Comparisons of LIPOXYGENASE3- and JASMONATE-RESISTANT4/6-silenced plants reveal that jasmonic acid and jasmonic acid-amino acid conjugates play different roles in herbivore resistance of Nicotiana attenuata

Abstract

Whereas jasmonic acid (JA) and its amino acid conjugates, particularly JA-isoleucine (Ile), are known to play important roles in plant-herbivore interactions, whether other compounds also function as signals independently of JA-Ile and whether conjugates elicit systemic responses are unknown. To answer these questions, we simultaneously silenced JASMONATE-RESISTANT4 (JAR4) and JAR6, two functionally redundant enzymes in Nicotiana attenuata that conjugate JA to amino acids to produce plants (irjar4/6) with low levels of JA-Ile, JA-leucine (Leu), and JA-valine (Val; <16% of wild type). As expected, irjar4/6 plants are more vulnerable to herbivore attack, but only JA-Ile -- not JA-Leu or JA-Val -- applications restored the resistance of irjar4/6 plants, suggesting that JA-Leu and JA-Val do not mediate herbivore defense responses. Interestingly, the direct defense traits of irjar4/6 plants are significantly higher than those in LIPOXYGENASE3 (LOX3)-silenced (aslox3) plants, which are impaired in JA biosynthesis, and JA-Ile treatment could not fully restore the resistance of aslox3 plants. We thus conclude that JA, its precursors, or other metabolites complement the function of JA-Ile by eliciting a panoply of induced defenses. Similarly, transcriptional profiling of wild-type, irjar4/6, and aslox3 plants with microarrays demonstrated that JA-Ile and JA play overlapping yet distinct roles in herbivore defense. Analysis of transcripts in distal tissues demonstrated that JAR activity is essential in eliciting systemic responses. However, attempts to recover JA-(13)C(6)-Ile in systemic leaves and roots after feeding wounded leaves with (13)C(6)-Ile were unsuccessful, suggesting that JA-Ile is not a long-distance signal, but is rather synthesized after the arrival of an unknown mobile signal to systemic tissues.

Figure 1.

Silencing JAR4 and JAR6 in irjar4/6 plants. A, Mean (±se) of JA-Ile/Leu and JA concentrations in four replicate +1 leaves (one leaf position older than the source-sink transition leaf) of wild-type and irjar4/6 (irjar4-2/6-2, irjar6-1/4-1) plants, as well as of plants independently silenced for JAR4 or JAR6 (inset; irjar4-1, irjar4-2, irjar6-1, irjar6-2) 1 h after JA treatment. B, Accumulation of JAR4 or JAR6 transcripts in irjar4/6 and wild-type plants. RNA was isolated from 10 leaves growing at node +1 of separate irjar4/6 plants or five pooled +1 leaves of wild-type plants. Leaves were harvested 1 h (for irjar6-1/4-1 and wild-type control) or 3 h (for irjar4-2/6-2 and wild-type control) after OS treatment. A duplicate gel stained with ethidium bromide was used as an RNA-loading control.

Figure 2.

Impaired direct defenses and resistance of irjar4/6 plant to M. sexta attack could be restored by treatment with JA-Ile but not JA-Leu or JA-Val. A, Mean (±se) levels of TPIs and nicotine in four replicate leaves growing at the +1 node of wild-type and two lines of irjar4/6 plants (irjar4-2/6-2 and irjar6-1/4-1), as well as plants independently silenced for JAR4 or JAR6 (inset; irjar4-1, irjar4-2, irjar6-1, irjar6-2). Rosette-stage plants were wounded and treated with 0.25 μmol JA (W + JA), JA-Ile (W + JA-Ile), JA-Val plus JA-Leu (W + JA-Val + JA-Leu), or water (W + W). Ninety-six hours after treatment, treated leaves were harvested for analysis. Asterisks represent significant difference between members of a pair (Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). B, Mean (±se) mass of 18 replicated M. sexta larvae after 9 d of feeding on wild-type and two lines of irjar4/6 plants (irjar4-2/6-2 and irjar6-1/4-1). Rosette-stage plants were wounded and treated with 0.25 μmol JA-Ile (W + JA-Ile), JA-Val (W + JA-Val), JA-Leu (W + JA-Leu), or water (W + W). One day later, one freshly hatched larva was placed on treated leaves of each treated plant. Asterisks represent significant difference between members of a pair (Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Silencing both JAR4 and JAR6 suppresses OS-elicited transcriptional response, which can be restored by JA-Ile supplementation. Leaves at node +1 of rosette-stage wild-type and irjar4/6 (irjar4-2/6-2) plants were wounded and treated with OS or 0.25 μmol JA-Ile. After 180 min, leaf material was harvested and RNA was extracted, transcribed into cDNA, and labeled with fluorescent dye (wild-type samples, Cy3; irjar4/6 samples, Cy5). For each treatment, three pools of wild-type samples and three pools of irjar4/6 samples were made, each consisting of three biological replicates. Each microarray was hybridized with one wild-type pool and one irjar4/6 pool of labeled cDNA. Three microarrays per treatment were hybridized and statistically analyzed. In the OS treatment (left bars), genes significantly (P < 0.05) higher (Cy3/Cy5 > 1.5) and lower (Cy3/Cy5 < −1.5) than those expressed in irjar4/6 plants are shown with red and green bars, respectively. In the JA-Ile treatment (right bars), genes not differently expressed (−1.5 < Cy3/Cy5 < 1.5) in two genotypes are shown with gray bars. Genes are identified by a two-letter genus/species designation followed by the gene name. St, Solanum tuberosum; Nt, Nicotiana tabacum; Ns, Nicotiana sylvestris; Sl, Solanum lycopersicum; Sa, Solanum americanum; AMY, β-amylase; NIA1, nitrate reductase; GPPH, α-glucan phosphorylase, H isozyme; ASN1, Asn synthase; DHYS, dehydroquinate synthase; PhD, prephenate dehydratase; EPSPS, 5-enolpyruvylshikimate-3-P synthase; 4CL, 4-coumarate-CoA ligase.

Figure 4.

JA-Ile is not the only active oxylipin signal eliciting antiherbivore defenses. A, Mean (±se) JA-Ile/Leu and JA concentrations of four replicate wild-type, aslox3, and irjar4/6 (irjar4-2/6-2) plants 1 h after OS treatment. B, Mean (±se) transcript levels of TPI and nicotine concentrations in four replicated wild-type, aslox3, or irjar4/6 (irjar4-2/6-2) plants 10 h (for TPI) or 72 h (for nicotine) after OS elicitation. C, Mean (±se) mass of 18 replicated M. sexta larvae feeding on wild-type, aslox3, and irjar4/6 plants (irjar4-2/6-2). Rosette-stage plants were wounded and treated with water (wounding + water) or 0.25 μmol JA-Ile (wounding + JA-Ile). One day later, one freshly hatched larva was placed on treated leaves of each treated plant. Asterisks represent significant differences between members of a pair (Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 5.

Genes differently expressed in aslox3 and irajr4/6 plants. Leaves growing at +1 node of rosette-stage aslox3 and irjar4/6 (irjar4-2/6-2) plants were OS elicited. After 180 min, leaf material was harvested and RNA was extracted, transcribed into cDNA, and labeled with fluorescent dye (aslox3 samples, Cy3; irjar4/6 samples, Cy5). Three pools of aslox3 samples and three pools of irjar4/6 samples were made, each consisting of three biological replicates. Each microarray was hybridized with one aslox3 pool and one irjar4/6 pool of labeled cDNA. Three microarrays per treatment were hybridized and statistically analyzed. Genes significantly (P < 0.05) higher (Cy3/Cy5 > 1.5) and lower (Cy3/Cy5 < −1.5) expressed in aslox3 plants are shown with red and green bars, respectively. Genes are identified by two-letter genus/species designations followed by the gene name. St, Solanum tuberosum; Nt, Nicotiana tabacum; Sl, Solanum lycopersicum; Sa, Solanum americanum.

Figure 6.

Herbivore-induced systemic responses are impaired in irjar4/6 plants. Mean (±se) transcript levels of TPI, THIONIN, and PAL1 in five replicated local or systemic leaves 10 h (for TPI and THIONIN) or 2 h (for PAL1) after OS elicitation. Node +1 leaves were wounded and treated with OS. The untreated systemic leaf is growing at node −4. Asterisks represent significant difference between members of a pair (Student's t test; * P, < 0.05; ** P, < 0.01).

Figure 7.

Labeled JA-Ile was not transported to systemic tissues. A, Numbering of leaf positions in a rosette-stage N. attenuata plant growing in hydroponic cultures and illustration of treatments. The leaf undergoing the source-sink transition was designated as growing at node 0. The treated local leaf growing at node +1, which is older by one leaf position than the source-sink transition leaf, was wounded with a pattern wheel and treated with OS containing 0.625 μmol ¹³C₆-labeled Ile. The untreated systemic leaf is growing at node −4. B, Mean (±se) of JA-Ile/Leu and ¹³C₆-labeled JA-Ile in four replicated treated leaves, systemic leaves, and roots.