Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana

Bekzod Khakimov; Vera Kuzina Poulsen; Pernille Østerbye Erthmann; Ery Odette Fukushima; Jörg Manfred Augustin; Carl Erik Olsen; Jelle Scholtalbers; Hanne Volpin; Sven Bode Andersen; Thure Pavlo Hauser; Toshiya Muranaka; Søren Bak

doi:10.1111/tpj.13012

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana

Bekzod Khakimov, Vera Kuzina Poulsen, Pernille Østerbye Erthmann, Ery Odette Fukushima, Jörg Manfred Augustin, Carl Erik Olsen, Jelle Scholtalbers, Hanne Volpin, Sven Bode Andersen, Thure Pavlo Hauser, Toshiya Muranaka, Søren Bak

50 Citations (Scopus)

Abstract

The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs). Significance Statement Saponins are widespread plant defence compounds, but in crucifers only a few species in the Barbarea genus can produce them. Here we used QTL analysis, transcriptomics, bioinformatics, and combinatorial biochemistry to identify genes in the saponin pathway of B. vulgaris, showed that they are located in different regions of the genome, and transferred the genes to tobacco, which reconstructed crucial steps of the saponin biosynthesis.

Original language	English
Journal	The Plant Journal
Volume	84
Issue number	3
Pages (from-to)	478-490
Number of pages	13
ISSN	1365-313X
DOIs	https://doi.org/10.1111/tpj.13012
Publication status	Published - Nov 2015

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Khakimov, B., Poulsen, V. K., Erthmann, P. Ø., Fukushima, E. O., Augustin, J. M., Olsen, C. E., Scholtalbers, J., Volpin, H., Andersen, S. B., Hauser, T. P., Muranaka, T., & Bak, S. (2015). Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana. The Plant Journal, 84(3), 478-490. https://doi.org/10.1111/tpj.13012

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana. / Khakimov, Bekzod; Poulsen, Vera Kuzina; Erthmann, Pernille Østerbye et al.
In: The Plant Journal, Vol. 84, No. 3, 11.2015, p. 478-490.

Research output: Contribution to journal › Journal article › Research › peer-review

Khakimov, B, Poulsen, VK, Erthmann, PØ, Fukushima, EO, Augustin, JM, Olsen, CE, Scholtalbers, J, Volpin, H, Andersen, SB, Hauser, TP, Muranaka, T & Bak, S 2015, 'Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana', The Plant Journal, vol. 84, no. 3, pp. 478-490. https://doi.org/10.1111/tpj.13012

@article{dbc65e0827d84e8198aa79af480aa451,

title = "Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana",

abstract = "The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs). Significance Statement Saponins are widespread plant defence compounds, but in crucifers only a few species in the Barbarea genus can produce them. Here we used QTL analysis, transcriptomics, bioinformatics, and combinatorial biochemistry to identify genes in the saponin pathway of B. vulgaris, showed that they are located in different regions of the genome, and transferred the genes to tobacco, which reconstructed crucial steps of the saponin biosynthesis.",

author = "Bekzod Khakimov and Poulsen, {Vera Kuzina} and Erthmann, {Pernille {\O}sterbye} and Fukushima, {Ery Odette} and Augustin, {J{\"o}rg Manfred} and Olsen, {Carl Erik} and Jelle Scholtalbers and Hanne Volpin and Andersen, {Sven Bode} and Hauser, {Thure Pavlo} and Toshiya Muranaka and S{\o}ren Bak",

year = "2015",

month = nov,

doi = "10.1111/tpj.13012",

language = "English",

volume = "84",

pages = "478--490",

journal = "The Plant Journal",

issn = "1365-313X",

publisher = "Wiley-Blackwell",

number = "3",

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TY - JOUR

T1 - Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana

AU - Khakimov, Bekzod

AU - Poulsen, Vera Kuzina

AU - Erthmann, Pernille Østerbye

AU - Fukushima, Ery Odette

AU - Augustin, Jörg Manfred

AU - Olsen, Carl Erik

AU - Scholtalbers, Jelle

AU - Volpin, Hanne

AU - Andersen, Sven Bode

AU - Hauser, Thure Pavlo

AU - Muranaka, Toshiya

AU - Bak, Søren

PY - 2015/11

Y1 - 2015/11

N2 - The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs). Significance Statement Saponins are widespread plant defence compounds, but in crucifers only a few species in the Barbarea genus can produce them. Here we used QTL analysis, transcriptomics, bioinformatics, and combinatorial biochemistry to identify genes in the saponin pathway of B. vulgaris, showed that they are located in different regions of the genome, and transferred the genes to tobacco, which reconstructed crucial steps of the saponin biosynthesis.

AB - The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs). Significance Statement Saponins are widespread plant defence compounds, but in crucifers only a few species in the Barbarea genus can produce them. Here we used QTL analysis, transcriptomics, bioinformatics, and combinatorial biochemistry to identify genes in the saponin pathway of B. vulgaris, showed that they are located in different regions of the genome, and transferred the genes to tobacco, which reconstructed crucial steps of the saponin biosynthesis.

U2 - 10.1111/tpj.13012

DO - 10.1111/tpj.13012

M3 - Journal article

C2 - 26333142

SN - 1365-313X

VL - 84

SP - 478

EP - 490

JO - The Plant Journal

JF - The Plant Journal

IS - 3

ER -

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana

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