Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems

Abstract

Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids, sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens. Oleoresin accumulates constitutively in resin ducts in the cortex and phloem (bark) of Norway spruce stems. De novo formation of traumatic resin ducts (TDs) is observed in the developing secondary xylem (wood) after insect attack, fungal elicitation, and mechanical wounding. Here, we characterize the methyl jasmonate-induced formation of TDs in Norway spruce by microscopy, chemical analyses of resin composition, and assays of terpenoid biosynthetic enzymes. The response involves tissue-specific differentiation of TDs, terpenoid accumulation, and induction of enzyme activities of both prenyltransferases and terpene synthases in the developing xylem, a tissue that constitutively lacks axial resin ducts in spruce. The induction of a complex defense response in Norway spruce by methyl jasmonate application provides new avenues to evaluate the role of resin defenses for protection of conifers against destructive pests such as white pine weevils (Pissodes strobi), bark beetles (Coleoptera, Scolytidae), and insect-associated tree pathogens.

Figure 1

Representative structures of terpenoids of Norway spruce (Picea abies L. Karst). A and D, Monoterpenes (10 carbon atoms). B, Sesquiterpenes (15 carbon atoms). C, Diterpene resin acids (20 carbon atoms). Monoterpenes are numbered corresponding to peak numbers in Figure 9.

Figure 2

Scheme of the pathways of terpenoid biosynthesis in conifers. The five-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are formed via two pathways, the mevalonate pathway in the cytosol/endoplasmic reticulum and the 2-C-methylerythritol-4-phosphate pathway (via 1-deoxyxylulose-5-phosphate) in plastids. Prenyltransferases (PTs) catalyze (1′-4) head-to-tail condensations of DMAPP with one, two, or three molecules of IPP to form geranyl diphosphate (GPP; GPP synthase), farnesyl diphosphate (FPP; FPP synthase), and geranylgeranyl diphosphate (GGPP; GGPP synthase), respectively. Terpene synthases (TPS; cyclases) of three classes (mono-TPS, sesqui-TPS, and di-TPS) convert the three prenyl diphosphate intermediates into the hundreds of cyclic and acyclic terpenoids characteristic of conifers.

Figure 3

Light microscopy of induced TD differentiation in cross sections of Norway spruce stems. A and E, Unsprayed controls show the absence of resin ducts in the constitutive xylem (X) and phloem (P), but large constitutive resin ducts (CD) are present in the cortex of control trees. B, Nine days after 10 mm methyl jasmonate (MeJA) treatment, early stage of induced development of TDs in the xylem next to the vascular cambium. C, Twenty-five days after MeJA treatment, showing accumulation of resin in the lumen of the fully differentiated TD visualized by staining with copper acetate. D, Two months after treatment, lumen (L) of TD with resin droplet (blue) and remainder of epithelial resin duct cells (E). F, Two months after treatment, showing a ring of fully formed traumatic ducts in the newly developed xylem.

Figure 4

Tissue-specific and dose-dependent changes in monoterpene and diterpene accumulation in wood and bark after treatment of trees with MeJA. A, Monoterpenoids, wood. B, Diterpenoids, wood. C, Monoterpenoids, bark. D, Diterpenoids, bark. Each concentration was tested on four trees. Analysis was performed in duplicate and results are presented as the mean with se. Wood denotes the entire xylem tissue of a stem section. Bark denotes all tissues outside the vascular cambium, including phloem, cortex, and periderm. Lower concentrations of MeJA of 100 to 500 μM induced chemical changes of wood resin terpenoids similar to those induced with 10 mm MeJA when Tween 20 was added at 0.1% (v/v) to the surface spray.

Figure 5

Time course of total monoterpenoid and diterpenoid content in wood and bark after treatment with MeJA. A, Monoterpenoids, wood. B, Diterpenoids, wood. C, Monoterpenoids, bark. D, Diterpenoids, bark. Data are presented as the means with se of duplicate or triplicate assays of extracts from treated (●) and control (○) trees.

Figure 6

Time course of total sesquiterpenoid content in bark after treatment with MeJA. Data are presented as the means with se of duplicate or triplicate assays of extracts from treated (●) and control (○) trees.

Figure 7

Time course of PT activity in wood and bark after treatment with MeJA. A, GPP synthase activity, wood. B, FPP synthase activity, wood. C, GGPP synthase activity, wood. D, GPP synthase activity, bark. E, FPP synthase activity, bark. F, GGPP synthase activity, bark. Values are the means of duplicate or triplicate assays of extracts from treated (●) and control (○) trees. A rapid increase of enzyme activity was found only for GGPP synthase in induced wood samples. The apparent increase in specific activities of GPP synthase and FPP synthase at d 35 reflect on a decrease of total protein in these samples. Ranges of duplicate assays were normally 1% to 25% of the mean but were 45% to 60% of the mean in control d 35 in A and B and in MeJA d 25 and 35 in B.

Figure 8

Time course of monoterpenoid synthase activity and diterpenoid synthase activity in wood and bark after treatment with MeJA. A, Monoterpenoid synthase activity, wood. B, Monoterpenoid synthase activity, bark. C, Diterpenoid synthase activity, wood. D, Diterpenoid synthase activity, bark. Data are the means of duplicate or triplicate assays of extracts from treated (●) and control (○) trees.

Figure 9

Analysis of products formed in vitro by constitutive and induced monoterpenoid synthase activity from wood tissue. A, Radio-GC traces for monoterpenoid synthase assay products from wood of control saplings. B, Radio-GC trace for monoterpenoid synthase assay products from wood of saplings treated with 10 mm MeJA. C, Thermal conductivity detector (TCD) trace for monoterpene standards. Standard for β-phellandrene not shown. Peak 1, α-Pinene; 2, camphene; 3, β-pinene; 4, Δ³-carene; 5, myrcene; 6, limonene; 7, β-phellandrene.

Figure 10

Analysis of products formed in vitro by induced diterpenoid synthase activity from wood tissue. A, Radio-GC trace for di-TPS assay products. B, GC-MS fragmentation pattern for major di-TPS assay product. C, GC-MS fragmentation pattern for authentic abietadiene standard.