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, 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  - JOUR
AU  - Brückner, Kathleen
AU  - Matekalo, Dragana
AU  - Manzano, David
AU  - Papaefthimiou, Dimitra
AU  - Pateraki, Irini
AU  - Scheler, Ulschan
AU  - Ferrer, Albert
AU  - de Vos, Ric C H
AU  - Kanellis, Angelos
AU  - Tissier, Alain
PY  - 2014
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4287
AB  - Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.
PB  - Elsevier
T2  - Phytochemistry
T1  - Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes
VL  - 101
DO  - 10.1016/j.phytochem.2014.01.021
SP  - 52
EP  - 64
ER  - 

@article{
author = "Brückner, Kathleen and Matekalo, Dragana and Manzano, David and Papaefthimiou, Dimitra and Pateraki, Irini and Scheler, Ulschan and Ferrer, Albert and de Vos, Ric C H and Kanellis, Angelos and Tissier, Alain",
year = "2014",
abstract = "Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.",
publisher = "Elsevier",
journal = "Phytochemistry",
title = "Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes",
volume = "101",
doi = "10.1016/j.phytochem.2014.01.021",
pages = "52-64"
}

Brückner, K., Matekalo, D., Manzano, D., Papaefthimiou, D., Pateraki, I., Scheler, U., Ferrer, A., de Vos, R. C. H., Kanellis, A.,& Tissier, A.. (2014). Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes. in Phytochemistry
Elsevier., 101, 52-64.
https://doi.org/10.1016/j.phytochem.2014.01.021

Brückner K, Matekalo D, Manzano D, Papaefthimiou D, Pateraki I, Scheler U, Ferrer A, de Vos RCH, Kanellis A, Tissier A. Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes. in Phytochemistry. 2014;101:52-64.
doi:10.1016/j.phytochem.2014.01.021 .

Brückner, Kathleen, Matekalo, Dragana, Manzano, David, Papaefthimiou, Dimitra, Pateraki, Irini, Scheler, Ulschan, Ferrer, Albert, de Vos, Ric C H, Kanellis, Angelos, Tissier, Alain, "Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes" in Phytochemistry, 101 (2014):52-64,
https://doi.org/10.1016/j.phytochem.2014.01.021 . .

TY  - THES
AU  - Božić, Dragana
PY  - 2014
UR  - http://eteze.bg.ac.rs/application/showtheses?thesesId=2848
UR  - https://fedorabg.bg.ac.rs/fedora/get/o:11054/bdef:Content/download
UR  - http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=1025012658
UR  - http://nardus.mpn.gov.rs/123456789/5284
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/2378
AB  - Karnozinska kiselina, fenolni diterpen, poseduje brojne biološke
aktivnosti zbog čega je potencijalno veoma značajna za farmaceutsku
industriju. Grčka žalfija (Salvia fruticosa Mill.) i ruzmarin (Rosmarinus
officinalis L.) bogati su prirodni izvori ovog jedinjenja. Iako je mnogo toga
poznato u vezi strukture i bioloških aktivnosti karnozinske kiseline,
veoma malo se ne zna o njenom biosintetskom putu u biljkama. Osnovni cilj
disertacije bio je izučavanje biosinteze karnozinske kiseline na
molekularnom nivou, izolacijom i funkcionalnom karakterizacijom gena
uključenih u ovaj proces. U tu svrhu, izvršena je analiza postojeće cDNK
biblioteke žlezdanih dlaka grčke žalfije, što je za rezultat imalo
identifikaciju dva gena, nazvanih SfCPS i SfKSL, koji potencijalno
kodiraju diterpen sintaze. Geni kandidati su izolovani i funkcionalno
okarakterisani u bakteriji Escherichia coli Mig., kvascima (Saccharomyces
cerevisiae Meyen ex E. C. Hansen) i duvanu (Nicotiana benthamiana Domin).
Heterologa ekpresija gena SfCPS i SfKSL rezultirala je sintezom
miltiradiena, što je utvrđeno GC-MS analizom i 1D i 2D NMR
spektroskopijom (1H, 13C, DEPT, COSY H-H, HMQC i HMBC). Za pronalaženje
preostalih gena odgovornih za biosintezu karnozinske kiseline, obavljeno
je sekvencioniranje transkriptoma grčke žalfije i ruzmarina, uz
korišćenje 454 GS FLX Titanium platforme. Na osnovu rezultata
fitohemijske analize LC-PDA-LTQ-Orbitrap FTMS metodom, žlezdane dlake
mladih listova blago stresiranih biljaka genotipa “Kavusi” grčke žalfije
i žlezdane dlake mlađih stadijuma razvoja komercijalnog genotipa
ruzmarina (B & T World Seeds) izabrane su kao biljni materijal za analizu
transkriptoma. Rezultat sekvencioniranja bio je anotacija velikog broja
novih sekvenci, a posebno značajna bila je identifikacija gena
metaboličkih puteva povezanih sa sekundarnim metabolizmom. Izolovano je
ukupno 24 gena koji su kodirali za enzime familije citohrom P450 (CYP)
monooksigenaze. Funkcija enzima CYP76Sf2 grčke žalfije, i CYP76Ro3 i
CYP76Ro4 ruzmarina, kao feruginol sintaza, utvrđena je esejima
funkcionalne karakterizacije u kvascima i duvanu. Kloniranje i
funkcionalna karakterizacija gena koji svojom zajedničkom aktivnošću
dovode do sinteze feruginola kod grčke žalfije i ruzmarina, pruža osnovu
budućim istraživanjima biosintetskog puta karnozinske kiseline. Uz to,
izvršena je lokalizacija biosintetskog puta karnozinske kiseline u
žlezdanim dlakama grčke žalfije i ruzmarina. Priloženi rezultati
doprinose boljem razumevanju metabolizma karnozinske kiseline, i time
omogućavaju primenu najsavremenijih biotehnoloških metoda za proizvodnju
ovog farmaceutski značajnog jedinjenja.
AB  - Carnosic acid is a phenolic diterpene potentially highly significant for the pharmaceutical industry, due to its numerous biological activities. Cretan sage (Salvia fruticosa Mill.) and Rosemary (Rosmarinus officinalis L.) are rich natural sources of this compound. Although the structure and biological activities of carnosic acid are widely known, its biosynthetic pathway in plants remains unexplored. The main aim of the dissertation was to investigate the carnosic acid biosynthesis at the molecular level, by isolating and functionally characterizing the genes involved in this process. For that purpose, the existing Cretan sage trichome cDNA library has been analyzed, which has resulted in the identification of two putative diterpene synthase genes, named SfCPS and SfKSL. The candidate genes were isolated and functionally characterized in Escherichia coli Mig., yeast (Saccharomyces cerevisiae Meyen ex E. C. Hansen) and Nicotiana benthamiana Domin. Heterologous expression of SfCPS and SfKSL genes has resulted in the synthesis of miltiradiene, which has been confirmed by GC-MS analysis and 1D and 2D NMR spectroscopy (1H, 13C, DEPT, COSY H-H, HMQC и HMBC). In order to retrieve the remaining genes responsible for the biosynthesis of carnosic acid, the glandular trichomes of Cretan sage and Rosemary have been sequenced using 454 GS FLX Titanium platform. Based on the results of the LC-PDA-LTQ-Orbitrap FTMS phytochemical analysis, glandular trichomes of Cretan sage young leaves (genotype Kavoussi) exposed to mild stress, and glandular trichomes on the Rosemary leaves of the younger developmental stage (commercial genotype B&T
World Seeds), were chosen for the transcriptome analysis...
PB  - Belgrade: University of Belgrade, Faculty of Biology
T2  - University of Belgrade, Faculty of Biology
T1  - Izolacija i funkcionalna karakterizacija gena uključenih u sintezu karnozinske kiseline
T1  - Isolation and functional characterization of the genes involved in the synthesis of carnosic acid.
SP  - 1
EP  - 216
UR  - https://hdl.handle.net/21.15107/rcub_nardus_5284
ER  - 

@phdthesis{
author = "Božić, Dragana",
year = "2014",
abstract = "Karnozinska kiselina, fenolni diterpen, poseduje brojne biološke
aktivnosti zbog čega je potencijalno veoma značajna za farmaceutsku
industriju. Grčka žalfija (Salvia fruticosa Mill.) i ruzmarin (Rosmarinus
officinalis L.) bogati su prirodni izvori ovog jedinjenja. Iako je mnogo toga
poznato u vezi strukture i bioloških aktivnosti karnozinske kiseline,
veoma malo se ne zna o njenom biosintetskom putu u biljkama. Osnovni cilj
disertacije bio je izučavanje biosinteze karnozinske kiseline na
molekularnom nivou, izolacijom i funkcionalnom karakterizacijom gena
uključenih u ovaj proces. U tu svrhu, izvršena je analiza postojeće cDNK
biblioteke žlezdanih dlaka grčke žalfije, što je za rezultat imalo
identifikaciju dva gena, nazvanih SfCPS i SfKSL, koji potencijalno
kodiraju diterpen sintaze. Geni kandidati su izolovani i funkcionalno
okarakterisani u bakteriji Escherichia coli Mig., kvascima (Saccharomyces
cerevisiae Meyen ex E. C. Hansen) i duvanu (Nicotiana benthamiana Domin).
Heterologa ekpresija gena SfCPS i SfKSL rezultirala je sintezom
miltiradiena, što je utvrđeno GC-MS analizom i 1D i 2D NMR
spektroskopijom (1H, 13C, DEPT, COSY H-H, HMQC i HMBC). Za pronalaženje
preostalih gena odgovornih za biosintezu karnozinske kiseline, obavljeno
je sekvencioniranje transkriptoma grčke žalfije i ruzmarina, uz
korišćenje 454 GS FLX Titanium platforme. Na osnovu rezultata
fitohemijske analize LC-PDA-LTQ-Orbitrap FTMS metodom, žlezdane dlake
mladih listova blago stresiranih biljaka genotipa “Kavusi” grčke žalfije
i žlezdane dlake mlađih stadijuma razvoja komercijalnog genotipa
ruzmarina (B & T World Seeds) izabrane su kao biljni materijal za analizu
transkriptoma. Rezultat sekvencioniranja bio je anotacija velikog broja
novih sekvenci, a posebno značajna bila je identifikacija gena
metaboličkih puteva povezanih sa sekundarnim metabolizmom. Izolovano je
ukupno 24 gena koji su kodirali za enzime familije citohrom P450 (CYP)
monooksigenaze. Funkcija enzima CYP76Sf2 grčke žalfije, i CYP76Ro3 i
CYP76Ro4 ruzmarina, kao feruginol sintaza, utvrđena je esejima
funkcionalne karakterizacije u kvascima i duvanu. Kloniranje i
funkcionalna karakterizacija gena koji svojom zajedničkom aktivnošću
dovode do sinteze feruginola kod grčke žalfije i ruzmarina, pruža osnovu
budućim istraživanjima biosintetskog puta karnozinske kiseline. Uz to,
izvršena je lokalizacija biosintetskog puta karnozinske kiseline u
žlezdanim dlakama grčke žalfije i ruzmarina. Priloženi rezultati
doprinose boljem razumevanju metabolizma karnozinske kiseline, i time
omogućavaju primenu najsavremenijih biotehnoloških metoda za proizvodnju
ovog farmaceutski značajnog jedinjenja., Carnosic acid is a phenolic diterpene potentially highly significant for the pharmaceutical industry, due to its numerous biological activities. Cretan sage (Salvia fruticosa Mill.) and Rosemary (Rosmarinus officinalis L.) are rich natural sources of this compound. Although the structure and biological activities of carnosic acid are widely known, its biosynthetic pathway in plants remains unexplored. The main aim of the dissertation was to investigate the carnosic acid biosynthesis at the molecular level, by isolating and functionally characterizing the genes involved in this process. For that purpose, the existing Cretan sage trichome cDNA library has been analyzed, which has resulted in the identification of two putative diterpene synthase genes, named SfCPS and SfKSL. The candidate genes were isolated and functionally characterized in Escherichia coli Mig., yeast (Saccharomyces cerevisiae Meyen ex E. C. Hansen) and Nicotiana benthamiana Domin. Heterologous expression of SfCPS and SfKSL genes has resulted in the synthesis of miltiradiene, which has been confirmed by GC-MS analysis and 1D and 2D NMR spectroscopy (1H, 13C, DEPT, COSY H-H, HMQC и HMBC). In order to retrieve the remaining genes responsible for the biosynthesis of carnosic acid, the glandular trichomes of Cretan sage and Rosemary have been sequenced using 454 GS FLX Titanium platform. Based on the results of the LC-PDA-LTQ-Orbitrap FTMS phytochemical analysis, glandular trichomes of Cretan sage young leaves (genotype Kavoussi) exposed to mild stress, and glandular trichomes on the Rosemary leaves of the younger developmental stage (commercial genotype B&T
World Seeds), were chosen for the transcriptome analysis...",
publisher = "Belgrade: University of Belgrade, Faculty of Biology",
journal = "University of Belgrade, Faculty of Biology",
title = "Izolacija i funkcionalna karakterizacija gena uključenih u sintezu karnozinske kiseline, Isolation and functional characterization of the genes involved in the synthesis of carnosic acid.",
pages = "1-216",
url = "https://hdl.handle.net/21.15107/rcub_nardus_5284"
}

Božić, D.. (2014). Izolacija i funkcionalna karakterizacija gena uključenih u sintezu karnozinske kiseline. in University of Belgrade, Faculty of Biology
Belgrade: University of Belgrade, Faculty of Biology., 1-216.
https://hdl.handle.net/21.15107/rcub_nardus_5284

Božić D. Izolacija i funkcionalna karakterizacija gena uključenih u sintezu karnozinske kiseline. in University of Belgrade, Faculty of Biology. 2014;:1-216.
https://hdl.handle.net/21.15107/rcub_nardus_5284 .

Božić, Dragana, "Izolacija i funkcionalna karakterizacija gena uključenih u sintezu karnozinske kiseline" in University of Belgrade, Faculty of Biology (2014):1-216,
https://hdl.handle.net/21.15107/rcub_nardus_5284 .

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  - CONF
AU  - Bogdanović, Milica
AU  - Todorović, Slađana
AU  - Banjanac, Tijana
AU  - Dragićević, Milan
AU  - Verstappen, Francel
AU  - Bouwmeester, Harro
AU  - Simonović, Ana
PY  - 2013
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6226
AB  - Chicory (Cichorium intybus L.) is a plant valued for its medicinal and culinary properties. C. intybus owes its healing effect, at least in part, to a group of secondary metabolites known as sesquiterpene lactones. These compounds are shown to have anti-feedant, anti-fungal, anti-bacterial, anti-protozoan, schistomicidal, molluscicidal and anti-cancer effects. Since they are present in relatively small amounts in the plant, there has been ongoing interest in ways to improve the production and yield of these compounds. One way of achieving this goal is Agrobacterium-mediated transformation, which is known to boost secondary metabolism. Chicory is readily transformed with A. rhizogenes to produce hairy-roots, from which whole plants can be regenerated. Several clones previously obtained through A. rhizogenes A4 transformation were analyzed by targeted LC-MS for the content of 8-deoxylactucin, lactucin, dihydrolactucin and lactupicrin. Three clones and one untransformed plant were chosen for the analysis, based on growth, biomass production and initial screening for these metabolites. Both leaves and roots of in vitro grown plants were analysed, in two growth phases: non-flowering rosette and flowering elongated plant. It was found that the selected clones indeed produce more sesquiterpene lactones than the control plants, and that this was dependent on the plant organ and growth phase. Roots in general contain more of these compounds than leaves, and roots of flowering clones have higher content of all target sesquiterpene lactones than control plants. Since these plants were transformed with the native Ri plasmid (pRiA4-GUS), expression of bacterial genes - rolA, rolB and rolC, which are known to affect secondary metabolism, was quantified, in an attempt to correlate elevated compound content with the rol genes’ transcript levels. Expression was highest for the rolC, and barely measurable for rolB. Higher transcript level of these genes was detected in roots, and all three clones had high expression of rolC in the roots of flowering plants, which coincides with sesquiterpene lactones presence.
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  - Sesquiterpene lactone production in transformed chicory
SP  - 93
EP  - 94
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6226
ER  - 

@conference{
author = "Bogdanović, Milica and Todorović, Slađana and Banjanac, Tijana and Dragićević, Milan and Verstappen, Francel and Bouwmeester, Harro and Simonović, Ana",
year = "2013",
abstract = "Chicory (Cichorium intybus L.) is a plant valued for its medicinal and culinary properties. C. intybus owes its healing effect, at least in part, to a group of secondary metabolites known as sesquiterpene lactones. These compounds are shown to have anti-feedant, anti-fungal, anti-bacterial, anti-protozoan, schistomicidal, molluscicidal and anti-cancer effects. Since they are present in relatively small amounts in the plant, there has been ongoing interest in ways to improve the production and yield of these compounds. One way of achieving this goal is Agrobacterium-mediated transformation, which is known to boost secondary metabolism. Chicory is readily transformed with A. rhizogenes to produce hairy-roots, from which whole plants can be regenerated. Several clones previously obtained through A. rhizogenes A4 transformation were analyzed by targeted LC-MS for the content of 8-deoxylactucin, lactucin, dihydrolactucin and lactupicrin. Three clones and one untransformed plant were chosen for the analysis, based on growth, biomass production and initial screening for these metabolites. Both leaves and roots of in vitro grown plants were analysed, in two growth phases: non-flowering rosette and flowering elongated plant. It was found that the selected clones indeed produce more sesquiterpene lactones than the control plants, and that this was dependent on the plant organ and growth phase. Roots in general contain more of these compounds than leaves, and roots of flowering clones have higher content of all target sesquiterpene lactones than control plants. Since these plants were transformed with the native Ri plasmid (pRiA4-GUS), expression of bacterial genes - rolA, rolB and rolC, which are known to affect secondary metabolism, was quantified, in an attempt to correlate elevated compound content with the rol genes’ transcript levels. Expression was highest for the rolC, and barely measurable for rolB. Higher transcript level of these genes was detected in roots, and all three clones had high expression of rolC in the roots of flowering plants, which coincides with sesquiterpene lactones presence.",
publisher = "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 = "Sesquiterpene lactone production in transformed chicory",
pages = "93-94",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6226"
}

Bogdanović, M., Todorović, S., Banjanac, T., Dragićević, M., Verstappen, F., Bouwmeester, H.,& Simonović, A.. (2013). Sesquiterpene lactone production in transformed chicory. 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
Belgrade: Serbian Plant Physiology Society., 93-94.
https://hdl.handle.net/21.15107/rcub_ibiss_6226

Bogdanović M, Todorović S, Banjanac T, Dragićević M, Verstappen F, Bouwmeester H, Simonović A. Sesquiterpene lactone production in transformed chicory. 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;:93-94.
https://hdl.handle.net/21.15107/rcub_ibiss_6226 .

Bogdanović, Milica, Todorović, Slađana, Banjanac, Tijana, Dragićević, Milan, Verstappen, Francel, Bouwmeester, Harro, Simonović, Ana, "Sesquiterpene lactone production in transformed chicory" 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):93-94,
https://hdl.handle.net/21.15107/rcub_ibiss_6226 .

TY  - CONF
AU  - Brückner, Kathleen
AU  - Matekalo, Dragana
AU  - Manzano, David
AU  - Papaefthimiou, Dimitra
AU  - Pateraki, Irini
AU  - Boronat, Albert
AU  - Datema, Erwin
AU  - de Vos, Ric C H
AU  - Ferrer, Albert
AU  - Kanellis, Angelos
AU  - Tissier, Alain
PY  - 2013
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4288
AB  - Plant secondary metabolites contribute substantially to the human pharmacopeae. To better exploit the medicinal plants that produce these compounds, it is necessary to understand how these compounds are synthesized within the plant. Identification of the biosynthetic pathway genes will allow both the breeding of plant varieties with improved productivity and/or metabolic profile, and the development of alternative production methods using biotechnology.
The phenolic diterpenes (PDs) carnosic acid (CA) and carnosol have high antioxidant activities and display potential for the treatment of neurodegenerative disorders. CA and carnosol are found in Rosmarinus and Salvia sp. which are known for their health promoting properties since ages.
As part of the EU-financed TERPMED project, PD biosynthesis is being investigated. To elucidate the pathway, the localization of the biosynthesis of PDs was determined. Glandular trichomes appear to contribute significantly to the biosynthesis of PDs and the transcriptome of glandular trichomes was thus determined by next generation sequencing of cDNA.
A set of gene candidates was selected according to alignment searches and analysis of trichome-specific gene expression profile. The search for terpene synthases yielded two types of candidates for the first steps of the pathway: a copalyl diphosphate synthase (CPS) and kaurene synthase-like (KSL) encoding genes. Genes encoding oxidases of cytochrome P450 clade were the most promising candidates for the downstream steps of the PDs pathway.
Finally, the first steps of the biosynthesis of PDs are reconstituted and further investigations on the role of P450 candidates in downstream steps are being carried out using N. benthamiana transient expression and heterologous expression in yeast.
PB  - Thessaloniki: Department of Pharmaceutical Science, Aristotle University of Thessaloniki
C3  - 11th International meeting Biosynthesis, Function and Biotechnology of Isoprenoids in Terrestrial and Marine Organisms, Book of Abstracts
T1  - Investigating phenolic diterpenes in rosemary (Rosmarinus officinalis) and sage (Salvia fruticosa)
SP  - 59
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4288
ER  - 

@conference{
author = "Brückner, Kathleen and Matekalo, Dragana and Manzano, David and Papaefthimiou, Dimitra and Pateraki, Irini and Boronat, Albert and Datema, Erwin and de Vos, Ric C H and Ferrer, Albert and Kanellis, Angelos and Tissier, Alain",
year = "2013",
abstract = "Plant secondary metabolites contribute substantially to the human pharmacopeae. To better exploit the medicinal plants that produce these compounds, it is necessary to understand how these compounds are synthesized within the plant. Identification of the biosynthetic pathway genes will allow both the breeding of plant varieties with improved productivity and/or metabolic profile, and the development of alternative production methods using biotechnology.
The phenolic diterpenes (PDs) carnosic acid (CA) and carnosol have high antioxidant activities and display potential for the treatment of neurodegenerative disorders. CA and carnosol are found in Rosmarinus and Salvia sp. which are known for their health promoting properties since ages.
As part of the EU-financed TERPMED project, PD biosynthesis is being investigated. To elucidate the pathway, the localization of the biosynthesis of PDs was determined. Glandular trichomes appear to contribute significantly to the biosynthesis of PDs and the transcriptome of glandular trichomes was thus determined by next generation sequencing of cDNA.
A set of gene candidates was selected according to alignment searches and analysis of trichome-specific gene expression profile. The search for terpene synthases yielded two types of candidates for the first steps of the pathway: a copalyl diphosphate synthase (CPS) and kaurene synthase-like (KSL) encoding genes. Genes encoding oxidases of cytochrome P450 clade were the most promising candidates for the downstream steps of the PDs pathway.
Finally, the first steps of the biosynthesis of PDs are reconstituted and further investigations on the role of P450 candidates in downstream steps are being carried out using N. benthamiana transient expression and heterologous expression in yeast.",
publisher = "Thessaloniki: Department of Pharmaceutical Science, Aristotle University of Thessaloniki",
journal = "11th International meeting Biosynthesis, Function and Biotechnology of Isoprenoids in Terrestrial and Marine Organisms, Book of Abstracts",
title = "Investigating phenolic diterpenes in rosemary (Rosmarinus officinalis) and sage (Salvia fruticosa)",
pages = "59",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4288"
}

Brückner, K., Matekalo, D., Manzano, D., Papaefthimiou, D., Pateraki, I., Boronat, A., Datema, E., de Vos, R. C. H., Ferrer, A., Kanellis, A.,& Tissier, A.. (2013). Investigating phenolic diterpenes in rosemary (Rosmarinus officinalis) and sage (Salvia fruticosa). in 11th International meeting Biosynthesis, Function and Biotechnology of Isoprenoids in Terrestrial and Marine Organisms, Book of Abstracts
Thessaloniki: Department of Pharmaceutical Science, Aristotle University of Thessaloniki., 59.
https://hdl.handle.net/21.15107/rcub_ibiss_4288

Brückner K, Matekalo D, Manzano D, Papaefthimiou D, Pateraki I, Boronat A, Datema E, de Vos RCH, Ferrer A, Kanellis A, Tissier A. Investigating phenolic diterpenes in rosemary (Rosmarinus officinalis) and sage (Salvia fruticosa). in 11th International meeting Biosynthesis, Function and Biotechnology of Isoprenoids in Terrestrial and Marine Organisms, Book of Abstracts. 2013;:59.
https://hdl.handle.net/21.15107/rcub_ibiss_4288 .

Brückner, Kathleen, Matekalo, Dragana, Manzano, David, Papaefthimiou, Dimitra, Pateraki, Irini, Boronat, Albert, Datema, Erwin, de Vos, Ric C H, Ferrer, Albert, Kanellis, Angelos, Tissier, Alain, "Investigating phenolic diterpenes in rosemary (Rosmarinus officinalis) and sage (Salvia fruticosa)" in 11th International meeting Biosynthesis, Function and Biotechnology of Isoprenoids in Terrestrial and Marine Organisms, Book of Abstracts (2013):59,
https://hdl.handle.net/21.15107/rcub_ibiss_4288 .

TY  - CONF
AU  - Todorović, Slađana
AU  - Banjanac, Tijana
AU  - Bogdanović, Milica
AU  - Dragićević, Milan
AU  - Simonović, Ana
AU  - Giba, Zlatko
AU  - Grubišić, Dragoljub
PY  - 2011
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6176
AB  - Many chicory varieties (Cichorium intybus L., Asteraceae) are important agricultural and medicinal plants used throughout Europe. The bitter taste of chicory is associated with the presence of large quantities of sesquiterpene lactones. Some of the guanolides isolated from chicory, play role as antifeedants and phytoalexins, and possess cytotoxic activity towards cultured cancer cells, while the root extracts have anti-inflammatory and hepatoprotective activities. Hairy roots obtained by A. rhizogenes transformation grow fast and do not require hormones in the medium. The greatest advantage of hairy roots is that they often exhibit greater biosynthetic capacity for secondary metabolite
production compared to the mother plants. The main scope of our investigation was to optimize protocols for transformation of two chicory cultivars, C. intybus blue and C. intybus catalogna, with A. rhizogenes bearing 35S-GUS reporter vector and to establish optimal conditions for hairy roots growth. Transformation was confirmed in 7 C. intybus blue and in 5 C. intybus catalogna clones by PCR with GUS-specific primers. The GUS expression was confirmed in 6 C. intybus blue and 4 C. intybus catalogna clones by RT-PCR and histochemical GUS staining. Two of the transformed clones of C. intybus catalogna explained the greatest biomass production and growth potential. The established transformation protocol will be the basis for future planned transformation experiments and for the large-scale production of target secundary metabolites using hairy root cultures.
PB  - Belgrade: Serbian Plant Physiology Society
C3  - Programme and Abstracts: 19th Symposium of the Serbian Plant Physiology Society; 2011 Jun 13-15; Banja Vrujci, Serbia
T1  - In vitro culture establishment and biomass production of Cichorium intybus L. hairy roots
SP  - 79
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6176
ER  - 

@conference{
author = "Todorović, Slađana and Banjanac, Tijana and Bogdanović, Milica and Dragićević, Milan and Simonović, Ana and Giba, Zlatko and Grubišić, Dragoljub",
year = "2011",
abstract = "Many chicory varieties (Cichorium intybus L., Asteraceae) are important agricultural and medicinal plants used throughout Europe. The bitter taste of chicory is associated with the presence of large quantities of sesquiterpene lactones. Some of the guanolides isolated from chicory, play role as antifeedants and phytoalexins, and possess cytotoxic activity towards cultured cancer cells, while the root extracts have anti-inflammatory and hepatoprotective activities. Hairy roots obtained by A. rhizogenes transformation grow fast and do not require hormones in the medium. The greatest advantage of hairy roots is that they often exhibit greater biosynthetic capacity for secondary metabolite
production compared to the mother plants. The main scope of our investigation was to optimize protocols for transformation of two chicory cultivars, C. intybus blue and C. intybus catalogna, with A. rhizogenes bearing 35S-GUS reporter vector and to establish optimal conditions for hairy roots growth. Transformation was confirmed in 7 C. intybus blue and in 5 C. intybus catalogna clones by PCR with GUS-specific primers. The GUS expression was confirmed in 6 C. intybus blue and 4 C. intybus catalogna clones by RT-PCR and histochemical GUS staining. Two of the transformed clones of C. intybus catalogna explained the greatest biomass production and growth potential. The established transformation protocol will be the basis for future planned transformation experiments and for the large-scale production of target secundary metabolites using hairy root cultures.",
publisher = "Belgrade: Serbian Plant Physiology Society",
journal = "Programme and Abstracts: 19th Symposium of the Serbian Plant Physiology Society; 2011 Jun 13-15; Banja Vrujci, Serbia",
title = "In vitro culture establishment and biomass production of Cichorium intybus L. hairy roots",
pages = "79",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6176"
}

Todorović, S., Banjanac, T., Bogdanović, M., Dragićević, M., Simonović, A., Giba, Z.,& Grubišić, D.. (2011). In vitro culture establishment and biomass production of Cichorium intybus L. hairy roots. in Programme and Abstracts: 19th Symposium of the Serbian Plant Physiology Society; 2011 Jun 13-15; Banja Vrujci, Serbia
Belgrade: Serbian Plant Physiology Society., 79.
https://hdl.handle.net/21.15107/rcub_ibiss_6176

Todorović S, Banjanac T, Bogdanović M, Dragićević M, Simonović A, Giba Z, Grubišić D. In vitro culture establishment and biomass production of Cichorium intybus L. hairy roots. in Programme and Abstracts: 19th Symposium of the Serbian Plant Physiology Society; 2011 Jun 13-15; Banja Vrujci, Serbia. 2011;:79.
https://hdl.handle.net/21.15107/rcub_ibiss_6176 .

Todorović, Slađana, Banjanac, Tijana, Bogdanović, Milica, Dragićević, Milan, Simonović, Ana, Giba, Zlatko, Grubišić, Dragoljub, "In vitro culture establishment and biomass production of Cichorium intybus L. hairy roots" in Programme and Abstracts: 19th Symposium of the Serbian Plant Physiology Society; 2011 Jun 13-15; Banja Vrujci, Serbia (2011):79,
https://hdl.handle.net/21.15107/rcub_ibiss_6176 .