Effect of Root Storage and Forcing on the Carbohydrate and Secondary Metabolite Composition of Belgian Endive (Cichorium intybus L. Var. foliosum)
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ABSTRACT:
SUBMITTER: van Arkel J
PROVIDER: S-EPMC9594316 | biostudies-literature | 2022 Sep
REPOSITORIES: biostudies-literature
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Project description:Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof.
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Project description:The interactions between the uptake of selenium (as selenite and selenate) and iodine (as iodate and iodide) by red chicory (Cichorium intybus L. var. foliosum Hegi) and their effects on selected morphological and physiological characteristics were investigated. Seedlings were transplanted to the field, and at the onset of head formation, the plants were foliar-sprayed with the following solutions: Milli-Q water (control), Se (IV), Se (VI), I (-I), I (V), Se (IV) + I (-I), Se (IV) + I (V), Se (VI) + I (-I) and Se (VI) + I (V). The different treatments had no significant effects on the yield (39.8-51.5 t ha-1) and mass (970-1200 g) of the chicory heads. The selenium content in Se-treated plants was up to 5.5-times greater than the control plants. The iodine content in the chicory leaves enriched with I was 3.5-times greater than the control plants. Iodide or iodate, applied together with selenite in the spray solution, increased the uptake of Se by chicory plants, while both forms of iodine, applied together with selenate, reduced the uptake of Se. Plants treated with I (V) had lower amounts of chlorophyll a and carotenoids than the control, while respiratory potential was higher than the control, which indicated the possible presence of stress in I (V)-treated plants. However, the potential photochemical efficiency of photosystem II was similar and close to the theoretical maximum (0.83) in the control and treated groups, which indicated that all of the plants were in good condition. Furthermore, the plant mass and yield were comparable in the control and treated groups. Molecular studies, like gene expression analysis, would represent a major upgrade of the present study by defining the mechanisms of Se and I uptake and their interactions and by enhancing the knowledge of the Se and I transporters.
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