A virusinduced gene silencing vigs system for functional. Structure of phytoene desaturase provides insights into herbicide. Phytoene desaturase lycopeneforming crti, fourstep phytoene desaturase ec 1. To improve the edition efficiency, two different single guide rnas grnas were associated to the cas9 nuclease for each target gene. A pcr copy of the gene to be deleted pds or zds that included 500 bases upstream and downstream of the gene. Pds silenced plants display photobleaching phenotype of leaves and impaired pigment formation in fruits. Gene editing the phytoene desaturase alleles of cavendish. Lecture inhibition of pigment synthesis bleaching herbicides. Phytoene desaturase pds is an essential plant carotenoid.
Crisprcas9mediated mutagenesis of phytoene desaturase in. Sequences software user requests ftp archive bulk data retrieval. Although the pds gene is often used as a marker for gene silencing in tomatoes, little is known about the role of pds in fruit ripening. Encodes phytoene desaturase phytoene dehydrogenase, an enzyme that catalyzes the desaturation of phytoene to zetacarotene during carotenoid biosynthesis. Phytoene desaturase pds is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides.
Sequence comparisons were carried out using blast programs altschul. Cloning and expression of citpds1, a gene encoding phytoene. The rcsb pdb also provides a variety of tools and resources. The recovery of the photobleached plants at 28 days post inoculation is attributed. For the phytoene desaturase crtp from synechococcus sp. Functional differences of bapds1 and bapds2 genes in chinese. To date, it is unknown whether lhpds can be silenced by trvbased vigs in lilium. Here, the enzyme phytoene desaturase pds transforms the phytoene into the next intermediate of synthesis, which already has a yellowish color.
Carotenoids are essential components of the photosynthetic apparatus. Norflurazon, a pyridazinone, and fluridone, a complex pyridone, block carotenoid biosynthesis by inhibiting the enzyme phytoene desaturase pds. Plant gene register phytoene desaturase from arabidopsis. The desaturase sequence occurs in the cis geometric isomer configuration, and the action of a carotene isomerase crtiso converts either cis neurosporene or poly cis lycopene to all trans lycopene prior to cyclization isaacson et al. Pds 15cisphytoene desaturase, chloroplasticchromoplastic. Transformation of the green alga haematococcus pluvialis. Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. 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. Pdf tobacco rattle virusinduced phytoene desaturase pds.
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. The twostep desaturation of phytoene to zetacarotene, carried out by the enzyme phytoene desaturase pds, is one of the. Phytoene desaturase is localized exclusively in the. In this study, we used the crisprcas9 gene editing system to deliver a selfcleaving polycistronic. The amplification product resulted in two fragments one of 1200. Structure of phytoene desaturase provides insights into. 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 cavendish dessert banana is a triploid aaa genome type and accounts for around 47% of global production. Nov 19, 2019 the strategy to mutate and knockout the phytoene desaturase gene pds by crisprcas9 has been widely applied to quickly demonstrate the feasibility of crisprcas9 since its mutation causes. Phytoene desaturase pds is a key enzyme involved in 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. Synthesis of carotenoids is initiated from the mevalonic acid pathway. The pds gene in plants was first isolated from the mutants of phytophthora in 1992 by perker et al. Silencing of the phytoene desaturase pds gene affects. The subsequent biosynthetic steps resulting in the different carotenoids also occurring in h. Nov, 2012 four double bonds are then introduced by two enzymes, phytoene desaturase pds and. Lycopene is a branching point in the pathway and is cyclized to form. We propose the use of a synthetic phytoene desaturaseencoding gene crtiop as a selectable marker. Transformation of the green alga haematococcus pluvialis with.
Primers were designed based on the musa acuminata aaa group cultivar cavendish phytoene desaturase pds mrna sequence genbank accession jq762260. As a test of these assignments, we separately deleted in synechocystis sp. The pathway involves specific polycisintermediates and results in the formation of 7,9,9. Isolation of the cavendish pds genomic dna sequence. The cdna of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined 1. This program was also used for separating phytofluene isomers from extracts of tangerine tomato fruits and pds assays.
Tobacco rattle virusinduced phytoene desaturase pds and. Pds assembles into homotetramers that interact monotopically with membranes. Intermediates in this part of the pathway are cisconfigured 12. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Being essentially sterile, genetic modification is perhaps the only pathway available to improve this cultivar. Pcc 6803 the genes that encode phytoene desaturase encoded by crtp and zetacarotene desaturase encoded by crtq zds. We substituted the arginine 304 present in the wild. The structure of pds initiating the polycis pathway of. Mode of action inhibition of pigment synthesis inhibition of phytoene desaturase herbicides with this mode of action inhibit phytoene desaturase pds, a key enzyme necessary to produce carotene and carotenoid pigments. They catalyze the conversion of colorless 15cisphytoene into a bright red lycopene in a biochemical pathway called the polytrans pathway.
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. This gene encodes phytoene desaturase which converts 15cis phytoene into zetacarotene. Pds activity acts as a rheostat of retrograde signaling during early chloroplast. Two marker genes, phytoene desaturase pds and mgchelatase h subunit chlh, were used to test the vigs system in s. The transgenic nature of the transformants can be confirmed by mere phenotypic scoring of the bleached and dwarf regenerants, as a result of pds gene silencing. Jan 25, 2006 the enzyme phytoene desaturase pds has been the main target for herbicides that inhibit the carotenoid biosynthetic pathway. The twostep desaturation of phytoene to zetacarotene, carried out by the enzyme phytoene desaturase pds, is. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve food security and nutritional value. Phytoene desaturase pds is one of the key ratelimiting enzymes in the carotenoid biosynthetic pathway, which catalyses the dehydrogenation of colourless phytoene to. Arabidopsis plants were transformed with an rnai construct targeting pds.
Here we report construction of phytoene desaturase pds intronhairpin rnai vector for its application as a scorable marker in plant genetic transformation. The maize pds sequence used for comparison was genbank u37285. In this study, we used the crisprcas9 gene editing system to deliver a selfcleaving polycistronic guide rna grna designed to target exon 1 of the phytoene desaturase pds gene in the. Three natural somatic mutations at codon 304 of the phytoene desaturase gene pds of hydrilla verticillata l. The pds gene encoding the phytoene desaturase enzyme is involved. Pds catalyzes the second step in carotenoid biosynthesis. Recombinant phytoene desaturase pds his6 from rice was purified to nearhomogeneity and shown to be enzymatically active in a biphasic, liposomebased assay system.
Construction of mutants the genes for phytoene desaturase pds and zetacarotene desaturase zds were deleted separately in the genome of wild type wt synechocystis sp. Its known that reduced levels of photoprotective carotenoids leads to rapid destruction of chlorophyll by photooxidation that results to white or bleached phenotypes. Tobacco rattle virusinduced phytoene desaturase pds. The phytoene desaturase pds is widely used as an indicator gene in the vigs system because silencing of the gene yields a photobleached phenotype cunningham and gantt, 1998. Silenced phytoene desaturase gene as a scorable marker for. Intragenic enhancers and suppressors of phytoene desaturase. In the current study, we demonstrate a successful mutation in phytoene desaturase ras pds of banana cv. The list of acronyms and abbreviations related to pds phytoene desaturase. Various software from the phenix suite and the ccp4 suite were used to build. On the structure and function of the phytoene desaturase. A lightsensitive, phytoene accumulating mutant, pds11, was isolated in chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase pds gene. Two arabidopsis thaliana carotene desaturases, phytoene.
Tobacco rattle virusinduced phytoene desaturase pds and mgchelatase h subunit chlh gene silencing in solanum pseudocapsicum l. Pds phytoene desaturase, all acronyms, viewed march 3, 2020. Phytoene undergoes a series of four desaturations to form lycopene via. Reliability of in silico modeling based on restriction. Efficient knockout of phytoene desaturase gene using. In contrast, the homology between the rcarotene desaturase gene and the pds phytoene desaturase genes from cyanobacteria and higher plants was comparable weak. Virusinduced gene silencing vigs is an attractive tool for determining gene function in plants. Phytoene desaturase pds introduces two double bonds and concomitantly isomerizes two neighboring double bonds from trans to cis. First, plants and cyanobacteria employ two different and very non. The protein contains fad as the sole proteinbound redoxcofactor. Four double bonds are then introduced by two enzymes, phytoene desaturase pds and.
Phytoene desaturase pds introduces two double bonds and. Genetic manipulation shows great promise to further boost the productivity of microalgaebased compounds. Users can perform simple and advanced searches based on annotations relating to sequence, structure and function. In this study, we optimized the conditions of application of this system on apple and explored its. Genomic dna 1 g was used as a template for pcr with the gotaq. Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations.
Williams leaf material using the ctab method stewart and via 1993. Frontiers efficient targeted mutagenesis in apple and. This gene encodes phytoene desaturase which converts 15cisphytoene into zetacarotene. These reactions are catalyzed by the enzymes phytoene desaturase pds and. The following desaturation steps leading to carotene are catalyzed by phytoene desaturase pds, the enzyme studied here. Phytoene synthase psy and phytoene desaturase pds are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. In this study, we investigated whether the pepper pds gene. Bananas are a staple food source and a major export commodity worldwide. Pds catalyzes the introduction of two double bonds into 15cisphytoene, yielding 9,15,9. Jul 21, 2017 here, the enzyme phytoene desaturase pds transforms the phytoene into the next intermediate of synthesis, which already has a yellowish color.
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. In this work, specific primers were used to amplify the phytoene desaturase pds gene of haematococcus sp. The transgenic nature of the transformants can be confirmed by mere phenotypic scoring of the bleached and dwarf regenerants, as a. The clustered regularly interspaced short palindromic repeats crisprcrisprassociated protein 9 cas9 has been reported for precise genome modification in many plants. Efficient knockout of phytoene desaturase gene using crispr. As a proof of concept, we chose to knockout the phytoene desaturase pds and terminal flower 1 tfl1 genes. Annotation program, plant protein annotation program. Tobacco rattle virusinduced phytoene desaturase pds and mg. To further characterize pds, we isolated the pds cdna from cauliflower bopds and confirmed its function by heterologous expression in a strain of e. The cdna of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined. Converts phytoene into zetacarotene via the intermediary of phytofluene by the symmetrical introduction of two.
Total genomic dna was isolated from banana cavendish cv. Plastid localization of the key carotenoid enzyme phytoene. Past research has shown that virusinduced phytoene desaturase pds gene silencing via agroinjection in the attached and detached fruit of tomato plants results in a paleyellow fruit phenotype. Left, the plantcyanobacterial system consisting of the two desaturases, phytoene desaturase pds and. Carotenoid biosynthesis in oriental melon cucumis melo l. A long hydrophobic tunnel is proposed to function in the sequential binding of phytoene and the electron acceptor plastoquinone.
Jul 09, 2018 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. A lightsensitive, phytoeneaccumulating mutant, pds11, was isolated in chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase pds gene. One of these genes, the phytoene desaturase pds gene, is the target for the bleaching herbicide norflurazon 5, 8, 25, 33. Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Pcc 6803 and replaced by a kanamycin resistance cassette. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining pds as a 15cis phytoene donor. 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. The strategy to mutate and knockout the phytoene desaturase gene pds by crisprcas9 has been widely applied to quickly demonstrate the feasibility of crisprcas9 since its mutation causes. Two pds genes were isolated from rasthali raspds1 and raspds2, and their. The following desaturation steps leading to zcarotene are catalyzed by phytoene desaturase pds, the enzyme studied here. Virusinduced phytoene desaturase pds gene silencing.
Royle have been reported to provide resistance to the herbicide fluridone. Desaturase definition of desaturase by medical dictionary. The pds and zds strains synthesize phytoene and zetacarotene, respectively, both of which absorb to shorter wavelength than betacarotene. As a member of the wwpdb, the rcsb pdb curates and annotates pdb data according to agreed upon standards. These molecules are visualized, downloaded, and analyzed by users who range from students to specialized scientists. In maize zea mays proteomic studies, the only carotenoid enzymes detected were pds and. Pdf gene editing the phytoene desaturase alleles of. Regulation of the synthesis of the enzymes is poorly understood. Two pds genes were isolated from rasthali raspds1 and raspds2, and their protein sequence analysis confirmed that both pds comprises conserved motifs for enzyme activity.
Silencing of the phytoene desaturase pds gene affects the. Two marker genes, phytoene desaturase pds and mgchelatase h subunit chlh, were used to test the vigs. Functional differences of bapds1 and bapds2 genes in. On the structure and function of the phytoene desaturase crti from. Maize phytoene desaturase and carotene desaturase catalyse a. Among other methods zinc finger nucleases or tal effector nucleases the crisprcas system proved to be the most effective, convenient and least expensive method. Epistasis in tomato color mutations involves regulation of. Recombinant phytoene desaturase pdshis6 from rice was purified to nearhomogeneity and shown to be enzymatically active in a biphasic, liposomebased assay system. Carotenoids are found in both cell types, yet other carotenoid biosynthetic enzymes were undetectable. Polya rna was purified from total rna with an structed with the upgma program based on the. This means that phytoene, a colorless carotenoid precursor, accumulates and that leads to the photodestruction of chlorophyll pigments bartels and watson, 1978.