An ecological analysis of the herbivory-elicited JA burst and its metabolism: plant memory processes and predictions of the moving target model

Abstract

Background: Rapid herbivore-induced jasmonic acid (JA) accumulation is known to mediate many induced defense responses in vascular plants, but little is known about how JA bursts are metabolized and modified in response to repeated elicitations, are propagated throughout elicited leaves, or how they directly influence herbivores.

Methodology/principal findings: We found the JA burst in a native population of Nicotiana attenuata to be highly robust despite environmental variation and we examined the JA bursts produced by repeated elicitations with Manduca sexta oral secretions (OS) at whole- and within-leaf spatial scales. Surprisingly, a 2(nd) OS-elicitation suppressed an expected JA burst at both spatial scales, but subsequent elicitations caused more rapid JA accumulation in elicited tissue. The baseline of induced JA/JA-Ile increased with number of elicitations in discrete intervals. Large veins constrained the spatial spread of JA bursts, leading to heterogeneity within elicited leaves. 1(st)-instar M. sexta larvae were repelled by elicitations and changed feeding sites. JA conjugated with isoleucine (JA-Ile) translates elicitations into defense production (e.g., TPIs), but conjugation efficiency varied among sectors and depended on NaWRKY3/6 transcription factors. Elicited TPI activity correlated strongly with the heterogeneity of JA/JA-Ile accumulations after a single elicitation, but not repeated elicitations.

Conclusions/significance: Ecologically informed scaling of leaf elicitation reveals the contribution of repeated herbivory events to the formation of plant memory of herbivory and the causes and importance of heterogeneity in induced defense responses. Leaf vasculature, in addition to transmitting long-distance damage cues, creates heterogeneity in JA bursts within attacked leaves that may be difficult for an attacking herbivore to predict. Such unpredictability is a central tenet of the Moving Target Model of defense, which posits that variability in itself is defensive.

Figure 1. Repeated elicitations of N. attenuata leaves at the scale of a feeding, early-instar M. sexta.

(A) 1 µl of undiluted M. sexta OS was applied to a needlepoint wound in a single middle sector of the leaf lamina. This elicitation was repeated 5 times (1 per h). Four laminal sectors were dissected and extracted (S_E*, S_T, S_B, and S_O) for jasmonate (JA) and TPI quantification. (B) OS elicitation results in rapid accumulation of JA, some of which is rapidly conjugated to Ile. Conjugation requires both NaTD and NaJAR4; silencing NaJAR4 and NaTD transcript accumulation decreases TPI activity , . Bioactive JA and JA-Ile can be hydroxylated at C-12 or C-11, or carboxylated at C-12. NaWRKY3 and NaWRKY6 mediate the accumulation and metabolism of JA by influencing processes upstream of JA biosynthesis .

Figure 2. Whole-leaf elicitation: a single OS elicitation induces a robust, transient JA burst even in a genetically heterogeneous native population of N. attenuata, but JA accumulations in response to repeated elicitations are not additive.

(A) A single whole-leaf OS elicitation of leaves on rosette stage plants from a native population growing at the Lytle Ranch Preserve, St. George, Utah, USA, resulted in a robust, transient JA burst similar to that observed in genetically homogeneous glasshouse-grown plants. Leaves were elicited by wounding with a pattern wheel and the resulting puncture wounds were immediately treated with either water (open squares, dotted line) or 10 µl of 1∶5 diluted M. sexta OS in water (solid triangles, solid line). (B) Repeated whole-leaf OS-elicitations (1 per h) of leaves from rosette stage, inbred plants grown in the glasshouse suggest competition between suppression and maintenance of JA accumulation. Arrows indicate time of each elicitation. All values represent mean±S.E. (n = 5).

Figure 3. Accumulation of JA in elicited sectors after repeated elicitations is not strictly additive, but baseline JA and JA-Ile increase in discrete steps.

(A) Observed patterns of OS-elicited JA accumulation (triangles, solid lines) differ from those predicted from the addition of repeated single elicitations (x's, dotted lines). Arrows indicate time of elicitation (1 per h). The JA burst was suppressed after the 2^nd elicitation, but returned after the 4^th and 5^th elicitations. (B) JA accumulation due to a single elicitation returned to a lower baseline level after the initial burst. The additive model in Panel A predicted that this baseline JA level would gradually increase to a saturated level as regular elicitations resulted in a regular pattern of JA bursts. However, observed baseline return levels of JA and JA-Ile 2 h after repeated elicitations (open bars) increased in discrete steps. All values represent JA or JA-Ile mean+S.E. (n = 4).

Figure 4. Accumulation of hydroxylated JA and JA-Ile (at C-12 or C-11) and carboxylated JA-Ile (at C-12) in elicited laminal sectors (S_E*) after repeated elicitations.

(A) Hydroxylated JA begins to accumulate about 60 min after OS-elicitation, when JA attains maximum levels. Increases in JA accumulation after repeated elicitations could result from limited or suppressed metabolism of JA. However, repeated elicitations result in immediate increases in hydroxylated JA (closed triangles, solid lines) compared to hydroxylated JA levels had the elicitations not occurred (open triangles, dashed lines), indicating that hydroxylation of JA is not limiting (at least until the 5^th elicitation). Arrows indicate time of elicitation (1 per h). Values represent relative 12/11-OH-JA mean±S.E. (n = 4) compared to a JA standard. (B) Relative accumulations of 12/11-OH-JA-Ile in response to 5 repeated elicitations in elicited laminal sector (S_E*). Repeated elicitations result in immediate increases in hydroxylated JA-Ile (closed triangles, solid line) compared to hydroxylated JA-Ile levels had the elicitations not occurred (open triangles, dashed lines). Arrows indicate time of elicitation (1 per h). Values represent relative 12/11-OH-JA-Ile mean±S.E. (n = 4), compared to a JA-Ile standard. (C) Relative accumulations of 12-COOH-JA-Ile in response to repeated elicitations in elicited laminal sector (S_E*). Repeated elicitations result in immediate increases in 12-COOH-JA-Ile (closed triangles, solid line) compared to 12-COOH-JA-Ile levels had the elicitations not occurred (open triangles, dashed lines). Arrows indicate time of elicitation (1 per h). Values represent relative 12/11-OH-JA mean±S.E. (n = 4), compared to a JA-Ile standard.

Figure 5. Spatial heterogeneity in JA accumulation and metabolism within an elicited leaf after repeated OS-elicitations.

(A) JA accumulation in the repeatedly elicited laminal sector (S_E*, closed circles on thick solid lines) was 3 times the amount in adjacent, non-elicited sectors (S_B, open triangles, dot-dash lines; and S_T, x's. thin solid lines). Sectors separated from the elicitation site by the mid-rib (S_O) rarely accumulated any JA (data not shown). Peaks and troughs in the JA kinetic are less distinct in non-elicited sectors on the elicited side of the midrib. (B) Kinetics of JA-Ile elicitation closely track the JA kinetics after repeated elicitations, but the ratio of JA-Ile to JA from all samples from S_B [open triangles, m = 0.0286±0.00138 (S.E.)] was significantly lower than those in S_E* [closed circles; 0.0414±0.00199 (S.E.); Student's t-test, P≪0.0001] and S_T [x's; 0.0388±0.00217 (S.E.); Student's t-test, P = 0.0001]. (C) Silencing the expression of both NaWRKY3 and NaWRKY6 significantly reduced elicited JA-Ile to JA ratios in elicited laminal sectors [ir-wrky3/6; open circles; m = 0.0187 (S.E.)±0.00258] compared to wild-type plants [closed circles; 0.0381±0.00100 (S.E.); Student's t-test, P≪0.0001] and ir-wrky6 plants [gray circles; 0.04285±0.00729 (S.E.); Student's t-test; P = 0.003]. Arrows indicate time of elicitation (1 per h).

Figure 6. Spread of the JA burst from elicited to adjacent unelicited laminal sectors within a leaf were highly variable and likely depended on the connectivity of minor vasculature across sectors.

(A) Of the 98 elicited leaves that accumulated JA in the elicited sectors (S_E*) approximately half did not accumulate more than 500 ng/g JA in either of the adjacent, non-elicited sectors located proximal or distal to S_E* (S_B and S_T), while the others accumulated JA in either S_B, S_T, or both adjacent sectors. Only 2 of the 112 leaves accumulated detectable quantities of JA in S_O (data not shown), the sector on the opposite side of the midrib from S_E*. (B) Crystal violet dye (2%) applied to the elicitation sites allowed the visualization of apoplastic transport within leaves. Dye either was (1) not taken up, or transported (2) towards the base of the leaf, (3) the tip of the leaf, or (4) in both directions.

Figure 7. OS-elicitation motivates movement of M. sexta larvae away from elicited laminal sectors.

1^st instar M. sexta were placed on the underside of N. attenuata leaf sectors that were (1) un-elicited (control), or elicited (2) 15 min or (3) 2 h prior to placement of caterpillars. Movement and feeding activity of larvae between and within sectors were recorded. (A) 24 h after elicitation, a larger percentage of larvae had moved to a new laminal sector (solid bars) if the leaf had been elicited 15 min prior to placement (17 of 29 larvae) than if the leaf was un-elicited (9 of 27 larvae; pair-wise χ² = 3.60, P = 0.06). However, if leaves were elicited 2 h prior, larval movement did not differ from that on control leaves (10 of 25 larvae moved to a new laminal sector). (B) Comparison of the total number of feeding sites established by individuals on control leaves (open bars) and leaves elicited 15 min prior to larval placement (solid bars) revealed that prior OS elicitation motivated larvae to initiate additional feeding sites on elicited leaves.

Figure 8. Early JA-Ile accumulations and later TPI activity in leaf sectors elicited once and five times reveals the proportionally of the responses.

Increases in TPI activity (right column) in response to a single elicitation (open circles on dotted lines) are proportional to early increases in JA-Ile accumulation (left column): TPI activity differs among laminal sectors, with S_E* having significantly higher TPI activity than S_O and S_B (Fisher's PLSD, P<0.05) and marginally significantly higher TPI activity than S_T (Fisher's PLSD, P = 0.10). However, TPI activities after 5 successive elicitations (1 per h; solid squares on solid lines) do not differ significantly among S_B, S_E*, and S_T (ANOVA, F = 0.48, P = 0.63), although all values are higher than those elicited by a single elicitation in the respective laminal sectors. In S_O, only 1 of the 68 analyzed sectors contained detectable JA-Ile and TPI activity did not differ from un-induced levels. Values represent JA-Ile or TPI activity mean±S.E. (n = 4 and 5, respectively).