Abstract
The native diploid tobacco Nicotiana attenuata produces abundant, potent anti-herbivore defense metabolites known as 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 family-1 UGT members. The transcript levels of three UGTs were highly correlated with the transcript levels two key HGL-DTG biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLS’s role in HGL-DTG biosynthesis was confirmed by virus-induced gene silencing. Silencing the Uridine diphosphate (UDP)-rhamnosyltransferase gene UGT91T1 demonstrated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. Plants with stable silencing of UGT74P3 and UGT74P5 were severely developmentally deformed, pointing to a phytotoxic effect of the aglycone. The application of synthetic 17-HGL and silencing of the UGTs in HGL-DTG-free plants confirmed this phytotoxic effect. Feeding assays with tobacco hornworm (Manduca sexta) larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation of 17-HGL is therefore a critical step that contributes to the resulting metabolites’ defensive function and solves the autotoxicity problem of this potent chemical defense.
Highlights
During the course of evolution, plants have developed versatile defense strategies against biotic stressors, such as herbivores and pathogens
To evaluate the defensive value of glycosylation and the potentialtoxicity of the 17-HGL aglycone for both plant cells and insect herbivores, we identified the uridine diphosphate glycosyltransferases (UGTs) responsible for the glycosylation of HGL-Diterpene glycosides (DTGs) by co-expression analysis of the 107 predicted UGTs in N. attenuata with two bait genes previously characterized for their involvement in the biosynthesis of 17-HGL-DTGs
We identified UGT candidates putatively responsible for the biosynthesis of HGL-DTGs by exploring gene co-expression in tissue-specific transcriptomes analyzed at 1, 5, 9, 13, 17, and 21 h after simulated herbivory treatment as described in the Supplemental Methods
Summary
During the course of evolution, plants have developed versatile defense strategies against biotic stressors, such as herbivores and pathogens. Among these defensive strategies are physical and chemical barriers such as trichomes (Fordyce and Agrawal, 2001), volatiles that attract predators.
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