Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot
Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific role...
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MDPI AG
2021-06-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/12/6516 |
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| author | Tomasz Oleszkiewicz Magdalena Klimek-Chodacka Michał Kruczek Kamila Godel-Jędrychowska Katarzyna Sala Anna Milewska-Hendel Maciej Zubko Ewa Kurczyńska Yiping Qi Rafal Baranski |
| author_facet | Tomasz Oleszkiewicz Magdalena Klimek-Chodacka Michał Kruczek Kamila Godel-Jędrychowska Katarzyna Sala Anna Milewska-Hendel Maciej Zubko Ewa Kurczyńska Yiping Qi Rafal Baranski |
| author_sort | Tomasz Oleszkiewicz |
| collection | DOAJ |
| description | Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of <i>psy</i> paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two <i>psy</i> paralogs in a model system and to reveal biochemical changes in the cell wall of <i>psy</i> knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (<i>Daucus carota</i>) callus cells in order to induce mutations in the <i>psy1</i> and <i>psy2</i> genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the <i>psy2</i> gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The <i>psy2</i> knockout also decreased the expression of the <i>psy1</i> paralog. Immunohistochemical staining of the <i>psy2</i> mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free <i>psy2</i> mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling. |
| first_indexed | 2024-03-10T10:18:16Z |
| format | Article |
| id | doaj.art-2c10b510f8b14d319904315245098e25 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T10:18:16Z |
| publishDate | 2021-06-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-2c10b510f8b14d319904315245098e252023-11-22T00:35:28ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-06-012212651610.3390/ijms22126516Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in CarrotTomasz Oleszkiewicz0Magdalena Klimek-Chodacka1Michał Kruczek2Kamila Godel-Jędrychowska3Katarzyna Sala4Anna Milewska-Hendel5Maciej Zubko6Ewa Kurczyńska7Yiping Qi8Rafal Baranski9Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, 31-425 Krakow, PolandDepartment of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, 31-425 Krakow, PolandDepartment of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, 31-425 Krakow, PolandInstitute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 40-032 Katowice, PolandInstitute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 40-032 Katowice, PolandInstitute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 40-032 Katowice, PolandInstitute of Materials Engineering, Faculty of Science and Technology, University of Silesia, 41-500 Chorzów, PolandInstitute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 40-032 Katowice, PolandDepartment of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USADepartment of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, 31-425 Krakow, PolandRecent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of <i>psy</i> paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two <i>psy</i> paralogs in a model system and to reveal biochemical changes in the cell wall of <i>psy</i> knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (<i>Daucus carota</i>) callus cells in order to induce mutations in the <i>psy1</i> and <i>psy2</i> genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the <i>psy2</i> gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The <i>psy2</i> knockout also decreased the expression of the <i>psy1</i> paralog. Immunohistochemical staining of the <i>psy2</i> mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free <i>psy2</i> mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.https://www.mdpi.com/1422-0067/22/12/6516arabinogalactan proteincallusCas9 proteinchromoplastsClustered Regularly Interspaced Short Palindromic RepeatsCRISPR |
| spellingShingle | Tomasz Oleszkiewicz Magdalena Klimek-Chodacka Michał Kruczek Kamila Godel-Jędrychowska Katarzyna Sala Anna Milewska-Hendel Maciej Zubko Ewa Kurczyńska Yiping Qi Rafal Baranski Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot International Journal of Molecular Sciences arabinogalactan protein callus Cas9 protein chromoplasts Clustered Regularly Interspaced Short Palindromic Repeats CRISPR |
| title | Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot |
| title_full | Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot |
| title_fullStr | Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot |
| title_full_unstemmed | Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot |
| title_short | Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot |
| title_sort | inhibition of carotenoid biosynthesis by crispr cas9 triggers cell wall remodelling in carrot |
| topic | arabinogalactan protein callus Cas9 protein chromoplasts Clustered Regularly Interspaced Short Palindromic Repeats CRISPR |
| url | https://www.mdpi.com/1422-0067/22/12/6516 |
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