Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.

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 inulina 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-15oxalates, 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.

Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.

“…Other approaches for gene down-regulation in chicory, such as RNAi, have previously been used. Bogdanovic et al (2020) used microRNA to down-regulate the expression of the CiGAS-short and CiGAS-long genes, but this approach gave only partial silencing and led to very variable levels of STL production compared with this study.…”

“…Expression analysis of the Ci GAS genes has shown that both the Ci GAS‐L and Ci GAS‐ short genes are expressed in chicory roots, while predominantly only the Ci GAS‐L is expressed in chicory leaves (Bogdanović et al ., 2019 ; Bouwmeester et al ., 2002 ). Silencing of the Ci GAS genes by a microRNA approach resulted in reduced expression of both the long and short germacrene A synthases and a partial decrease of STL levels (Bogdanovic et al ., 2020 ). However, it is still not known which of the Ci GAS genes play a role in different tissues and which Ci GAS genes need to be inactivated before a strong effect on STL production can be observed.…”

Analysis of root volatiles and functional characterization of a root-specific germacrene A synthase inArtemisia pallens

“…Isoforms of GAS have been reported in several plant species, including Sunflower (Göpfert et al 2010), chicory (Bogdanović et al 2020), Barnadesia spinosa (Nguyen et al 2016), and Lettuce (Bennett et al 2002). The presence of such GAS isoforms within the same plant species highlights the complexity of terpene biosynthesis and the potential functional specialization of GAS isoforms in different biochemical pathways.…”

Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus

“…The biosynthetic pathway that forms bitter guaianolide SL metabolites has been studied in a number of plants of the Asteraceae family: C. intybus ( Bouwmeester et al., 2002 ; Nguyen et al., 2010 ; Cankar et al., 2011 ; Liu et al., 2011 ; Bogdanović et al., 2019 ; Bogdanović et al., 2020 ; Cankar et al., 2021 ; Cankar et al., 2022 ), C. endiva ( Zhang et al., 2022 ), Helianthus annuus and Lactuca sativa ( Nguyen et al., 2010 ; Ikezawa et al., 2011 ), Tanacetum parthenium ( Liu et al., 2014 ; Liu et al., 2018 ), Artemisia annua ( Majdi et al., 2016 ) , Saussurea lappa and Barnadesia spinosa ( Nguyen et al., 2010 ), among others. The biosynthesis starts with the cyclization of farnesyl pyrophosphate (FPP) by the terpene synthase (TPS) GERMACRENE A SYNTHASE (GAS) to form germacrene A ( De Kraker et al., 1998 ; Bouwmeester et al., 2002 ) ( Figure 1 ).…”