Phylogenetic analyses of the genes involved in carotenoid biosynthesis in algae

Carotenoids play a crucial role in absorbing light energy for photosynthesis,as well as in protecting chlorophyll from photodamage.In contrast to the Streptophyta,few studies have examined carotenoid biosynthetic pathways in algae,owing to a shortage of datasets.As part of the 1000 Plants Project,we sequenced and assembled the transcriptomes of 41 marine macroalgal species,including 22 rhodophytes and 19 phaeophytes,and then combined the datasets with publicly available data from Gen Bank（National Center for Biotechnology Information） and the U.S.Department of Energy Joint Genome Institute.As a result,we identified 68 and 79 fulllength homologs in the Rhodophyta and Phaeophyceae,respectively,of seven inferred carotenoid biosynthetic genes,including the genes for phytoene synthase（PSY）,phytoene desaturase（PDS）,ζ-carotene desaturase（ZDS）,ζ-carotene isomerase（Z-ISO）,prolycopene isomerase（crt ISO）,lycopene β-cyclase（LCYB）,and lycopene ε-cyclase（LCYE）.We found that the evolutionary history of the algal carotenoid biosynthetic pathway was more complex than that of the same pathway in the Streptophyta and,more specifically,that the evolutionary history involved endosymbiotic gene transfer,gene duplication,and gene loss.Almost all of the eukaryotic algae that we examined had inherited the seven carotenoid biosynthesis genes via endosymbiotic gene transfer.Moreover,PSY,crt ISO,and the ancestral lycopene cyclase gene（LCY） underwent duplication events that resulted in multiple gene copies,and the duplication and subsequent divergence of LCYB and LCYE specialized and complicated the cyclization of lycopene.Our findings also verify that the loss of LCYE in both the microphytic rhodophytes and phaeophytes explains the differences in their carotenoid patterns,when compared to the macrophytic rhodophytes.These analyses provide a molecular basis for further biochemical and physiological validation in additional algal species and should help elucidate the origin and evolution of carotenoid biosynthetic pathways.

Carotenoids: New Applications of “Old” Pigments

Carotenoids represent a large group of mainly red,orange,and yellow natural metabolites mainly involved in regulation of many metabolic processes.Carotenoids are beneficial for human health.Current study describes the importance,chemical composition and functioning of carotenoids.It is well known that carotenoids support pigments acting in light absorbance mechanisms during photosynthesis,and are known to protect the chlorophyll molecules from oxidative stress and reactive oxygen species(ROS)damage.Carotenoids are involved in signaling processes in plants,responses to environmental stresses,pollination,germination and reproduction,and development regulation.As nutrients of strong antioxidant activity that is primarily linked to their polyene molecular structure,the carotenoids are reported as immune-enhancement and anticancer agents,which are also involved in prevention of eye-,gastric and neurocognitive disorders,and in regulation of obesity and anti-ageing.Concerning the wide prospective applications of carotenoids as pharmaceuticals and nutraceuticals,there are some critical aspects associated with carotenoids’bioavailability and challenges in their bioengineering.This mostly refers to the needs for identification and cloning of genes responsible for carotenoid biosynthesis and transformation and related development of transgenic carotenoid-rich crops.In the recent years,technologies of micro-and nanoencapsulation have addressed the needs of carotenoid entrapping to enhance their bioavailability,solubility and chemical stability,and to ensure the target delivery and manifestation of their strong antioxidant and other biological activity.Among standard and some advanced analytic tools for carotenoid determination(e.g.,High performance liquid chromatography-HPLC,Liquid chromatography–mass spectrometry-LC-MS,Ultra high performance liquid chromatography-UHPLC,High-performance thin-layer chromatography-HPTLC and others),the vibrational spectroscopy techniques,primarily Raman spectroscopy coupled with chemometric modeling,opened a new era in carotenoid research and application.

Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A

Citrus fruits are rich in carotenoids.In the carotenoid biosynthetic pathway,lycopene β-cyclase（LCYb,EC：1.14.-.-） is a key regulatory enzyme in the catalysis of lycopene to β-carotene,an important dietary precursor of vitamin A for human nutrition.Two closely related lycopene β-cyclase cDNAs,designated CsLCYb1 and CsLCYb2,were isolated from the pulp of orange fruits（Citrus sinensis）.The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopeneaccumulating genotype Cara Cara than that in common genotype Washington,and this might be correlated with lycopene accumulation in Cara Cara fruit.The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system,but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene.In tomato transformation studies,expression of CsLCYb1 under the control of the cauliflower mosaic virus（CaMV） 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene,and the ripe fruits displayed a bright orange colour.However,the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation.In fruits of the CsLCYb1 transgenic plants,most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g-1DW.Unexpectedly,most transgenic tomatoes showed a reduction in total carotenoid accumulation,and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits.Collectively,these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency,and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.

Mutation of the PHYTOENE DESATURASE 3 gene causes yellowish-white petals in Brassica napus

Oilseed rape (Brassica napus) with yellow flowers is an attractive ornamental landscape plant during the flowering period,and the development of different petal colors has become a breeding objective.Although yellowish flower color is a common variant observed in field-grown oilseed rape,the genetics behind this variation remains unclear.We obtained a yellowish-white flower (ywf) mutant from Zhongshuang 9 (ZS9) by ethyl methanesulfonate mutagenesis (EMS) treatment.Compared with ZS9,ywf exhibited a lower carotenoid content with a reduced and defective chromoplast ultrastructure in the petals.Genetic analysis revealed that the yellowish-white trait was controlled by a single recessive gene.Using bulked-segregant analysis sequencing (BSA-seq) and kompetitive allele-specific PCR(KASP),we performed map-based cloning of the ywf locus on chromosome A08 and found that ywf harbored a C-to-T substitution in the coding region,resulting in a premature translation termination.YWF,encoding phytoene desaturase 3 (PDS3),was highly expressed in oilseed rape petals and involved in carotenoid biosynthesis.Pathway enrichment analysis of the transcriptome profiles from ZS9 and ywf indicated the carotenoid biosynthesis pathway to be highly enriched.Further analyses of differentially expressed genes and carotenoid components revealed that the truncated Bna A08.PDS3 resulted in decreased carotenoid biosynthesis in the mutant.These results contribute to an understanding of the carotenoid biosynthesis pathway and manipulation of flower-color variation in B.napus.

Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets

Vitamin A deficiency remains a severe global health issue,which creates a need to biofortify crops with provitamin A carotenoids(PACs).Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored.Here,we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves,Arabidopsis seeds,and citrus callus cells,using a fungal(Neurospora crassa)carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs,including β-carotene.This strategy led to the accumulation of significant amounts of phytoene and γ-and β-carotene,in addition to fungal,health-promoting carotenes with 13 conjugated double bonds,such as the PAC torulene,in the cytosol.Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production.Engineered carotenes accumulate in cytosolic lipid droplets(CLDs),which represent a novel sequestering sink for storing these pigments in plant cytosol.Importantly,β-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidialβ-carotene.Moreover,engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of β-apocarotenoids,including retinal,the aldehyde corresponding to vitamin A.Collectively,our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues,especially in lipid-storing seeds,and thus paves the way for further optimization of carotenoid biofortification in crops.

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.

Characterization of carotenoid biosynthetic pathway genes in the pea aphid(Acyrthosiphon pisum)revealed by heterologous complementation and RNA interference assays

Carotenoids are involved in many essential physiological functions and are produced from geranylgeranyl pyrophosphate through synthase,desaturase,and cyclase activities.In the pea aphid(Acyrthosiphon pisum),the duplication of carotenoid biosynthetic genes,including carotenoid synthases/cyclases(ApCscA-C)and desaturases(ApCdeA-D),through horizontal gene transfer from fungi has been detected,and ApCdeB has known dehydrogenation functions.However,whether other genes contribute to aphid carotenoid biosynthesis,and its specific regulatory pathway,remains unclear.In the current study,functional analyses of seven genes were performed using heterologous complementation and RNA interference assays.The bifunctional enzymes ApCscA-C were responsible for the synthase of phytoene,and ApCscC may also have a cyclase activity.ApCdeA,ApCdeC,and ApCdeD had diverse dehydrogenation functions.ApCdeA catalyzed the enzymatic conversion of phytoene to neurosporene(three-step product),ApCdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene(two-step product),and ApCdeD catalyzed the enzymatic conversion of phytoene to lycopene(four-step product).Silencing of ApCscs reduced the expression levels of ApCdes,and silencing these carotenoid biosynthetic genes reduced the α-,β-,and γ-carotene levels,as well as the total carotenoid level.The results suggest that these genes were activated and led to carotenoid biosynthesis in the pea aphid.