Jasmonate and ppHsystemin regulate key Malonylation steps in the biosynthesis of 17-Hydroxygeranyllinalool Diterpene Glycosides, an abundant and effective direct defense against herbivores in Nicotiana attenuata

Abstract

We identified 11 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) that occur in concentrations equivalent to starch (mg/g fresh mass) in aboveground tissues of coyote tobacco (Nicotiana attenuata) and differ in their sugar moieties and malonyl sugar esters (0-2). Concentrations of HGL-DTGs, particularly malonylated compounds, are highest in young and reproductive tissues. Within a tissue, herbivore elicitation changes concentrations and biosynthetic kinetics of individual compounds. Using stably transformed N. attenuata plants silenced in jasmonate production and perception, or production of N. attenuata Hyp-rich glycopeptide systemin precursor by RNA interference, we identified malonylation as the key biosynthetic step regulated by herbivory and jasmonate signaling. We stably silenced N. attenuata geranylgeranyl diphosphate synthase (ggpps) to reduce precursors for the HGL-DTG skeleton, resulting in reduced total HGL-DTGs and greater vulnerability to native herbivores in the field. Larvae of the specialist tobacco hornworm (Manduca sexta) grew up to 10 times as large on ggpps silenced plants, and silenced plants suffered significantly more damage from herbivores in N. attenuata's native habitat than did wild-type plants. We propose that high concentrations of HGL-DTGs effectively defend valuable tissues against herbivores and that malonylation may play an important role in regulating the distribution and storage of HGL-DTGs in plants.

Figure 1.

Identity and Biosynthesis of HGL-DTGs. (A) Newly identified and previously described compounds from leaves of N. attenuata differing in their sugar and malonyl groups are organized into the following biosynthetic groups: (1) precursor, (2) core, (3) singly malonylated, and (4) dimalonylated compounds. Rh, rhamnose; Glc, glucose. (B) Biosynthetic pathway: HGL is glycosylated at the C-3 and C-17 hydroxyl groups to lyciumoside I, the precursor for all HGL-DTGs. Additional glycosylations of lyciumoside I (G2a, b, and c) or lyciumoside IV (G2d and e) produce larger core HGL-DTGs that may be malonylated once (M1) or twice (M2). 1*, 2*, 3*, and 4* refer to the group in (A).

Figure 2.

N. attenuata Accumulates HGL-DTGs during Growth and Differentially Accumulates Them to Young and Reproductive Tissues. Plants were left unelicited (Control), or one leaf per plant was elicited with wounding and M. sexta oral secretions (W+OS). Aboveground tissues were harvested 3 d after treatment and pooled by tissue type. In a previous experiment, no HGL-DTGs were found in roots (data not shown). Note that some compounds have been multiplied by constants to fit the scale of the graph and that scales change among graphs. *P < 0.05 in a Student's t test. n.d., not detected. (A) HGL-DTGs (average + se) accumulate as plants grow from the rosette stage (34 d old) and elongate (44 d old) through the initiation of flowering; total HGL-DTG pools are no different in young (54 d old) versus old (68 d) flowering plants (n = 4 plants). (B) Accumulation of HGL-DTGs in a young flowering plant. Singly malonylated compounds accumulate in reproductive tissues, whereas doubly malonylated compounds accumulate in leaves. The greatest amounts of HGL-DTGs are found in young and reproductive tissues. W+OS elicitation of a leaf changes the composition of HGL-DTGs in that leaf, likely by reallocation from the rest of the plant because a single treatment has no significant effect on total HGL-DTG pools in (A).

Figure 3.

ggpps Transcript Accumulation in Tissues of Young Flowering Plants (54 d old) Relative to N. attenuata actin (average + se; n = 4 Plants). Different letters indicate significant differences (P < 0.05) in Bonferroni-corrected post-hoc tests following an ANOVA.

Figure 4.

The Different Biosynthetic Groups of HGL-DTGs Display Characteristic Dynamic Patterns over 1 Week of Growth in Rosette-Stage Plants. Plants were left unelicited (Control), or five leaves per plant were elicited with W+OS. Elicited leaf positions were harvested and pooled from each plant (one pooled sample per plant, n = 5) at one of five time points: 1, 4, and 14 h (night), and 3, 5, and 7 d. The precursor lyciumoside I (average + se) attains maximum concentration during the night at 14 h and then decreases as other compounds accumulate. In W+OS-elicited leaves, the sharp decline in precursor levels following their 14 h peak likely supply substrates for the subsequent accumulation of core compounds, which do not peak, but are maintained at a constant level as they provide substrate for the synthesis of malonylated compounds. The elevation in malonylated compounds begins concurrently with the elevation in core compounds and precursor levels in W+OS-elicited leaves, but at 7 d in unelicited leaves. All compounds reach stable elicited levels beginning at 3 d after W+OS elicitation (indicated by the dotted rectangle) with the exception of malonylated compounds derived from nicotianoside III, which continue to increase. Note that scales change among graphs. Asterisks indicate significant differences between W+OS-treated and control samples in Student's t tests within each time point (*P ≤ 0.05, ** P < 0.01, and *** P < 0.001).

Figure 5.

Malonylation of HGL-DTGs Is Regulated by JA Signaling and Tuned by ppHS Signaling. (A) to (C) Experiment 1: wild type, IRlox3, and IRcoi1. (D) to (F) Experiment 2: IRsys and the wild type. Elongated N. attenuata plants were left unelicited (Control), or one leaf per plant was elicited with W+OS to mimic herbivory, with 150 μ g MJ in lanolin paste to supplement JA production. Elicited leaves were harvested at 3 d. Plants impaired in JA biosynthesis (IRlox3) and perception (IRcoi1) or in systemin-mediated signaling (IRsys) were compared with wild-type plants in two separate experiments using identical methods. In this figure, we compare levels of each HGL-DTG among silenced lines and the wild type, which have received the same treatment. Lanolin paste (lan) was used to control for solvent effects for the lan+MJ treatment, and the effects of treatments within genotypes are shown in Supplemental Figure 3 and Supplemental Tables 4 and 5 online. Asterisks indicate significant differences from the wild type in one-way ANOVAs (wild type, IRlox3, and IRcoi1; [A] to [C]) or Student's t tests (wild type and IRsys; [D] to [F]) within each treatment: *P ≤ 0.05, ** P < 0.01, and *** P < 0.001 in Bonferroni-corrected tests. (Student's t tests were also subjected to the Bonferroni comparison as the same data were used to analyze the effect of treatment on HGL-DTG accumulation within genotypes.) Each graph represents the full biosynthetic pathway from glycosylation through malonylation for one set of HGL-DTGs. For example, in (A), IRcoi1 plants accumulate the precursor lyciumoside I (average + se; n = 5 plants) and are impaired in their ability to convert it to the core compound lyciumoside IV at a wild-type rate, especially after lan+MJ treatment; IRlox3 plants accumulate lyciumoside IV, especially after W+OS elicitation. Both IRcoi1 and IRlox3 are unable to malonylate lyciumoside IV in control and W+OS-elicited leaves. Malonylation is recovered in IRlox3 and not in IRcoi1 by lan+MJ. By contrast, in (D), IRsys plants are able to synthesize wild-type levels of lyciumosides IV and nicotianosides I and II, but like IRcoi1, accumulate lyciumoside I in unelicited tissue; however, in (E) and (F), IRsys plants are unable to accumulate wild-type levels of other dimalonylated HGL-DTGs (nicotianosides V and VII) after lan+MJ treatment. Note that some compounds have been multiplied by constants to fit the scale of the graph and that scales change among graphs. LOQ, below limit of quantification.

Figure 6.

Total HGL-DTG Accumulation (Average + se; n = 5 Plants) Requires JA Perception as Mediated by COI1. Total HGL-DTGs were measured after no treatment (control) or treatment with W+OS or lan+MJ in wild-type, IRlox3, and IRcoi1 leaves in one experiment and in wild-type versus IRsys leaves in a replicate experiment conducted under the same conditions. Asterisks indicate significant differences from the wild type in a one-way ANOVA followed by Bonferroni-corrected tests (wild type, IRlox3, and IRcoi1; [A]) or Student's t tests (the wild type and IRsys; [B]) within each treatment: *P ≤ 0.05, ** P < 0.01, and *** P < 0.001. (A) Experiment 1: IRlox3 accumulates nearly wild-type levels of HGL-DTGs after W+OS elicitation due to buildup of core compounds (Figure 5) and is completely restored to wild-type levels by lan+MJ treatment; neither W+OS nor lan+MJ treatment restores total HGL-DTG levels in IRcoi1. (B) Experiment 2: IRsys plants are not impaired in the accumulation of total HGL-DTGs.

Figure 7.

Total HGL-DTGs Have a Significant Effect on the Growth of the Specialist Herbivore M. sexta. (A) Mass of M. sexta larvae feeding on two independently silenced IRggpps lines versus the wild type (average ± se; final n = 7 to 15 plants with one larva per plant; see Methods). Larvae grow significantly larger on both lines of IRggpps: growth on IRggpps line 1 was significantly faster by day 4 (P = 0.018) and on both IRggpps lines by day 7 (line 1, P = 0.011; line 2, P = 0.016) as determined by a repeat-measures ANOVA followed by Bonferroni-corrected tests for each day. For clarity, significance is shown only for day 13: ** P < 0.01 and *** P < 0.001. (B) to (E) Total HGL-DTGs (B) and concentrations of individual compounds ([C] to [E]) in mature young rosette leaves of the wild type and both lines of IRggpps 3 d after treatment with lan or lan+MJ (average + se). Note that some compounds have been multiplied by constants to fit the scale of the graph and that scales change among graphs. Asterisks indicate significantly lower levels of HGL-DTGs in IRggpps in one-way ANOVAs within treatment (*P < 0.05, ** P < 0.01, and *** P < 0.001 in Bonferroni-corrected tests). LOQ, below limit of quantification.

Figure 8.

HGL-DTGs Reduce Damage from Generalist Herbivores in Nature. (A) The most abundant herbivore in the 2008 field season, mirids caused significantly more damage on IRggpps line 1 than on wild-type plants in the field by May 16th (average + se; n = 9 pairs; *P < 0.05 in paired t tests). (B) Grasshoppers caused significantly more damage on IRggpps line 1 by May 16th (average + se, *P < 0.05 in Wilcoxon's sign-rank test). (C) Flea beetles tended to cause more damage on IRggpps line 1 at the beginning of damage measurements on May 8th; differences are not statistically significant (average + se, Wilcoxon's sign-rank test). Scale for less abundant herbivores in (B) and (C): damage level 0 = 0% total canopy damage; level 1, <1%; level 2, < 5%; level 3, 5 to 10%. (D) to (G) Total HGL-DTGs (D) and concentrations of individual compounds (average + se; [E] to [G]) in undamaged systemic leaves harvested from pairs of the wild type and IRggpps line 1 on May 15th. Note that some compounds have been multiplied by constants to fit the scale of the graph and that scales change. *P < 0.05 and ** P < 0.01 in paired t tests. LOQ, below limit of quantification.

Figure 9.

Proposed Model for the Regulation of HGL-DTG Biosynthesis and Effect on Herbivores in N. attenuata. A simplified biosynthetic pathway is shown for HGL-DTGs from the 17-hydroxygeranylinalool skeleton to the higher molecular weight HGL-DTGs and their mono- and dimalonylated forms. Herbivore attack and larval oral secretions (OS) elicits JA biosynthesis (requiring lox3) and perception (requiring coi1), which strongly regulate malonylation of HGL-DTGs, and JA perception accelerates glycosylation. ppHS may fine-tune JA-mediated malonylation by altering either the perception of JAs or the positive feedback of JAs on their own biosynthesis. The accumulated HGL-DTGs function as a profoundly effective defense against herbivores.