Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata

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.

Figure 1

HGL-DTG biosynthetic pathway. Components of the diverse HGL-DTGs structures previously identified and annotated in the leaves of N. attenuata that differ with respect to their sugar and malonyl group compositions.

Figure 2

Metabolite profiling and morphologies of N. attenuata plants transiently silenced in the expression of HGL-DTG-predicted UGTs by VIGS. A, EIC for identified HGL-DTGs in leaves of 37-day-old elongated N. attenuata plants silenced in UGT91T1, UGT74P3, or UGT74P5 transcript accumulation as well as in the EV controls (pTV00). HGL-DTGs were categorized into rhamnosylated, non-rhamnosylated, and intermediates with one or two glucose moieties to facilitate visualization. The 17-hydroxygeranyllinallool (17-HGL) aglycone was only detected in transiently silenced pTVUGT74P3 and pTVUGT74P5 lines. B, Heatmap visualization of the patterns of deregulation in control plants or in plants treated with 150 µg methyl jasmonate (MeJA) in 20 µL lanolin paste (N = 5). The color gradient visualizes fold changes in individual HGL-DTGs for each of the VIGs constructs compared to the average in the pTV00 EV VIGS plants. C, Morphological alterations observed in pTV00, pTVUGT74P3, and pTVUGT74P5 transiently transformed plants ranged from necrotic spots and tissues to necrotic apical meristem and flower buds frequently stalled in the opening process. Additional phenotypic details are provided in Supplemental Figure 7, a–c. D and E, Morphological alterations of the corolla tube, corolla limb, style, ovary and nectary (average ± se; N = 20). Asterisks indicate significant differences between the EV control and pTVUGT74P3 or pTVUGT74P5 VIGS plants (*P ≤ 0.05, ** P < 0.01, *** P < 0.001).

Figure 3

Metabolite profiling and morphologies of N. obtusifolia plants transiently silenced in the expression of HGL-DTG-predicted UGTs by VIGS. A, EIC for the identified HGL-DTGs of 37-day-old elongated N. obtusifolia plants silenced in NoUGT91T1-like, NoUGT74P4 and NoUGt74P6 transcript accumulation as well as in the EV (pTV00) controls. Rhamnosylated, non-rhamnosylated, and intermediate HGL-DTGs with one or two glucose moieties were color-categorized as in Figure 3. The 17-HGL aglycone was only detected in pTVNoUGT74P4, pTVNoUGT74P6, and the double construct pTVNoUGT74P4/UGT74P6 VIGS plants. B, Heatmap visualization of deregulations in the leaf HGL-DTG profiles of transiently transformed pTVNoUGT91T1-like, pTVNoUGT74P4, pTVNoUGT74P6, or pTVNoUGT74P4/UGT74P6 plants (N = 3–7). The color gradient visualizes fold changes in individual HGL-DTGs for each of the VIGS constructs compared to the average in the pTV00 EV VIGS plants. C, Morphological alterations in pTV00, pTVNoUGT91T1-like, pTVNoUGT74P4, pTVNoUGT74P6, or pTVNoUGT74P4/UGT74P6 ranged from necrotic spots to a high percentage of stalled flower buds. Additional phenotypic details are reported in Supplemental Figure 9.

Figure 4

Recombinant UGT74P3, UGT74P4, and UGT74P5 proteins glucosylate the 17-HGL aglycone. Enzyme activity assays of the recombinant UGT74P3, UGT74P4, and UGT74P5 proteins expressed in E. coli BL21 DE3 cells. Chromatograms (EIC traces for aglycone m/z 329.2475, HGL-DTGs with one glucose moiety m/z 491.3003 and HGL-DTGs with two glucose moieties m/z 653.3507) of analyses of four UGT recombinant proteins incubated for 3 h in 50 mM Tris HCL pH 7.0 with 5 mM 17-HGL in the presence of 5 mM UDP-glucose. Additionally, activity assays combining UGT74P5 with UGT74P3 or UGT74P4 were performed, but the results did not differ from the results of the enzyme assays using only UGT74P3 or UGT74P4.

Figure 5

Metabolite profiling and morphological characterization of stably silenced N. attenuata plants. A, EICs for identified HGL-DTGs in leaves of 42-day-old elongated N. attenuata plants silenced in GGPPS, UGT91T1, UGT74P3, and UGT74P5 transcript accumulation as well as WT control plants. HGL-DTGs were categorized into rhamnosylated, non-rhamnosylated HGL-DTG, and intermediates with one or two glucose moieties to facilitate visualization. The 17-HGL aglycone was only detected in IRugt74p5 and IRugt74p3/ugt74p5 plants. B, Heatmap visualization of deregulations in the leaf HGL-DTG profile of IRugt91t1, IRugt74p5, IRugt74p3/ugt74p5, and IRggpps (N = 5). The color gradient visualizes fold changes in individual HGL-DTGs for each of the stably transformed lines compared to the average in the WT plants. C, Morphological alterations in IRugt74p5 and IRugt74p3/ugt74p5 with milder phenotypes ranged from necrotic spots and tissues, altered leaf shape and thickness, to apical meristem necrosis and a high percentage of stalled flower buds and overall highly stunted growth. Additional details of these phenotypes are shown in Supplemental Figure 14, a–c. D, 1-year-old independent T0-transformants silenced in the expression of UGT74P3, UGT74P5, and UGT74P3/UGT74P5. Strong morphological alterations ranging from stunted growth, succulent leaves, stalled flower buds to a “broom” like appearance were consistently detected among T0-transformants. Viable seeds were produced by a few transformants.

Figure 6

Silencing efficiency for the three 17-HGL-DTG biosynthetic UGTs in IRugt91t1, IRugt74p5, IRugt74p3/ugt74p5 plants. Relative transcript abundance of UGT91T1, UGT74P3, and UGT74P5 in leaves of stably transformed N. attenuata plants (average ± se; N = 4). Asterisks indicate significant differences between the WT control and stable transformants (*P ≤ 0.05, ** P < 0.01, *** P < 0.001).

Figure 7

Abolishing 17-HGL aglycone synthesis by silencing NaGGPPS abrogates morphological alterations resulting from the silencing of UGT74P3 and UGT74P5. A, Morphological alterations of 42-day-old N. attenuata plants stably transformed to silence NaGGPPS expression (IRggpps) and WT after inoculation with A. tumefaciens harboring pTVUGT74P5, pTVUGT74P3, and the EV control (pTV00) VIGS constructs. B, Stem height, number of side branches and rosette diameter in WT and IRggpps (average ± se; N = 10). Different letters indicate significant differences between EV control and transient silenced lines (*P ≤ 0.05).

Figure 8

Application of 17-HGL aglycone results in necrotic lesions that phenocopy those observed in IRugt74p5 and IRugt74p3/ugt74p5 plants. A, Concentrations of HGL in leaf material of WT, IRggpps, IRugt74p5, IRugt74p3/ugt74p5, and IRudp91t1. B, Necrotic leaf tissue of 32-day-old elongated WT and 48-day-old flowering IRggpps plants treated with DMSO, DMSO + 140 nmol HGL, DMSO + 280 nmol HGL, and DMSO + 9800 nmol HGL after 1 day. C, Percentage of damaged leaf area in WT (N = 3) and IRggpps (average ± se; N = 5). Asterisks indicate significant differences between Control (DMSO) and treated (+HGL) leaves (*P ≤ 0.05, ** P < 0.01, *** P < 0.001).

Figure 9

Performance assays show reduced growth of M. sexta fed on transformed lines impaired in HGL-DTG glycosylation. A, Mass of M. sexta larvae feeding on leaf disk material of four stably transformed plants impaired in glucosylation (IRugt74p5, IRugt74p3/ugt74p5) and rhamnosylation (IRugt91t1) of HGL-DTGs as well as the formation of their precursor geranylgeranyl diphosphate (IRggpps; average ± se; n = 24–30). Larvae grow significantly larger on IRggpps (P = 0.005) and are significantly smaller on IRugt74p3/ugt74p5 (P = 0.005) and IRugt74p5 (P = 0.006) by day 6, as determined by Mann–Whitney–Wilcox pairwise tests. For clarity, significance is only shown for day 12: *P < 0.05, **P < 0.01, ***P < 0.001. B, Mass of leaf disks from transgenic plants consumed by caterpillars (average ± se; n = 26–30 leaf disks with one larva). Larvae fed IRugt74p5, IRugt74p3/ugt74p5, and IRugt91t1 consumed significantly less leaf disk material than larvae fed WT tissue, as determined by Mann-Whitney-Wilcox Pairwise Tests. *P < 0.05, **P < 0.01, ***P < 0.001.