TY  - CONF
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Ćuković, Katarina
AU  - van Arkel, Jeroen
AU  - Bosch, Dirk
AU  - Cankar, Katarina
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6155
AB  - Root chicory (Cichorium intybus var. sativum), an industrial crop species used for the production of a fructose polymer inulin, has been shown to contain a mixture of bitter tasting sesquiterpene lactones (STLs), that are currently discarded as waste. For several STLs found in plants of the Asteraceae family including chicory, interesting bioactivities have been demonstrated, including potent anti-cancer, anti-malarial, anti-inflammatory, anti-fungal and anti-bacterial activity. This activity is mainly attributed to guaianolide STLs; in chicory the most abundant STLs are lactucin, lactucopicrin, and 8-deoxylactucin, found predominantly in their oxalated forms in the latex of the plant. Several steps in the biosynthetic pathway of these compounds have been unraveled recently. However, the enzymes involved in the formation of STL oxalates, the most abundant form of STLs in chicory, have not yet been identified. Candidate genes for the chicory oxalate-CoA ligase (CiOxL) and chicory STL oxalyl transferases (CiOxT) putatively involved in the STL-oxalate formation were identified. Next, introduction of CRISPR/Cas reagents into chicory by Agrobacterium tumefaciens-mediated stable transformation was used to inactivate gene candidates putatively involved in STL-oxalate formation, and several chicory lines with edited genes were successfully regenerated. Detailed genotyping of mutant lines revealed the presence of indels leading to frame-shift predominantly, varying from 1 to 44 base pairs in length. Detailed genotyping also confirmed previous observations that plants transformed via Agrobacterium often showed chimerism, and a mixture of different on-target edits in one plant was observed. Leaves of plants carrying mutations in CiOxL or CiOxT were characterized by LC-MS to determine changes in terpene profile. The analysis showed that the production of STLs was reduced or eliminated in leaves of several CiOxL and CiOxT4 plants. Surprisingly, not only the oxalated terpenes were reduced but also the non-oxalated STLs, perhaps due to feedback regulation or toxicity of non-oxalated forms. These results contribute to further elucidation of the STL pathway in chicory and show that Agrobacterium-mediated plant transformation with CRISPR/Cas reagents requires detailed genotyping for characterization of genome edited plants.
PB  - COST Action CA18111
C3  - Book of abstracts: 4th PlantEd Conference; 2023 Sep 18-20; Porto, Portugal
T1  - CRISPR/Cas targeted inactivation of guaianolide oxalate formation in chicory
SP  - 74
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6155
ER  - 

@conference{
author = "Bogdanović, Milica and Todorović, Slađana and Ćuković, Katarina and van Arkel, Jeroen and Bosch, Dirk and Cankar, Katarina",
year = "2023",
abstract = "Root chicory (Cichorium intybus var. sativum), an industrial crop species used for the production of a fructose polymer inulin, has been shown to contain a mixture of bitter tasting sesquiterpene lactones (STLs), that are currently discarded as waste. For several STLs found in plants of the Asteraceae family including chicory, interesting bioactivities have been demonstrated, including potent anti-cancer, anti-malarial, anti-inflammatory, anti-fungal and anti-bacterial activity. This activity is mainly attributed to guaianolide STLs; in chicory the most abundant STLs are lactucin, lactucopicrin, and 8-deoxylactucin, found predominantly in their oxalated forms in the latex of the plant. Several steps in the biosynthetic pathway of these compounds have been unraveled recently. However, the enzymes involved in the formation of STL oxalates, the most abundant form of STLs in chicory, have not yet been identified. Candidate genes for the chicory oxalate-CoA ligase (CiOxL) and chicory STL oxalyl transferases (CiOxT) putatively involved in the STL-oxalate formation were identified. Next, introduction of CRISPR/Cas reagents into chicory by Agrobacterium tumefaciens-mediated stable transformation was used to inactivate gene candidates putatively involved in STL-oxalate formation, and several chicory lines with edited genes were successfully regenerated. Detailed genotyping of mutant lines revealed the presence of indels leading to frame-shift predominantly, varying from 1 to 44 base pairs in length. Detailed genotyping also confirmed previous observations that plants transformed via Agrobacterium often showed chimerism, and a mixture of different on-target edits in one plant was observed. Leaves of plants carrying mutations in CiOxL or CiOxT were characterized by LC-MS to determine changes in terpene profile. The analysis showed that the production of STLs was reduced or eliminated in leaves of several CiOxL and CiOxT4 plants. Surprisingly, not only the oxalated terpenes were reduced but also the non-oxalated STLs, perhaps due to feedback regulation or toxicity of non-oxalated forms. These results contribute to further elucidation of the STL pathway in chicory and show that Agrobacterium-mediated plant transformation with CRISPR/Cas reagents requires detailed genotyping for characterization of genome edited plants.",
publisher = "COST Action CA18111",
journal = "Book of abstracts: 4th PlantEd Conference; 2023 Sep 18-20; Porto, Portugal",
title = "CRISPR/Cas targeted inactivation of guaianolide oxalate formation in chicory",
pages = "74",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6155"
}

Bogdanović, M., Todorović, S., Ćuković, K., van Arkel, J., Bosch, D.,& Cankar, K.. (2023). CRISPR/Cas targeted inactivation of guaianolide oxalate formation in chicory. in Book of abstracts: 4th PlantEd Conference; 2023 Sep 18-20; Porto, Portugal
COST Action CA18111., 74.
https://hdl.handle.net/21.15107/rcub_ibiss_6155

Bogdanović M, Todorović S, Ćuković K, van Arkel J, Bosch D, Cankar K. CRISPR/Cas targeted inactivation of guaianolide oxalate formation in chicory. in Book of abstracts: 4th PlantEd Conference; 2023 Sep 18-20; Porto, Portugal. 2023;:74.
https://hdl.handle.net/21.15107/rcub_ibiss_6155 .

Bogdanović, Milica, Todorović, Slađana, Ćuković, Katarina, van Arkel, Jeroen, Bosch, Dirk, Cankar, Katarina, "CRISPR/Cas targeted inactivation of guaianolide oxalate formation in chicory" in Book of abstracts: 4th PlantEd Conference; 2023 Sep 18-20; Porto, Portugal (2023):74,
https://hdl.handle.net/21.15107/rcub_ibiss_6155 .

TY  - JOUR
AU  - Salvagnin, Umberto
AU  - Unkel, Katharina
AU  - Sprink, Thorben
AU  - Bundock, Paul
AU  - Sevenier, Robert
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Cankar, Katarina
AU  - Hakkert, Johanna Christina
AU  - Schijlen, Elio
AU  - Nieuwenhuis, Ronald
AU  - Hingsamer, Maria
AU  - Kulmer, Veronika
AU  - Kernitzkyi, Michael
AU  - Bosch, Dirk
AU  - Martens, Stefan
AU  - Malnoy, Mickael
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5794
AB  - Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.
PB  - Lausanne: Frontiers Media SA
T2  - Frontiers in Plant Science
T1  - A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L
VL  - 14
DO  - 10.3389/fpls.2023.1111110
SP  - 1111110
ER  - 

@article{
author = "Salvagnin, Umberto and Unkel, Katharina and Sprink, Thorben and Bundock, Paul and Sevenier, Robert and Bogdanović, Milica and Todorović, Slađana and Cankar, Katarina and Hakkert, Johanna Christina and Schijlen, Elio and Nieuwenhuis, Ronald and Hingsamer, Maria and Kulmer, Veronika and Kernitzkyi, Michael and Bosch, Dirk and Martens, Stefan and Malnoy, Mickael",
year = "2023",
abstract = "Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.",
publisher = "Lausanne: Frontiers Media SA",
journal = "Frontiers in Plant Science",
title = "A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L",
volume = "14",
doi = "10.3389/fpls.2023.1111110",
pages = "1111110"
}

Salvagnin, U., Unkel, K., Sprink, T., Bundock, P., Sevenier, R., Bogdanović, M., Todorović, S., Cankar, K., Hakkert, J. C., Schijlen, E., Nieuwenhuis, R., Hingsamer, M., Kulmer, V., Kernitzkyi, M., Bosch, D., Martens, S.,& Malnoy, M.. (2023). A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. in Frontiers in Plant Science
Lausanne: Frontiers Media SA., 14, 1111110.
https://doi.org/10.3389/fpls.2023.1111110

Salvagnin U, Unkel K, Sprink T, Bundock P, Sevenier R, Bogdanović M, Todorović S, Cankar K, Hakkert JC, Schijlen E, Nieuwenhuis R, Hingsamer M, Kulmer V, Kernitzkyi M, Bosch D, Martens S, Malnoy M. A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. in Frontiers in Plant Science. 2023;14:1111110.
doi:10.3389/fpls.2023.1111110 .

Salvagnin, Umberto, Unkel, Katharina, Sprink, Thorben, Bundock, Paul, Sevenier, Robert, Bogdanović, Milica, Todorović, Slađana, Cankar, Katarina, Hakkert, Johanna Christina, Schijlen, Elio, Nieuwenhuis, Ronald, Hingsamer, Maria, Kulmer, Veronika, Kernitzkyi, Michael, Bosch, Dirk, Martens, Stefan, Malnoy, Mickael, "A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L" in Frontiers in Plant Science, 14 (2023):1111110,
https://doi.org/10.3389/fpls.2023.1111110 . .

TY  - CONF
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Ćuković, Katarina
AU  - Dragićević, Milan
AU  - Beekwilder, Jules
AU  - Cankar, Katarina
AU  - Simonović, Ana
PY  - 2020
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5438
AB  - Chicory (Cichorium intybus L.) is a valuable crop grown mostly in northern France, Belgium and the Netherlands, whose roots, leaves and etiolated shoots are used as vegetable, coffee replacement and remedy for several conditions including inflammation. Roots of chicory are an important source of inulin, a prebiotic fiber and a sweetener. Chicory also contains bioactive terpene compounds that protect the plant against herbivores, but are also responsible for medicinal properties of the plant, as they have anti-microbial, anti-cancer and anti-inflammatory activity. Biosynthesis of plant bioactive metabolites can be modified using new plant breeding techniques, especially new gene editing techniques like CRISPR/Cas9 to improve their health-promoting qualities. Chicory was transformed with Agrobacterium tumefaciens carrying CRISPR/Cas9 constructs to knock-out expression of key genes in the sesquiterpene biosynthesis pathway. Regenerated plants were selected on media with kanamycin and further tested by PCR for the presence of transgenes. Obtained plants were also tested for mutations in target genes, by PCR with primers surrounding the mutation site detecting larger indels and by Sanger sequencing detecting smaller indels. Further analysis was performed detecting heteroduplex DNA fragments as a result of either hetero- or homozygous mutation events.
PB  - East Sarajevo: Faculty of Agriculture
C3  - Book of abstracts: XI International Scientific Agriculture Symposium: Agrosym 2020; 2020 Oct 8-9; Jahorina, Republika Srpska, Bosnia and Herzegovina
T1  - Metabolic engeneering in chicory by CRISPR/Cas9 editing
SP  - 152
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5438
ER  - 

@conference{
author = "Bogdanović, Milica and Todorović, Slađana and Ćuković, Katarina and Dragićević, Milan and Beekwilder, Jules and Cankar, Katarina and Simonović, Ana",
year = "2020",
abstract = "Chicory (Cichorium intybus L.) is a valuable crop grown mostly in northern France, Belgium and the Netherlands, whose roots, leaves and etiolated shoots are used as vegetable, coffee replacement and remedy for several conditions including inflammation. Roots of chicory are an important source of inulin, a prebiotic fiber and a sweetener. Chicory also contains bioactive terpene compounds that protect the plant against herbivores, but are also responsible for medicinal properties of the plant, as they have anti-microbial, anti-cancer and anti-inflammatory activity. Biosynthesis of plant bioactive metabolites can be modified using new plant breeding techniques, especially new gene editing techniques like CRISPR/Cas9 to improve their health-promoting qualities. Chicory was transformed with Agrobacterium tumefaciens carrying CRISPR/Cas9 constructs to knock-out expression of key genes in the sesquiterpene biosynthesis pathway. Regenerated plants were selected on media with kanamycin and further tested by PCR for the presence of transgenes. Obtained plants were also tested for mutations in target genes, by PCR with primers surrounding the mutation site detecting larger indels and by Sanger sequencing detecting smaller indels. Further analysis was performed detecting heteroduplex DNA fragments as a result of either hetero- or homozygous mutation events.",
publisher = "East Sarajevo: Faculty of Agriculture",
journal = "Book of abstracts: XI International Scientific Agriculture Symposium: Agrosym 2020; 2020 Oct 8-9; Jahorina, Republika Srpska, Bosnia and Herzegovina",
title = "Metabolic engeneering in chicory by CRISPR/Cas9 editing",
pages = "152",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5438"
}

Bogdanović, M., Todorović, S., Ćuković, K., Dragićević, M., Beekwilder, J., Cankar, K.,& Simonović, A.. (2020). Metabolic engeneering in chicory by CRISPR/Cas9 editing. in Book of abstracts: XI International Scientific Agriculture Symposium: Agrosym 2020; 2020 Oct 8-9; Jahorina, Republika Srpska, Bosnia and Herzegovina
East Sarajevo: Faculty of Agriculture., 152.
https://hdl.handle.net/21.15107/rcub_ibiss_5438

Bogdanović M, Todorović S, Ćuković K, Dragićević M, Beekwilder J, Cankar K, Simonović A. Metabolic engeneering in chicory by CRISPR/Cas9 editing. in Book of abstracts: XI International Scientific Agriculture Symposium: Agrosym 2020; 2020 Oct 8-9; Jahorina, Republika Srpska, Bosnia and Herzegovina. 2020;:152.
https://hdl.handle.net/21.15107/rcub_ibiss_5438 .

Bogdanović, Milica, Todorović, Slađana, Ćuković, Katarina, Dragićević, Milan, Beekwilder, Jules, Cankar, Katarina, Simonović, Ana, "Metabolic engeneering in chicory by CRISPR/Cas9 editing" in Book of abstracts: XI International Scientific Agriculture Symposium: Agrosym 2020; 2020 Oct 8-9; Jahorina, Republika Srpska, Bosnia and Herzegovina (2020):152,
https://hdl.handle.net/21.15107/rcub_ibiss_5438 .

TY  - JOUR
AU  - Bogdanović, Milica
AU  - Cankar, Katarina
AU  - Dragićević, Milan
AU  - Bouwmeester, Harro
AU  - Beekwilder, Jules
AU  - Simonović, Ana
AU  - Todorović, Slađana
PY  - 2020
UR  - https://www.tandfonline.com/doi/full/10.1080/21645698.2019.1681868
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/3520
AB  - Chicory (Cichorium intybus L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin - a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms - GAS long (encoded by CiGASlo) and GAS short (encoded by CiGASsh). AmiRNA silencing was used to obtain plants with reduced GAS gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.
T2  - GM Crops & Food
T1  - Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.
IS  - 1
VL  - 11
DO  - 10.1080/21645698.2019.1681868
SP  - 54
EP  - 66
ER  - 

@article{
author = "Bogdanović, Milica and Cankar, Katarina and Dragićević, Milan and Bouwmeester, Harro and Beekwilder, Jules and Simonović, Ana and Todorović, Slađana",
year = "2020",
abstract = "Chicory (Cichorium intybus L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin - a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms - GAS long (encoded by CiGASlo) and GAS short (encoded by CiGASsh). AmiRNA silencing was used to obtain plants with reduced GAS gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.",
journal = "GM Crops & Food",
title = "Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.",
number = "1",
volume = "11",
doi = "10.1080/21645698.2019.1681868",
pages = "54-66"
}

Bogdanović, M., Cankar, K., Dragićević, M., Bouwmeester, H., Beekwilder, J., Simonović, A.,& Todorović, S.. (2020). Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.. in GM Crops & Food, 11(1), 54-66.
https://doi.org/10.1080/21645698.2019.1681868

Bogdanović M, Cankar K, Dragićević M, Bouwmeester H, Beekwilder J, Simonović A, Todorović S. Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.. in GM Crops & Food. 2020;11(1):54-66.
doi:10.1080/21645698.2019.1681868 .

Bogdanović, Milica, Cankar, Katarina, Dragićević, Milan, Bouwmeester, Harro, Beekwilder, Jules, Simonović, Ana, Todorović, Slađana, "Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L." in GM Crops & Food, 11, no. 1 (2020):54-66,
https://doi.org/10.1080/21645698.2019.1681868 . .

TY  - JOUR
AU  - Bogdanović, Milica
AU  - Cankar, Katarina
AU  - Todorović, Slađana
AU  - Dragićević, Milan
AU  - Simonović, Ana
AU  - van Houwelingen, Adele
AU  - Schijlen, Elio
AU  - Schipper, Bert
AU  - Gagneul, David
AU  - Hendriks, Theo
AU  - Quillet, Marie-Christine
AU  - Bouwmeester, Harro
AU  - Bosch, Dirk
AU  - Beekwilder, Jules
PY  - 2019
UR  - https://www.sciencedirect.com/science/article/pii/S092666901831063X?via%3Dihub
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/3212
AB  - Chicory (Cichorium intybus L.) produces bitter sesquiterpene lactones (STLs). Some enzymes in the biosynthetic pathway towards these compounds have been characterized. However, the genomic organization and tissue specificity of their biosynthesis is largely unknown. Concentrations of two sesquiterpene lactones and expression of genes involved in the first dedicated biosynthetic step were measured in different chicory tissues. BAC clones containing different genes encoding germacrene A synthase were sequenced, and revealed several tightly linked paralogs. Promoters of genes encoding two germacrene A synthases were fused to GFP and expressed in plants regenerated from transformed chicory hairy root cultures. Highest expression was observed in the epidermis of leaves and external root tissue. This work opens the possibility to select for chicory germplasm diversified in STL content, and to study their role in chicory in defence and physiology.
T2  - Industrial Crops and Products
T1  - Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L. (Asteraceae)
VL  - 129
DO  - 10.1016/J.INDCROP.2018.12.011
SP  - 253
EP  - 260
ER  - 

@article{
author = "Bogdanović, Milica and Cankar, Katarina and Todorović, Slađana and Dragićević, Milan and Simonović, Ana and van Houwelingen, Adele and Schijlen, Elio and Schipper, Bert and Gagneul, David and Hendriks, Theo and Quillet, Marie-Christine and Bouwmeester, Harro and Bosch, Dirk and Beekwilder, Jules",
year = "2019",
abstract = "Chicory (Cichorium intybus L.) produces bitter sesquiterpene lactones (STLs). Some enzymes in the biosynthetic pathway towards these compounds have been characterized. However, the genomic organization and tissue specificity of their biosynthesis is largely unknown. Concentrations of two sesquiterpene lactones and expression of genes involved in the first dedicated biosynthetic step were measured in different chicory tissues. BAC clones containing different genes encoding germacrene A synthase were sequenced, and revealed several tightly linked paralogs. Promoters of genes encoding two germacrene A synthases were fused to GFP and expressed in plants regenerated from transformed chicory hairy root cultures. Highest expression was observed in the epidermis of leaves and external root tissue. This work opens the possibility to select for chicory germplasm diversified in STL content, and to study their role in chicory in defence and physiology.",
journal = "Industrial Crops and Products",
title = "Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L. (Asteraceae)",
volume = "129",
doi = "10.1016/J.INDCROP.2018.12.011",
pages = "253-260"
}

Bogdanović, M., Cankar, K., Todorović, S., Dragićević, M., Simonović, A., van Houwelingen, A., Schijlen, E., Schipper, B., Gagneul, D., Hendriks, T., Quillet, M., Bouwmeester, H., Bosch, D.,& Beekwilder, J.. (2019). Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L. (Asteraceae). in Industrial Crops and Products, 129, 253-260.
https://doi.org/10.1016/J.INDCROP.2018.12.011

Bogdanović M, Cankar K, Todorović S, Dragićević M, Simonović A, van Houwelingen A, Schijlen E, Schipper B, Gagneul D, Hendriks T, Quillet M, Bouwmeester H, Bosch D, Beekwilder J. Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L. (Asteraceae). in Industrial Crops and Products. 2019;129:253-260.
doi:10.1016/J.INDCROP.2018.12.011 .

Bogdanović, Milica, Cankar, Katarina, Todorović, Slađana, Dragićević, Milan, Simonović, Ana, van Houwelingen, Adele, Schijlen, Elio, Schipper, Bert, Gagneul, David, Hendriks, Theo, Quillet, Marie-Christine, Bouwmeester, Harro, Bosch, Dirk, Beekwilder, Jules, "Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L. (Asteraceae)" in Industrial Crops and Products, 129 (2019):253-260,
https://doi.org/10.1016/J.INDCROP.2018.12.011 . .

TY  - CONF
AU  - Cankar, Katarina
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Dragićević, Milan
AU  - Simonović, Ana
AU  - van Houwelingen, Adele
AU  - Schijlen, Elio
AU  - van de Geest, Henri
AU  - Hendriks, Theo
AU  - Quillet, MC
AU  - Bouwmeester, Harro
AU  - Bosch, Dirk
AU  - Beekwilder, Jules
PY  - 2016
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5441
AB  - Sesquiterpene lactones (STLs) give chicory its characteristic bitter taste and are highly concentrated in the latex channels of chicory taproot. The study of STLs is interesting to control the bitterness of industrial and consumption chicory, including witloof. The major STLs in chicory are lactucin, deoxylactucin, and lactucopicrin and oxalate and sulphate conjugates thereof. Formation of chicory STLs requires the biosynthesis of costunolide. Two germacrene A synthases, two germacrene A oxidases and a costunolide synthase have been described from chicory. In this work, the genetic organization of the genes involved in STL biosynthesis in chicory linkage groups is described. A number of genomic regions hosting STL biosynthetic genes have been addressed by NGS sequencing. Gene expression in different tissues is addressed by following their expression
using an RNAseq approach and by promoter activity studies using GFP. Finally, the downregulation of STL gene expression in chicory is achieved in hairy root culture regenerants expressing amiRNAs, and the consequences for STL content in different tissues is tested. These results will contribute to successful breeding strategies for chicory germplasm with well-defined STL content and bitterness.
PB  - Ghent : Phytochemical Society of Europe
C3  - Book of abstracts: Plant omics and biotechnology for human health: POBHH; 2016 Nov 21-24; Gent, Belgium
T1  - Tissue specific expression and genomic organization of sesquiterpene lactone biosynthetic genes in chicory
SP  - 65
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5441
ER  - 

@conference{
author = "Cankar, Katarina and Bogdanović, Milica and Todorović, Slađana and Dragićević, Milan and Simonović, Ana and van Houwelingen, Adele and Schijlen, Elio and van de Geest, Henri and Hendriks, Theo and Quillet, MC and Bouwmeester, Harro and Bosch, Dirk and Beekwilder, Jules",
year = "2016",
abstract = "Sesquiterpene lactones (STLs) give chicory its characteristic bitter taste and are highly concentrated in the latex channels of chicory taproot. The study of STLs is interesting to control the bitterness of industrial and consumption chicory, including witloof. The major STLs in chicory are lactucin, deoxylactucin, and lactucopicrin and oxalate and sulphate conjugates thereof. Formation of chicory STLs requires the biosynthesis of costunolide. Two germacrene A synthases, two germacrene A oxidases and a costunolide synthase have been described from chicory. In this work, the genetic organization of the genes involved in STL biosynthesis in chicory linkage groups is described. A number of genomic regions hosting STL biosynthetic genes have been addressed by NGS sequencing. Gene expression in different tissues is addressed by following their expression
using an RNAseq approach and by promoter activity studies using GFP. Finally, the downregulation of STL gene expression in chicory is achieved in hairy root culture regenerants expressing amiRNAs, and the consequences for STL content in different tissues is tested. These results will contribute to successful breeding strategies for chicory germplasm with well-defined STL content and bitterness.",
publisher = "Ghent : Phytochemical Society of Europe",
journal = "Book of abstracts: Plant omics and biotechnology for human health: POBHH; 2016 Nov 21-24; Gent, Belgium",
title = "Tissue specific expression and genomic organization of sesquiterpene lactone biosynthetic genes in chicory",
pages = "65",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5441"
}

Cankar, K., Bogdanović, M., Todorović, S., Dragićević, M., Simonović, A., van Houwelingen, A., Schijlen, E., van de Geest, H., Hendriks, T., Quillet, M., Bouwmeester, H., Bosch, D.,& Beekwilder, J.. (2016). Tissue specific expression and genomic organization of sesquiterpene lactone biosynthetic genes in chicory. in Book of abstracts: Plant omics and biotechnology for human health: POBHH; 2016 Nov 21-24; Gent, Belgium
Ghent : Phytochemical Society of Europe., 65.
https://hdl.handle.net/21.15107/rcub_ibiss_5441

Cankar K, Bogdanović M, Todorović S, Dragićević M, Simonović A, van Houwelingen A, Schijlen E, van de Geest H, Hendriks T, Quillet M, Bouwmeester H, Bosch D, Beekwilder J. Tissue specific expression and genomic organization of sesquiterpene lactone biosynthetic genes in chicory. in Book of abstracts: Plant omics and biotechnology for human health: POBHH; 2016 Nov 21-24; Gent, Belgium. 2016;:65.
https://hdl.handle.net/21.15107/rcub_ibiss_5441 .

Cankar, Katarina, Bogdanović, Milica, Todorović, Slađana, Dragićević, Milan, Simonović, Ana, van Houwelingen, Adele, Schijlen, Elio, van de Geest, Henri, Hendriks, Theo, Quillet, MC, Bouwmeester, Harro, Bosch, Dirk, Beekwilder, Jules, "Tissue specific expression and genomic organization of sesquiterpene lactone biosynthetic genes in chicory" in Book of abstracts: Plant omics and biotechnology for human health: POBHH; 2016 Nov 21-24; Gent, Belgium (2016):65,
https://hdl.handle.net/21.15107/rcub_ibiss_5441 .

TY  - CONF
AU  - Cankar, Katarina
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Dragićević, Milan
AU  - Simonović, Ana
AU  - van Houwelingen, Adele
AU  - Schijlen, Elio
AU  - van de Geest, Henri
AU  - Hendriks, Theo
AU  - Quillet, Marie-Christine
AU  - Bouwmeester, Harro
AU  - Bosch, Dirk
AU  - Beekwilder, Jules
PY  - 2015
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6164
PB  - Vancouver: University of British Columbia
C3  - 12th International Meeting on Biosynthesis, Function and Synthetic Biology of Isoprenoids: TERPNET 2015; 2015 Jun 1-5; Vancouver, Canada
T1  - Genomic organization and expression of sesquiterpene lactone biosynthetic genes in chicory
SP  - 102
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6164
ER  - 

@conference{
author = "Cankar, Katarina and Bogdanović, Milica and Todorović, Slađana and Dragićević, Milan and Simonović, Ana and van Houwelingen, Adele and Schijlen, Elio and van de Geest, Henri and Hendriks, Theo and Quillet, Marie-Christine and Bouwmeester, Harro and Bosch, Dirk and Beekwilder, Jules",
year = "2015",
publisher = "Vancouver: University of British Columbia",
journal = "12th International Meeting on Biosynthesis, Function and Synthetic Biology of Isoprenoids: TERPNET 2015; 2015 Jun 1-5; Vancouver, Canada",
title = "Genomic organization and expression of sesquiterpene lactone biosynthetic genes in chicory",
pages = "102",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6164"
}

Cankar, K., Bogdanović, M., Todorović, S., Dragićević, M., Simonović, A., van Houwelingen, A., Schijlen, E., van de Geest, H., Hendriks, T., Quillet, M., Bouwmeester, H., Bosch, D.,& Beekwilder, J.. (2015). Genomic organization and expression of sesquiterpene lactone biosynthetic genes in chicory. in 12th International Meeting on Biosynthesis, Function and Synthetic Biology of Isoprenoids: TERPNET 2015; 2015 Jun 1-5; Vancouver, Canada
Vancouver: University of British Columbia., 102.
https://hdl.handle.net/21.15107/rcub_ibiss_6164

Cankar K, Bogdanović M, Todorović S, Dragićević M, Simonović A, van Houwelingen A, Schijlen E, van de Geest H, Hendriks T, Quillet M, Bouwmeester H, Bosch D, Beekwilder J. Genomic organization and expression of sesquiterpene lactone biosynthetic genes in chicory. in 12th International Meeting on Biosynthesis, Function and Synthetic Biology of Isoprenoids: TERPNET 2015; 2015 Jun 1-5; Vancouver, Canada. 2015;:102.
https://hdl.handle.net/21.15107/rcub_ibiss_6164 .

Cankar, Katarina, Bogdanović, Milica, Todorović, Slađana, Dragićević, Milan, Simonović, Ana, van Houwelingen, Adele, Schijlen, Elio, van de Geest, Henri, Hendriks, Theo, Quillet, Marie-Christine, Bouwmeester, Harro, Bosch, Dirk, Beekwilder, Jules, "Genomic organization and expression of sesquiterpene lactone biosynthetic genes in chicory" in 12th International Meeting on Biosynthesis, Function and Synthetic Biology of Isoprenoids: TERPNET 2015; 2015 Jun 1-5; Vancouver, Canada (2015):102,
https://hdl.handle.net/21.15107/rcub_ibiss_6164 .

TY  - CONF
AU  - Dragićević, Milan
AU  - Bogdanović, Milica
AU  - Cankar, Katarina
AU  - Beekwilder, Jules
AU  - Bouwmeester, Harro
AU  - Todorović, Slađana
AU  - Simonović, Ana
PY  - 2015
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6160
AB  - Germacrene A synthase (GAS) catalyzes an important step in guaianolide biosynthesis converting farnesyl pyrophosphate to germacrene A – universal sesquiterpene precursor in chicory. The function of two genes coding for GAS – CiGASlo and CiGASsh, was studied by gene silencing. Chicory was transformed with amiRNA constructs designed to specifically silence either of the two genes, and regenerated transgenic plants were tested for changes in gene expression and sesquiterpene lactones content. Silencing was successful in most of the clones, which had reduced gene expression. However, the amiRNA constructs were not specific for CiGASlo or CiGASsh, so that both genes were silenced in most transgenic plants. To verify that the gene silencing had impact on sesquiterpene lactone levels, three representative guaianolide oxalates (lactucin-15-oxalate, deoxilactucin-15-oxalate and lactucopicrin-15-oxalate) were identified and quantified by UPLC-MS/MS in roots and shoots of transgenic and control plants. Data analyzes showed unequal segregation of targeted compounds in the plant tissues, and implied that the synthesis of these compounds highly depends on the expression of GAS genes in the shoot and that these metabolites are pooled in the shoot, while the root guaianolide oxalates content is regulated on a level different from GAS expression. Nevertheless gene silencing reduced the targeted metabolites levels significantly. It was concluded that both genes are involved in guaianolide biosynthesis, and that their complex pattern of expression in different parts of the plant determines the amount of guaianolides and their derivatives.
PB  - Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”
C3  - Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia
T1  - Study of chicory germacrene-A synthase function in guaianolide biosynthesis through amiRNA-induced gene silencing
SP  - 20
EP  - 20
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6160
ER  - 

@conference{
author = "Dragićević, Milan and Bogdanović, Milica and Cankar, Katarina and Beekwilder, Jules and Bouwmeester, Harro and Todorović, Slađana and Simonović, Ana",
year = "2015",
abstract = "Germacrene A synthase (GAS) catalyzes an important step in guaianolide biosynthesis converting farnesyl pyrophosphate to germacrene A – universal sesquiterpene precursor in chicory. The function of two genes coding for GAS – CiGASlo and CiGASsh, was studied by gene silencing. Chicory was transformed with amiRNA constructs designed to specifically silence either of the two genes, and regenerated transgenic plants were tested for changes in gene expression and sesquiterpene lactones content. Silencing was successful in most of the clones, which had reduced gene expression. However, the amiRNA constructs were not specific for CiGASlo or CiGASsh, so that both genes were silenced in most transgenic plants. To verify that the gene silencing had impact on sesquiterpene lactone levels, three representative guaianolide oxalates (lactucin-15-oxalate, deoxilactucin-15-oxalate and lactucopicrin-15-oxalate) were identified and quantified by UPLC-MS/MS in roots and shoots of transgenic and control plants. Data analyzes showed unequal segregation of targeted compounds in the plant tissues, and implied that the synthesis of these compounds highly depends on the expression of GAS genes in the shoot and that these metabolites are pooled in the shoot, while the root guaianolide oxalates content is regulated on a level different from GAS expression. Nevertheless gene silencing reduced the targeted metabolites levels significantly. It was concluded that both genes are involved in guaianolide biosynthesis, and that their complex pattern of expression in different parts of the plant determines the amount of guaianolides and their derivatives.",
publisher = "Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”",
journal = "Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia",
title = "Study of chicory germacrene-A synthase function in guaianolide biosynthesis through amiRNA-induced gene silencing",
pages = "20-20",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6160"
}

Dragićević, M., Bogdanović, M., Cankar, K., Beekwilder, J., Bouwmeester, H., Todorović, S.,& Simonović, A.. (2015). Study of chicory germacrene-A synthase function in guaianolide biosynthesis through amiRNA-induced gene silencing. in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia
Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”., 20-20.
https://hdl.handle.net/21.15107/rcub_ibiss_6160

Dragićević M, Bogdanović M, Cankar K, Beekwilder J, Bouwmeester H, Todorović S, Simonović A. Study of chicory germacrene-A synthase function in guaianolide biosynthesis through amiRNA-induced gene silencing. in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia. 2015;:20-20.
https://hdl.handle.net/21.15107/rcub_ibiss_6160 .

Dragićević, Milan, Bogdanović, Milica, Cankar, Katarina, Beekwilder, Jules, Bouwmeester, Harro, Todorović, Slađana, Simonović, Ana, "Study of chicory germacrene-A synthase function in guaianolide biosynthesis through amiRNA-induced gene silencing" in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia (2015):20-20,
https://hdl.handle.net/21.15107/rcub_ibiss_6160 .

TY  - CONF
AU  - Bogdanović, Milica
AU  - Dragićević, Milan
AU  - Cankar, Katarina
AU  - Beekwilder, Jules
AU  - Bouwmeester, Harro
AU  - Subotić, Angelina
AU  - Simonović, Ana
AU  - Todorović, Slađana
PY  - 2015
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6159
AB  - Chicory (Cichorium intybus L.) is rich in sesquiterpene lactones, bitter compounds whose biosynthesis involves several recently characterized genes, namely germacrene A synthase (GAS), in chicory present as long and short form, and germacrene A oxidase (GAO, CYP71AV8). Promoters of these genes have been studied by cloning promoter regions to drive the expression of eGFP, and obtaining stable chicory transformants carrying promoter constructs. Due to incompatibility of eGFP with another fluorescent marker – DsRED, which was used for selection of transformed clones, promoter activity was detected in transgenic plants by RT-PCR and qRT-PCR techniques. Most of the obtained chicory clones were expressing GFP, in roots, leaves, stems and flowers, suggesting that cloned promoters were functional in different chicory organs. The promoters were characterized by different strength - GAO and GAS long promoters were stronger than both GAS short promoters, judging by eGFP expression level. The promoters also showed partial tissue specificity – GAS long was active in roots, leaves, stems and flowers, while GAS short promoters were mainly active in chicory roots.
PB  - Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”
C3  - Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia
T1  - Germacrene A synthase and oxidase promoter analysis in chicory
SP  - 19
EP  - 19
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6159
ER  - 

@conference{
author = "Bogdanović, Milica and Dragićević, Milan and Cankar, Katarina and Beekwilder, Jules and Bouwmeester, Harro and Subotić, Angelina and Simonović, Ana and Todorović, Slađana",
year = "2015",
abstract = "Chicory (Cichorium intybus L.) is rich in sesquiterpene lactones, bitter compounds whose biosynthesis involves several recently characterized genes, namely germacrene A synthase (GAS), in chicory present as long and short form, and germacrene A oxidase (GAO, CYP71AV8). Promoters of these genes have been studied by cloning promoter regions to drive the expression of eGFP, and obtaining stable chicory transformants carrying promoter constructs. Due to incompatibility of eGFP with another fluorescent marker – DsRED, which was used for selection of transformed clones, promoter activity was detected in transgenic plants by RT-PCR and qRT-PCR techniques. Most of the obtained chicory clones were expressing GFP, in roots, leaves, stems and flowers, suggesting that cloned promoters were functional in different chicory organs. The promoters were characterized by different strength - GAO and GAS long promoters were stronger than both GAS short promoters, judging by eGFP expression level. The promoters also showed partial tissue specificity – GAS long was active in roots, leaves, stems and flowers, while GAS short promoters were mainly active in chicory roots.",
publisher = "Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”",
journal = "Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia",
title = "Germacrene A synthase and oxidase promoter analysis in chicory",
pages = "19-19",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6159"
}

Bogdanović, M., Dragićević, M., Cankar, K., Beekwilder, J., Bouwmeester, H., Subotić, A., Simonović, A.,& Todorović, S.. (2015). Germacrene A synthase and oxidase promoter analysis in chicory. in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia
Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”., 19-19.
https://hdl.handle.net/21.15107/rcub_ibiss_6159

Bogdanović M, Dragićević M, Cankar K, Beekwilder J, Bouwmeester H, Subotić A, Simonović A, Todorović S. Germacrene A synthase and oxidase promoter analysis in chicory. in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia. 2015;:19-19.
https://hdl.handle.net/21.15107/rcub_ibiss_6159 .

Bogdanović, Milica, Dragićević, Milan, Cankar, Katarina, Beekwilder, Jules, Bouwmeester, Harro, Subotić, Angelina, Simonović, Ana, Todorović, Slađana, "Germacrene A synthase and oxidase promoter analysis in chicory" in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia (2015):19-19,
https://hdl.handle.net/21.15107/rcub_ibiss_6159 .

TY  - CONF
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Subotić, Angelina
AU  - Dragićević, Milan
AU  - Cankar, Katarina
AU  - Beekwilder, Jules
AU  - Simonović, Ana
PY  - 2015
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6158
AB  - Reporter genes are indispensible tools in plant biotechnology, used to assess transformation efficiency, select transgenic tissues, study promoters, etc. Advantages of fluorescent markers include non-destructive visualization, simplicity of use, non-toxicity, and stability in most cellular contexts. GFP and DsRED are widely used fluorescent reporters and are considered compatible. Unlike animal systems, plant tissues are notoriously known for autofluorescence, spanning most of the visible spectrum. This limits the application of fluorescent proteins in plants and requires special equipment and a set of control samples, to discern autofluorescence from transgene expression. In the course of study of guaianolide biosynthesis in chicory, several vectors were constructed containing DsRED as co-transformation reporter and GFP as a reporter fused to promoters of the studied genes. To observe DsRED and GFP fluorescence in transgenic chicory tissues, we have used: a system for macro visualization consisting of LED light sources and emission filters, fluorescent stereobinocular, fluorescent microscope and confocal microscope. These methods revealed strong green autofluorescence in newly formed roots, calli, lignified cell walls and parenchyma cells. The intensity of autofluorescence was especially high at large magnification and could obstruct GFP visualization. DsRED fluorescence, on the other hand, was easily discerned from any kind of plant autofluorescence. Another problem was signal crossover from DsRED into GFP channel. DsRED has a small emission peak in the green part of the spectrum, originating from its chromophore maturation. Thus it was impossible to separate fluorescent emission of these two markers present in the same tissue without using spectral deconvolution techniques.
PB  - Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”
C3  - Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia
T1  - Problems in detecting activity of fluorescent reporter genes – case of DsRED and GFP
SP  - 16
EP  - 16
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6158
ER  - 

@conference{
author = "Bogdanović, Milica and Todorović, Slađana and Subotić, Angelina and Dragićević, Milan and Cankar, Katarina and Beekwilder, Jules and Simonović, Ana",
year = "2015",
abstract = "Reporter genes are indispensible tools in plant biotechnology, used to assess transformation efficiency, select transgenic tissues, study promoters, etc. Advantages of fluorescent markers include non-destructive visualization, simplicity of use, non-toxicity, and stability in most cellular contexts. GFP and DsRED are widely used fluorescent reporters and are considered compatible. Unlike animal systems, plant tissues are notoriously known for autofluorescence, spanning most of the visible spectrum. This limits the application of fluorescent proteins in plants and requires special equipment and a set of control samples, to discern autofluorescence from transgene expression. In the course of study of guaianolide biosynthesis in chicory, several vectors were constructed containing DsRED as co-transformation reporter and GFP as a reporter fused to promoters of the studied genes. To observe DsRED and GFP fluorescence in transgenic chicory tissues, we have used: a system for macro visualization consisting of LED light sources and emission filters, fluorescent stereobinocular, fluorescent microscope and confocal microscope. These methods revealed strong green autofluorescence in newly formed roots, calli, lignified cell walls and parenchyma cells. The intensity of autofluorescence was especially high at large magnification and could obstruct GFP visualization. DsRED fluorescence, on the other hand, was easily discerned from any kind of plant autofluorescence. Another problem was signal crossover from DsRED into GFP channel. DsRED has a small emission peak in the green part of the spectrum, originating from its chromophore maturation. Thus it was impossible to separate fluorescent emission of these two markers present in the same tissue without using spectral deconvolution techniques.",
publisher = "Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”",
journal = "Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia",
title = "Problems in detecting activity of fluorescent reporter genes – case of DsRED and GFP",
pages = "16-16",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6158"
}

Bogdanović, M., Todorović, S., Subotić, A., Dragićević, M., Cankar, K., Beekwilder, J.,& Simonović, A.. (2015). Problems in detecting activity of fluorescent reporter genes – case of DsRED and GFP. in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia
Belgrade: Serbian Plant Physiology Society: Institute for Biological Research “Siniša Stanković”., 16-16.
https://hdl.handle.net/21.15107/rcub_ibiss_6158

Bogdanović M, Todorović S, Subotić A, Dragićević M, Cankar K, Beekwilder J, Simonović A. Problems in detecting activity of fluorescent reporter genes – case of DsRED and GFP. in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia. 2015;:16-16.
https://hdl.handle.net/21.15107/rcub_ibiss_6158 .

Bogdanović, Milica, Todorović, Slađana, Subotić, Angelina, Dragićević, Milan, Cankar, Katarina, Beekwilder, Jules, Simonović, Ana, "Problems in detecting activity of fluorescent reporter genes – case of DsRED and GFP" in Book of Abstracts: 2nd International Conference on Plant Biology, 21th Symposium of the Serbian Plant Physiology Society, and CОST Action FA1106 Quality Fruit Workshop; 2015 Jun 17-20; Petnica, Serbia (2015):16-16,
https://hdl.handle.net/21.15107/rcub_ibiss_6158 .

TY  - CONF
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Dragićević, Milan
AU  - Cankar, Katarina
AU  - Beekwilder, Jules
AU  - Bouwmeester, Harro
AU  - Simonović, Ana
PY  - 2013
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6228
AB  - Chicory (Cichorium intybus L.) is rich in sesquiterpene lactones, compounds known for their bitter taste and medicinal properties. Most enzymes involved in the biosynthetic pathway of these secondary metabolites have recently been discovered and characterized. The first step in their biosynthesis is catalyzed by germacrene A synthase (GAS), in chicory present in two forms - long and short, and several P450 mono-oxygenases. So far, promoters of these genes have not been studied, and little is known about the spatial and temporal regulation of their expression. To address this issue, four vectors for plant transformation containing promoter-reporter gene fusions were designed and constructed by Gateway cloning, including one for GAS long, two for GAS short, and one for the cytochrome P450, germacrene A oxidase. As a marker for co-transformation, DsRED, a red fluorescent protein, was used, while the studied promoters were inserted to drive GFP/GUS fusion, to allow for visualization of promoter activity. Integrity and function of the constructs were checked by agroinfiltration in lettuce (Lactuca sativa Cv. Olof) - a transient transformation assay. Infiltration was performed with Agrobacterium tumefaciens, carrying the promoter constructs. Transformation success was checked five days after infiltration by fluorescent stereomicroscopy, and both DsRED and GFP were detected, indicating that the chicory promoters were active in lettuce. DsRED had strong and uniform fluorescence in all samples, but GFP fluorescence varied among plants infiltrated with different constructs. The GAS long promoter had strongest expression, followed by the P450 and the two rather weak GAS short promoters. The fluorescence was visible only in the infiltrated parts of the leaves, in tissues between leaf veins, but not in the veins themselves. Both abaxial and adaxial leaf sides were fluorescing. There were no differences observed between spatial distribution of DsRED and GFP: all infiltrated parts showed both markers. Since these vectors were confirmed to be functional, stable transformants of chicory will be generated by transformation using A. rhizogenes carrying the same promoter constructs.
PB  - Serbian Plant Physiology Society, Institute for Biological Research "Siniša Stanković", University of Belgrade
PB  - Belgrade: Serbian Plant Physiology Society
C3  - Programme and Abstracts: 1st International Conference on Plant Biology and 20th Symposium of the Serbian Plant Physiology Society; 2013 Jun 4-7; Subotica, Belgrade
T1  - Vector construction for promoter analyses in chicory and fluorescence evaluation by agroinfiltration
SP  - 60
EP  - 61
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6228
ER  - 

@conference{
author = "Bogdanović, Milica and Todorović, Slađana and Dragićević, Milan and Cankar, Katarina and Beekwilder, Jules and Bouwmeester, Harro and Simonović, Ana",
year = "2013",
abstract = "Chicory (Cichorium intybus L.) is rich in sesquiterpene lactones, compounds known for their bitter taste and medicinal properties. Most enzymes involved in the biosynthetic pathway of these secondary metabolites have recently been discovered and characterized. The first step in their biosynthesis is catalyzed by germacrene A synthase (GAS), in chicory present in two forms - long and short, and several P450 mono-oxygenases. So far, promoters of these genes have not been studied, and little is known about the spatial and temporal regulation of their expression. To address this issue, four vectors for plant transformation containing promoter-reporter gene fusions were designed and constructed by Gateway cloning, including one for GAS long, two for GAS short, and one for the cytochrome P450, germacrene A oxidase. As a marker for co-transformation, DsRED, a red fluorescent protein, was used, while the studied promoters were inserted to drive GFP/GUS fusion, to allow for visualization of promoter activity. Integrity and function of the constructs were checked by agroinfiltration in lettuce (Lactuca sativa Cv. Olof) - a transient transformation assay. Infiltration was performed with Agrobacterium tumefaciens, carrying the promoter constructs. Transformation success was checked five days after infiltration by fluorescent stereomicroscopy, and both DsRED and GFP were detected, indicating that the chicory promoters were active in lettuce. DsRED had strong and uniform fluorescence in all samples, but GFP fluorescence varied among plants infiltrated with different constructs. The GAS long promoter had strongest expression, followed by the P450 and the two rather weak GAS short promoters. The fluorescence was visible only in the infiltrated parts of the leaves, in tissues between leaf veins, but not in the veins themselves. Both abaxial and adaxial leaf sides were fluorescing. There were no differences observed between spatial distribution of DsRED and GFP: all infiltrated parts showed both markers. Since these vectors were confirmed to be functional, stable transformants of chicory will be generated by transformation using A. rhizogenes carrying the same promoter constructs.",
publisher = "Serbian Plant Physiology Society, Institute for Biological Research "Siniša Stanković", University of Belgrade, Belgrade: Serbian Plant Physiology Society",
journal = "Programme and Abstracts: 1st International Conference on Plant Biology and 20th Symposium of the Serbian Plant Physiology Society; 2013 Jun 4-7; Subotica, Belgrade",
title = "Vector construction for promoter analyses in chicory and fluorescence evaluation by agroinfiltration",
pages = "60-61",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6228"
}

Bogdanović, M., Todorović, S., Dragićević, M., Cankar, K., Beekwilder, J., Bouwmeester, H.,& Simonović, A.. (2013). Vector construction for promoter analyses in chicory and fluorescence evaluation by agroinfiltration. in Programme and Abstracts: 1st International Conference on Plant Biology and 20th Symposium of the Serbian Plant Physiology Society; 2013 Jun 4-7; Subotica, Belgrade
Serbian Plant Physiology Society, Institute for Biological Research "Siniša Stanković", University of Belgrade., 60-61.
https://hdl.handle.net/21.15107/rcub_ibiss_6228

Bogdanović M, Todorović S, Dragićević M, Cankar K, Beekwilder J, Bouwmeester H, Simonović A. Vector construction for promoter analyses in chicory and fluorescence evaluation by agroinfiltration. in Programme and Abstracts: 1st International Conference on Plant Biology and 20th Symposium of the Serbian Plant Physiology Society; 2013 Jun 4-7; Subotica, Belgrade. 2013;:60-61.
https://hdl.handle.net/21.15107/rcub_ibiss_6228 .

Bogdanović, Milica, Todorović, Slađana, Dragićević, Milan, Cankar, Katarina, Beekwilder, Jules, Bouwmeester, Harro, Simonović, Ana, "Vector construction for promoter analyses in chicory and fluorescence evaluation by agroinfiltration" in Programme and Abstracts: 1st International Conference on Plant Biology and 20th Symposium of the Serbian Plant Physiology Society; 2013 Jun 4-7; Subotica, Belgrade (2013):60-61,
https://hdl.handle.net/21.15107/rcub_ibiss_6228 .

TY  - JOUR
AU  - Majdi, Mohammad
AU  - Liu, Qing
AU  - Karimzadeh, Ghasem
AU  - Malboobi, Mohammad Ali
AU  - Beekwilder, Jules
AU  - Cankar, Katarina
AU  - de Vos, Ric
AU  - Todorović, Slađana
AU  - Simonović, Ana
AU  - Bouwmeester, Harro
PY  - 2011
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/1257
AB  - Feverfew (Tanacetum parthenium) is a perennial medicinal herb and is a rich source of sesquiterpene lactones. Parthenolide is the main sesquiterpene lactone in feverfew and has attracted attention because of its medicinal potential for treatment of migraine and cancer. In the present work the parthenolide content in different tissues and developmental stages of feverfew was analyzed to study the timing and localization of parthenolide biosynthesis. The strongest accumulating tissue was subsequently used to isolate sesquiterpene synthases with the goal to isolate the gene encoding the first dedicated step in parthenolide biosynthesis. This led to the isolation and charachterization of a germacrene A synthase (TpGAS) and an (E)-beta-caryophyllene synthase (TpCarS). Transcript level patterns of both sesquiterpene synthases were analyzed in different tissues and glandular trichomes. Although TpGAS was expressed in all aerial tissues, the highest expression was observed in tissues that contain high concentrations of parthenolide and in flowers the highest expression was observed in the biosynthetically most active stages of flower development. The high expression of TpGAS in glandular trichomes which also contain the highest concentration of parthenolide, suggests that glandular trichomes are the secretory tissues where parthenolide biosynthesis and accumulation occur. (C) 2011 Elsevier Ltd. All rights reserved.
T2  - Phytochemistry
T1  - Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.)
IS  - 14-15
VL  - 72
EP  - 1750
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_1257
ER  - 

@article{
author = "Majdi, Mohammad and Liu, Qing and Karimzadeh, Ghasem and Malboobi, Mohammad Ali and Beekwilder, Jules and Cankar, Katarina and de Vos, Ric and Todorović, Slađana and Simonović, Ana and Bouwmeester, Harro",
year = "2011",
abstract = "Feverfew (Tanacetum parthenium) is a perennial medicinal herb and is a rich source of sesquiterpene lactones. Parthenolide is the main sesquiterpene lactone in feverfew and has attracted attention because of its medicinal potential for treatment of migraine and cancer. In the present work the parthenolide content in different tissues and developmental stages of feverfew was analyzed to study the timing and localization of parthenolide biosynthesis. The strongest accumulating tissue was subsequently used to isolate sesquiterpene synthases with the goal to isolate the gene encoding the first dedicated step in parthenolide biosynthesis. This led to the isolation and charachterization of a germacrene A synthase (TpGAS) and an (E)-beta-caryophyllene synthase (TpCarS). Transcript level patterns of both sesquiterpene synthases were analyzed in different tissues and glandular trichomes. Although TpGAS was expressed in all aerial tissues, the highest expression was observed in tissues that contain high concentrations of parthenolide and in flowers the highest expression was observed in the biosynthetically most active stages of flower development. The high expression of TpGAS in glandular trichomes which also contain the highest concentration of parthenolide, suggests that glandular trichomes are the secretory tissues where parthenolide biosynthesis and accumulation occur. (C) 2011 Elsevier Ltd. All rights reserved.",
journal = "Phytochemistry",
title = "Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.)",
number = "14-15",
volume = "72",
pages = "1750",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_1257"
}

Majdi, M., Liu, Q., Karimzadeh, G., Malboobi, M. A., Beekwilder, J., Cankar, K., de Vos, R., Todorović, S., Simonović, A.,& Bouwmeester, H.. (2011). Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.). in Phytochemistry, 72(14-15).
https://hdl.handle.net/21.15107/rcub_ibiss_1257

Majdi M, Liu Q, Karimzadeh G, Malboobi MA, Beekwilder J, Cankar K, de Vos R, Todorović S, Simonović A, Bouwmeester H. Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.). in Phytochemistry. 2011;72(14-15):null-1750.
https://hdl.handle.net/21.15107/rcub_ibiss_1257 .

Majdi, Mohammad, Liu, Qing, Karimzadeh, Ghasem, Malboobi, Mohammad Ali, Beekwilder, Jules, Cankar, Katarina, de Vos, Ric, Todorović, Slađana, Simonović, Ana, Bouwmeester, Harro, "Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.)" in Phytochemistry, 72, no. 14-15 (2011),
https://hdl.handle.net/21.15107/rcub_ibiss_1257 .