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CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots

Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have bee...

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Autores principales: Cankar, Katarina, Hakkert, Johanna Christina, Sevenier, Robert, Campo, Eva, Schipper, Bert, Papastolopoulou, Christina, Vahabi, Khabat, Tissier, Alain, Bundock, Paul, Bosch, Dirk
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9465254/
https://www.ncbi.nlm.nih.gov/pubmed/36105709
http://dx.doi.org/10.3389/fpls.2022.940003
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author Cankar, Katarina
Hakkert, Johanna Christina
Sevenier, Robert
Campo, Eva
Schipper, Bert
Papastolopoulou, Christina
Vahabi, Khabat
Tissier, Alain
Bundock, Paul
Bosch, Dirk
author_facet Cankar, Katarina
Hakkert, Johanna Christina
Sevenier, Robert
Campo, Eva
Schipper, Bert
Papastolopoulou, Christina
Vahabi, Khabat
Tissier, Alain
Bundock, Paul
Bosch, Dirk
author_sort Cankar, Katarina
collection PubMed
description Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory.
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spelling pubmed-94652542022-09-13 CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots Cankar, Katarina Hakkert, Johanna Christina Sevenier, Robert Campo, Eva Schipper, Bert Papastolopoulou, Christina Vahabi, Khabat Tissier, Alain Bundock, Paul Bosch, Dirk Front Plant Sci Plant Science Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory. Frontiers Media S.A. 2022-08-29 /pmc/articles/PMC9465254/ /pubmed/36105709 http://dx.doi.org/10.3389/fpls.2022.940003 Text en Copyright © 2022 Cankar, Hakkert, Sevenier, Campo, Schipper, Papastolopoulou, Vahabi, Tissier, Bundock and Bosch. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Cankar, Katarina
Hakkert, Johanna Christina
Sevenier, Robert
Campo, Eva
Schipper, Bert
Papastolopoulou, Christina
Vahabi, Khabat
Tissier, Alain
Bundock, Paul
Bosch, Dirk
CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots
title CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots
title_full CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots
title_fullStr CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots
title_full_unstemmed CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots
title_short CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots
title_sort crispr/cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9465254/
https://www.ncbi.nlm.nih.gov/pubmed/36105709
http://dx.doi.org/10.3389/fpls.2022.940003
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