorless C40 carotenoid and a promising antioxidant, has been developed. To make this base strain, first, the crtI gene encoding phytoene desaturase was deleted to block the conversion of phytoene to other carotenoids such as lycopene and γ-carotene. This engineered strain produced 0.413 ± 0.023 mg/l of phytoene from 10 g/l of fructose. Further enhanced production of phytoene up to 4.46 ± 0.19 mg/l was achieved by overexpressing the crtB gene encoding phytoene synthase and the dxs genes encoding 1-deoxy-D-xylulose-5-phosphate synthase gene, and by deleting the crtD gene. High cell-density culture of our final engineered strain allowed production of 10.3 ± 0.85 mg/l of phytoene with the yield and productivity of 1.04 ± 0.05 mg/g and 0.143 ± 0.012 mg/l/h, respectively, from 10 g/l of fructose. Furthermore, the antioxidant potential of phytoene produced by the final engineered strain was confirmed by in vitro DPPH radical-scavenging assay.

The responses of two genes encoding phytoene synthase (Psy) and phytoene desaturase (Pds) to nitrogen limitation and salinity up-shock with special emphasis on carotenogenesis in Dunaliella parva

Green alga Dunaliella parva can adapt to environmental stresses such as salinity up-shock and nitrogen limitation which are associated with carotenoid accumulation. However, the underlying molecular basis related to the change of carotenoid content needs further study. In the present study, we cloned two carotenoid biosynthesis genes coding for phytoene synthase and phytoene desaturase. In addition, the effects of salinity up-shock and nitrogen limitation on carotenoid content and expression of these two genes were studied in D. parva FACHB-815. The results showed that both salinity up-shock and nitrogen limitation had important effect on carotenoid yield and expression of these two genes. Cloning and expression analysis of these two genes will contribute to the understanding of molecular basis in stress-induced carotenoid accumulation, which is favorable for performing metabolic and genetic engineering to improve traits of D. parva.

Loss of Carotenoids Impacts Membrane Protein and Lipid Distribution in Pantoea sp. YR343

Bacterial cell membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties to the membrane and allows it to respond to environmental conditions. Pantoea sp. YR343 is a plant-associated Gram-negative proteobacteria that produces carotenoids, including zeaxanthin. We recently reported that Pantoea sp. YR343 mutants lacking the crtB gene encoding phytoene synthase, which catalyzes the first step in carotenoid biosynthesis, failed to produce carotenoids and displayed enhanced sensitivity to reactive oxygen species. The crtB mutant also displayed unexpected defects in biofilm formation and root colonization compared to wildtype cells. Studies using in silico molecular simulations suggest that carotenoids can act as modulators of membrane biophysical properties, leading to the possibility that these phenotypes may result from defects in membrane organization. We hypothesize that carotenoids modulate membrane dynamics by influencing the composition and organization of membrane proteins and lipids. Fluorescence anisotropy measurements on vesicles generated from lipids extracted from either wildtype or crtB mutant cells revealed that the wildtype membrane vesicles were significantly more rigid than mutant membrane vesicles. In contrast, anisotropy measurements on whole cells indicated that wildtype cells were less rigid than crtB mutant cells. These observations were confirmed by atomic force microscopy. Mass spectrometric based analysis of membrane proteins and lipids revealed differences in membrane protein and lipid profiles between the mutant and wildtype cells. Among the different classes of proteins, signaling and transport protein classes were the most effected in the mutant. Similarly, unsaturated and cyclopropane fatty acids classes differed in both cell types. These data suggest that loss of carotenoids in cell membranes changes the abundance and distribution of membrane proteins and lipids which may be the result of, or help compensate for, changes in membrane biophysical properties.

Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota).

Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization.

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343.

Regulation of Carotenoid Biosynthesis by Shade Relies on Specific Subsets of Antagonistic Transcription Factors and Cofactors.

Carotenoids are photosynthetic pigments essential for the protection against excess light. During deetiolation, their production is regulated by a dynamic repression-activation module formed by PHYTOCHROME-INTERACTING FACTOR1 (PIF1) and LONG HYPOCOTYL5 (HY5). These transcription factors directly and oppositely control the expression of the gene encoding PHYTOENE SYNTHASE (PSY), the first and main rate-determining enzyme of the carotenoid pathway. Antagonistic modules also regulate the responses of deetiolated plants to vegetation proximity and shade (i.e. to the perception of far-red light-enriched light filtered through or reflected from neighboring plants). These responses, aimed to adapt to eventual shading from plant competitors, include a reduced accumulation of carotenoids. Here, we show that PIF1 and related photolabile PIFs (but not photostable PIF7) promote the shade-triggered decrease in carotenoid accumulation. While HY5 does not appear to be required for this process, other known PIF antagonists were found to modulate the expression of the Arabidopsis (Arabidopsis thaliana) PSY gene and the biosynthesis of carotenoids early after exposure to shade. In particular, PHYTOCHROME-RAPIDLY REGULATED1, a transcriptional cofactor that prevents the binding of true transcription factors to their target promoters, was found to interact with PIF1 and hence directly induce PSY expression. By contrast, a change in the levels of the transcriptional cofactor LONG HYPOCOTYL IN FAR RED1, which also binds to PIF1 and other PIFs to regulate shade-related elongation responses, did not impact PSY expression or carotenoid accumulation. Our data suggest that the fine-regulation of carotenoid biosynthesis in response to shade relies on specific modules of antagonistic transcriptional factors and cofactors.