Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and accumulation of terpenoid synthase and putative octadecanoid pathway transcripts in Sitka spruce

Abstract

Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (-)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (-)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system.

Figure 1.

Accumulation of monoterpenoids, sesquiterpenoids, and diterpenoids in outer and inner stem tissues of Sitka spruce after feeding by white pine weevil or MeJA treatment. Terpenoids were analyzed from samples harvested 20 d after treatment of sapling trees with 0.1% Tween 20 (Tween), treatment with 0.01% MeJA in 0.1% Tween (MeJA), or after feeding by white pine weevils (Weevil). Untreated trees served as controls (Control). Each bar represents the mean total + se of three samples.

Figure 2.

Transcript accumulation of mono-TPS, sesqui-TPS, and di-TPS in outer stem tissues of Sitka spruce after feeding by white pine weevils or MeJA treatment. RNA for northern hybridizations was isolated from outer stem samples harvested over a time course of 32 d after treatment of sapling trees with 0.1% Tween 20 (Tween), treatment with 0.01% MeJA in 0.1% Tween (MeJA), or after feeding by white pine weevils (Weevil). Mono-TPS probes (Supplemental Table III) are PaTPS-Myr, PaMTS, PaTPS-Lin, PaTPS-Car, PaTPS-Pin, and PaTPS-Lim. Sesqui-TPS probes are PaTPS-Far, PaTPS-Bis, and PaTPS-Lon. Di-TPS probes are PaTPS-LAS and PaTPS-Iso. Ethidium bromide-stained major ribosomal RNA bands serve for evaluation of equal gel loading.

Figure 3.

Accumulation of TPS transcripts in inner and outer stem tissues after MeJA treatment. RNA for northern hybridizations was isolated from inner and outer stem samples harvested over a time course of 4 d after treatment with 0.01% MeJA in 0.1% Tween. Ethidium bromide-stained major ribosomal RNA bands serve for evaluation of equal gel loading.

Figure 4.

Terpenoid accumulation and TPS transcripts in distal, noninfested stem sections upon local feeding by weevils. A, Terpenoids were analyzed in inner and outer tissues of stem sections below the site of weevil feeding at day 20. Data represent mean + se from three samples. B, Northern hybridizations of total RNA from outer stem tissue below the site of weevil exposure harvested over a time course of 2 d after initiation of weevil feeding. C, Northern hybridizations of total RNA from outer stem tissue above the site of weevil exposure. Ethidium bromide-stained major ribosomal RNA bands serve for evaluation of equal gel loading.

Figure 5.

Accumulation of monoterpenoids, sesquiterpenoids, and diterpenoids in young and mature needles after feeding by weevils on stems or after treatment of trees with MeJA. Terpenoids were analyzed from samples harvested 20 d after treatment of sapling trees with 0.1% Tween 20 (Tween), treatment with 0.01% MeJA in 0.1% Tween (MeJA), or after exposure of trees to weevils (Weevil). Each bar represents the mean total + se from three samples.

Figure 6.

Terpenoid volatile emission from Sitka spruce saplings upon treatment with MeJA. A, Time course of volatile emissions of trees treated with 0.1% Tween 20 (Tween Control) and 0.01% MeJA in 0.1% Tween 20 (MeJA). Volatile collections started 9 h after treatment of trees to allow for evaporation of excessive amounts of MeJA. Trees were placed into collection chambers 6 h before onset of volatile collections. Data represent mean + se from three independent volatile collection experiments. Black and white bars on top of the graph represent day and night periods. B, Representative chromatogram of terpenoids collected from MeJA-treated trees and from (C) Tween 20-treated control trees on the 4th day after treatment between 6 am and 12 pm, 69 to 75 h after treatment. Peaks were identified as: 1, (−)-α-pinene; 2, myrcene; 3, (+)-β-pinene; 4, (−)-β-pinene; 5, sabinene; 6, (−)-linalool; 7, (+)-linalool; 8, (E,E)-α-farnesene; 9, (Z)-α-bisabolene; and IS, internal standard.

Figure 7.

Terpenoid volatile emission from Sitka spruce saplings upon feeding by white pine weevils. A, Time course of volatile emissions of trees with five weevils feeding per tree (Weevil), and time course of volatile emissions from control trees with weevils separated from trees by mesh bag (Control). Volatile collections started 6 h after onset of weevil feeding at the time when trees were placed into the collection system. Black and white bars on top of the graph represent day and night periods. Data represent mean + se from three independent volatile collection experiments. B, Representative chromatogram of terpenoids collected from weevil infested trees on the 1st day, 6 to 12 h after begin of weevil feeding. C, Representative chromatogram of terpenoids collected from a weevil-treated tree on the 4th day between 6 am and 12 pm, 66 to 72 h after onset of treatment. D, Representative chromatogram of terpenoids collected from a control tree independent of time point during the course of the volatile collection experiment. Peaks were identified as: 1, (−)-α-pinene; 2, myrcene; 3, (+)-β-pinene; 4, (−)-β-pinene; 5, sabinene; 6, terpinolene; 7, (−)-linalool; 8, (+)-linalool; 9, (E,E)-α-farnesene; 10, (Z)-α-bisabolene; and IS, internal standard.

Figure 8.

Time course of (−)-linalool emission from Sitka spruce saplings upon feeding by white pine weevils. Time course of (−)-linalool emissions of trees with five weevils feeding per tree. Volatile collections started 6 h after onset of weevil feeding at the time when trees were placed into the collection system. Black and white bars on top represent day and night periods. Data represent mean + se from three independent volatile collection experiments. No emission of linalool was detected with control trees.

Figure 9.

Transcript accumulation of mono-TPS, sesqui-TPS, and di-TPS in needles of Sitka spruce after treatment with MeJA. RNA for northern hybridizations was isolated from needles over a time course of 32 d after treatment of sapling trees with 0.1% Tween 20 (Tween) or treatment with 0.01% MeJA in 0.1% Tween (MeJA). The mono-TPS probes (Supplemental Table III) are PaTPS-Myr, PaMTS, PaTPS-Lin, PaTPS-Car, PaTPS-Pin, and PaTPS-Lim. Sesqui-TPS probes are PaTPS-Far, PaTPS-Bis, and PaTPS-Lon. Di-TPS probes are PaTPS-LAS and PaTPS-Iso. Ethidium bromide-stained major ribosomal RNA bands serve for evaluation of equal gel loading.

Figure 10.

Transcript accumulation of genes of octadecanoid biosynthesis in stems of Sitka spruce after feeding by white pine weevil or treatment with MeJA. RNA for northern hybridizations was isolated from outer stem samples harvested over a time course of 32 d after treatment of sapling trees with 0.1% Tween 20 (Tween), treatment with 0.01% MeJA in 0.1% Tween (MeJA), or after feeding by white pine weevils (Weevil). EST cDNA clones used as probes are described in Supplemental Table VI. PLA, phospholipaseA; LOX, lipoxygenase; AOS, allene oxide synthase; AOC, allene oxide cyclase; OPDAR, 12-oxo-phytodienoic acid reductase. Ethidium bromide-stained major ribosomal RNA bands serve for evaluation of equal gel loading.

Figure 11.

Transcript accumulation of genes of octadecanoid biosynthesis in needles of Sitka spruce after treatment with MeJA. RNA for northern hybridizations was isolated from needles over a time course of 32 d after treatment of sapling trees with 0.1% Tween 20 (Tween) or treatment with 0.01% MeJA in 0.1% Tween (MeJA). EST cDNA clones used as probes are described in Supplemental Table VI. PLA, phospholipaseA; LOX, lipoxygenase; AOS, allene oxide synthase; AOC, allene oxide cyclase; OPDAR, 12-oxo-phytodienoic acid reductase. Ethidium bromide-stained major ribosomal RNA bands serve for evaluation of equal gel loading.