Effects on isoprenoids, plastids, and intermediary metabolism w paul d. Pdf regulation of phytoene desaturase expression is. Efficient crisprcas9 genome editing of phytoene desaturase. Phytoene desaturase pds is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as. A single amino acid substitution, l505f, enhanced its desaturation.
Mutations in phytoene desaturase gene in fluridone. Bleaching herbicide flurtamone interferes with phytoene. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining pds as a 15cisphytoene donor. Pseudescherichia vulneris escherichia vulneris status. But the genetic toolbox for this alga is not available. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. Structure of phytoene desaturase provides insights into herbicide binding and reaction mechanisms involved in carotene desaturation graphical abstract highlights d the structure of pds initiating the polycis pathway of carotene desaturation d the herbicidal inhibitor nor. The twostep desaturation of phytoene to zetacarotene, carried out by the enzyme phytoene desaturase pds, is one of the. Disruption of phytoene desaturase gene using transient expression of cas9. Crtitype phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cistans isomerases for the same purpose. The bacterial phytoene desaturaseencoding gene crti mdpi. Pdf disruption of phytoene desaturase gene using transient.
The bacterial phytoene desaturaseencoding gene crti. Frontiers efficient crisprcas9 genome editing of phytoene. A phytoene desaturase gene crti found in the photosynthesis gene cluster of this bacterium was expressed in an escherichia coli strain that can produce. A preparation comprising the carotenoids phytoene and phytofluene in an amount which, in combination, is effective in preventing various kinds of damage resulting from oxidation and exposure to uv light is provided.
Polyclonal antibodies raised against the recombinant protein allowed the detection of soluble and tightly membrane. Plos one, nov 2019 julian koschmieder, mirjam fehlingkaschek, patrick schaub, sandro. Carotenogenesis gene cluster and phytoene desaturase. Regulation of phytoene desaturase expression is independent of leaf pigment content in arabidopsis thaliana. Determine phytoene desaturase gene copy number in hydrillaverticillata tu huynh, dr. The phytoene desaturation step in carotenoid biosynthesis has also been. Structure of phytoene desaturase provides insights into herbicide binding and reaction mechanisms involved in carotene desaturation anton brausemann,1 sandra gemmecker,2 julian koschmieder,2 sandro ghisla,3 peter beyer,2,4,5, and oliver einsle1,4. Formation anddecrease ofchl is lightdependent in cress seedlings, whereasinhibition ofcarotenoidsbyflurtamoneis not. Phytoene desaturase pds introduces two double bonds and concomitantly isomerizes two neighboring double bonds from trans to cis. Silenced phytoene desaturase gene as a scorable marker for.
Phytoene desaturase is localized exclusively in the chloroplast and upregulated at the mrna level during accumulation of secondary carotenoids in haematococcus pluvialis volvocales, chlorophyceae 1, 2. The unicellular green alga chlorella zofingiensis has been proposed as a promising producer of natural astaxanthin, a commercially important ketocarotenoid. In bacteria and fungi, a second type of carotene desaturase, termed crti, also. Psy3, a new member of the phytoene synthase gene family conserved in the poaceae and regulator of abioticstressinduced root carotenogenesis. They catalyze the conversion of colorless 15cis phytoene into a bright red lycopene in a biochemical pathway called the polytrans pathway. The mutant e17 could grow well and produce normal levels of colored carotenoids in the presence of 0. Phytoene desaturase is the key enzyme involved in the biosynthesis pathway of lutein. Jul 22, 2008 the unicellular green alga dunaliella is outstanding for its ability of massive accumulation of carotenoids. Phytoene desaturase, a target for herbicidal inhibitors. However, since the interest in these compounds in the context of many applications is expanding, it. This enzyme converts phytoene into lycopene via the intermediaries of phytofluene, zetacarotene and neurosporene by the introduction of four double bonds.
Regulation of phytoene desaturase expression is independent. Wo20000654a2 a carotenoid composition containing phytoene. Gene editing the phytoene desaturase alleles of cavendish. Phytoene desaturases are membranebound enzymes localized in plastids and introduce two double bonds into their colorless substrate phytoene by dehydrogenation and isomerize. The gene encoding phytoene desaturase in the deinoxanthin synthesis pathway of deinococcus radiodurans was identified and characterized. Cloning and characterization of the gene for phytoene. Being naturally present in common foods, their intake at dietary levels is not expected to present a safety concern. In the present study, an efficient transformation system was established for c. The results reported set the groundwork for future studies of gene function and trait development using the crisprcas9 geneediting technology in this important staple food crop. Further analysis showed that high level of phytoene was accumulated in the pds3 mutant. Synthesis of carotenoids in higher plants occurs in the plastids, but all of the required enzymes are coded for in the nuclear genome and are posttranscriptionally imported into the plastid compartment. The second cluster is located on scaffold 11 and harbors two putatively intact phytoene desaturases and one cyclasesynthase along with one phytoene desaturase pseudogene.
We have cloned and analyzed the genomic dna sequence of pds from tomato. The twostep desaturation of phytoene to zetacarotene, carried out by the enzyme phytoene desaturase pds, is. Somatic mutations in amino acid 304 of hydrilla pds are known to confer herbicide resistance. Desaturase definition of desaturase by medical dictionary. Abscisic acid deficiency caused by phytoene desaturase silencing is associated with dwarfing syndrome in citrus article in plant cell reports may 2019 with 53 reads how we measure reads. A cdna coding for the carotenoid biosynthetic enzyme phytoene desaturase from narcissus pseudonarcissus was cloned and the corresponding protein expressed in insect cells using the baculovirus system. Disruption of phytoene desaturase gene results in albino and dwarf. Here we report construction of phytoene desaturase pds intronhairpin rnai vector for its application as a scorable marker in plant genetic transformation. Identification and functional analysis of a phytoene. We propose the use of a synthetic phytoene desaturaseencoding gene crtiop as a selectable marker and the bleaching herbicide norflurazon.
A putative promoter sequence has been identified by comparison with the cdna sequence of pds. Oct 18, 2017 crisprcas9 has become a powerful genomeediting tool for introducing genetic changes into crop species. Phytoene desaturase, crti, of the purple photosynthetic bacterium. This enzyme catalyses the following chemical reaction. This conversion introduces two additional double bonds at positions 11 and 11 of the carbon chain and isomerizes two adjacent already existing double bonds at positions 9 and 9 from trans to cis. Genetic manipulation shows great promise to further boost the productivity of microalgaebased compounds.
Gene editing using crisprcas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement and its use has been reported in a growing number of important food crops, including recently fragaria. The transformation system utilized a modified norflurazonresistant phytoene desaturase. Pdf engineering of an endogenous phytoene desaturase gene as. The desaturation and isomerization of phytoene to lycopene occurs in four consecutive. Algal pds is inhibited by some herbicides, leading to the bleaching of the cells due to destruction of chl. The preimmune serum did not affect the carotenogenic reactions. A long hydrophobic tunnel is proposed to function in the sequential binding of phytoene and the electron acceptor plastoquinone. Phytoene desaturase from arabidopsis phytoene desaturase from arabidopsis p. Plant gene register phytoene desaturase from arabidopsis.
On the structure and function of the phytoene desaturase crti. Regulation of the synthesis of the enzymes is poorly understood. A construct phsecmpds carrying both grnas and the cas9 protein was delivered by pegmediated transformation in protoplasts. Phytoene desaturase catalyzes the insertion of two double bonds into the first carotene in a series leading to. Mechanistically, crti is an oxidase that transfers the electrons from phytoene directly to oxygen, forming water. This extends the triene chromophore of phytoene to formvia the pentaene intermediate phytofluenethe light yellow. The protein contains fad as the sole proteinbound redoxcofactor. The preparation is essentially colorless and may be used as a topical cosmetic or pharmaceutical preparation as well as an additive in food preparations.
Structure of phytoene desaturase provides insights into. Because fluridone is an inhibitor of the enzyme phytoene desaturase pds, the gene for pds pds was cloned from fluridonesusceptible and resistant hydrilla biotypes. This involves functional complementation of phytoene desaturation by heterologous expression of different genes coding for this enzyme originating from rhodobacter capsulatus, erwinia uredovora, and synechococcus and the enzymatic characterization of purified enzymes. The colorless carotenoids phytoene and phytofluene are comparatively understudied compounds found in common foods e. In our study, phytoene desaturase gene of melon cmpds was selected as target for the crisprcas9 system with two designed grnas, targeting exons 1 and 2.
Request pdf abscisic acid deficiency caused by phytoene desaturase silencing is associated with dwarfing syndrome in citrus key message in citrus, abscisic aciddeficiency was associated with. Aquatic plant management and the impact of emerging herbicide resistance issues. This property renders crti a valuable gene to engineer provitamin aformation to help combat vitamin a malnutrition, such as with golden rice. In vitro inhibition studies of phytoene desaturase by. Defects in phytoene desaturase pds gene function result in a distinctive albino phenotype 38, and recent studies in populus 23, apple 24, grapes 25 and cassava 26 have targeted the pds gene to demonstrate successful genome editing in these crops. Engineering of an endogenous phytoene desaturase gene as a.
These results give strong evidence that chl decrease in thepresenceofflurtamoneis asecondaryeffectmostpossibly. Recombinant phytoene desaturase pdshis6 from rice was purified to nearhomogeneity and shown to be enzymatically active in a biphasic, liposomebased assay system. Oligomeric assembly, membrane association and preliminary 3danalysis. Cloning and characterization of the phytoene desaturase pds. Phytoene desaturase is a common target site for bleaching herbicides bramley, 1993b. In the present study, nicotiana benthamiana phytoene desaturase nbpds gene was. The unicellular microalga, chlorella protothecoides cs41, had been selected for the heterotrophic production of high concentrations of lutein. Phytoene desaturases pds, the subject of this work, prevail in plants and cyanobacteria. Pdf silencing of the phytoene desaturase pds gene affects the. Phytoene desaturase pds is a key enzyme in the carotenogenic pathway and its disruption causes albinism and dwarfing by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Pds assembles into homotetramers that interact monotopically with membranes. Crisprcas9mediated mutagenesis of phytoene desaturase in. In order to inform application of the technology in fragaria, we targeted the visible endogenous marker gene pds phytoene desaturase in diploid fragaria vesca ssp. Phytoene desaturase 3,4didehydrolycopeneforming ec 1.
The cdna of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined. Disruption of phytoene desaturase gene results in albino. Phytoene is then sequentially desaturated by phytoene desaturase crti. To elucidate the carotenoids synthesis pathway in this alga, phytoene desaturase pds gene cdna together with its dna sequences were isolated and their structures and functions analyzed.
In order to develop capacity for crisprcas9 technology in the tropical staple cassava manihot esculenta, the phytoene desaturase mepds gene was targeted in two cultivars using constructs carrying grnas targeting two sequences within mepds exon. Light activation of rosa cellular metabolism cell division and growth hrac mode of action classification 2020 a free copy of this poster can be downloaded at. Immunological detection of phytoene desaturase in algae. The antiserum against the phytoene desaturase fusion pro. The gene pds encodes phytoene desaturase, a key enzyme in carotenoid biosynthesis that converts phytoene to. Matt hale department of biology, texas christian university 1. Abscisic acid deficiency caused by phytoene desaturase. Bramleya,3 a school of biological sciences, royal holloway, university of london, egham, surrey, tw20 oex, united kingdom.
We propose the use of a synthetic phytoene desaturaseencoding gene crtiop as a selectable marker. That is, the headtohead condensation of two molecules of geranylgeranyl diphosphate c 20 pp is catalyzed by phytoene synthase crtb. This property renders crti a valuable gene to engineer. The phytoene formation process is conserved in various c 40 carotenogenesis pathways. Phytoene desaturase is localized exclusively in the. Phytoene desaturase lycopeneforming crti, fourstep phytoene desaturase ec 1. The pathway involves specific polycisintermediates and results in the formation of 7,9,9979tetracislycopene. The desaturation and isomerization of phytoene to lycopene occurs in. Past research has shown that virusinduced phytoene desaturase pds gene silencing via agroinjection in the attached. Phytoene synthase psy and phytoene desaturase pds are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. As such, the pds gene has been used as a convenient indicator for crisprcas9mediated gene knockouts in a variety of plant species fan et al. Crti is a bacterial phytoene desaturase that is distinct from the cyanobacterialplant carotene desaturation system. Currently used marker systems either require cumbersome assays and specialized equipment, or have biosafety concerns. Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene pds3 encodes one of the important enzymes in the.
The transgenic nature of the transformants can be confirmed by mere phenotypic scoring of the bleached and dwarf regenerants, as a. Phytoene desaturase pds can be an necessary place carotenoid biosynthetic enzyme and a prominent focus on of specific inhibitors, such as for example norflurazon, acting seeing that bleaching herbicides. The list of acronyms and abbreviations related to pds phytoene desaturase. One amino acid substitution in phytoene desaturase makes. Mar 10, 2010 a stable chlorella zofingiensis mutant e17 produced by chemical mutagen was characterized with respect to growth, astaxanthin biosynthesis, and phytoene desaturation. Mutations in phytoene desaturase gene in fluridoneresistant. Pdf on may 1, 20, jin liu and others published engineering of an endogenous phytoene desaturase gene as a dominant selectable. Structure of phytoene desaturase provides insights into herbicide.
We propose the use of a synthetic phytoene desaturase encoding gene crtiop as a selectable marker and the. Two putative phytoene desaturase homologues dr0861 and dr0810 were identified by analysis of conserved amino acid regions, and the former displayed the highest. The phytoenerelated desaturases are the key enzymes in the carotenoid biosynthetic pathway. The bacterial phytoene desaturaseencoding gene crti is. In tomato pds is comprised of 15 exons that, together with the introns occupy over 8 kb. Recombinant phytoene desaturase pdshis 6 from rice was purified to nearhomogeneity and shown to be enzymatically active in a biphasic, liposomebased assay system. Specific point mutations in pds confer resistance to the herbicide norflurazon, suggesting that mutated pds could be used as a dominant selectable marker for genetic engineering of algae, for which very few. However, to our knowledge, there is no report of crisprcas9mediated genome editing in melon cucumis melo. Effect of constitutive expression of bacterial phytoene. Crisprcas9 has become a powerful genomeediting tool for introducing genetic changes into crop species. With the exception of the fadbinding domain, crti shares no sequence homology with plant desaturases. On the structure and function of the phytoene desaturase. Competitive inhibition of synechococcus phytoene desaturase using decyl plastoquinone as cofactor and phytoene as substrate at 0. Left, the plantcyanobacterial system consisting of the two desaturases, phytoene desaturase pds and fcarotene desaturase zds.
They catalyze the conversion of colorless 15cisphytoene into a bright red lycopene in a biochemical pathway called the polytrans pathway. Functional evaluation of structural implications plos one, nov 2019 julian koschmieder, mirjam fehlingkaschek, patrick schaub, sandro ghisla, anton brausemann, jens timmer, peter beyer. Truesdale,a,1 dongmei yu,a,2 christopher gerrish,a and peter m. The desaturase enzymes are membrane bound within the plastid and have been reported to be associated with the chloroplast envelope of spinach leaves lutkebrinkhaus et al. Past research has shown that virusinduced phytoene desaturase pds gene silencing via agroinjection in the attached and detached fruit of. The fulllength pds cdna of 2290 bp genbank accession no. A phytoene desaturase pds gene was cloned and characterized from the unicellular green microalgachlamydomonas reinhardtii. Structure and function characterization of the phytoene. Jin liu i henri gerken i junchao huang i feng chen. In several fungi this key enzyme is responsible for lightdark regulation of carotenogenesis 1. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining pds as a 15cis phytoene donor. This involves functional complementation of phytoene desaturation by heterologous expression of different genes coding for this enzyme originating from.
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