Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.     

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

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.     

Immunofluorescence localization of levopimaradiene/abietadiene synthase in methyl jasmonate treated stems of Sitka spruce (Picea sitchensis) shows activation of diterpenoid biosynthesis in cortical and developing traumatic resin ducts

Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species (Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by insects, fungi or treatment with methyl jasmonate (MeJA). Using immunofluorescence localization we tested if previously formed CRDs respond to MeJA treatment with increased capacity for diterpenoid biosynthesis. We also tested the dynamics of diterpene synthase localization in the cambial zone. Immunofluorescence localization was performed using an antibody against a diterpene synthase, levopimaradiene/abietadiene synthase (LAS), in stem cross-sections of untreated and 0.1% MeJA-treated 4-year old Sitka spruce (P. sitchensis) trees. No fluorescence signal was observed in untreated stem cross-sections; however, signal was present 2 days after treatment with MeJA exclusively in the epithelial cells of CRDs. Fluorescence steadily increased in the CRD epithelial cells 4 and 8 days after treatment. At 8 days, additional fluorescence was observed in developing epithelial cells of traumatic resin ducts TRDs in the cambial zone. These results confirm that resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in CRD epithelial cells early upon treatment with MeJA, and immature developing TRD epithelial cells produce diterpene synthase enzyme. Overall, the results of this work improve our understanding of spatial and temporal patterns of induced diterpene resin acid biosynthesis in conifers.     

Terpenoids are transported in the xylem sap of Norway spruce

Norway spruce is a conifer storing large amounts of terpenoids in resin ducts of various tissues. Parts of the terpenoids stored in needles can be emitted together with de novo synthesized terpenoids. Since previous studies provided hints on xylem transported terpenoids as a third emission source, we tested if terpenoids are transported in xylem sap of Norway spruce. We further aimed at understanding if they might contribute to terpenoid emission from needles. We determined terpenoid content and composition in xylem sap, needles, bark, wood and roots of field grown trees, as well as terpenoid emissions from needles. We found considerable amounts of terpenoids—mainly oxygenated compounds—in xylem sap. The terpenoid concentration in xylem sap was relatively low compared with the content in other tissues, where terpenoids are stored in resin ducts. Importantly, the terpenoid composition in the xylem sap greatly differed from the composition in wood, bark or roots, suggesting that an internal transport of terpenoids takes place at the sites of xylem loading. Four terpenoids were identified in xylem sap and emissions, but not within needle tissue, suggesting that these compounds are likely derived from xylem sap. Our work gives hints that plant internal transport of terpenoids exists within conifers; studies on their functions should be a focus of future research.     

Diterpene resin acid biosynthesis in loblolly pine (Pinus taeda): functional characterization of abietadiene/levopimaradiene synthase (PtTPS-LAS) cDNA and subcellular targeting of PtTPS-LAS and abietadienol/abietadienal oxidase (PtAO, CYP720B1)

Diterpene resin acids are prominent defense compounds against insect pests and pathogens in conifers. Biochemical and molecular analyses in grand fir (Abies grandis), Norway spruce (Picea abies), and loblolly pine (Pinus taeda) have identified two classes of genes and enzymes that generate much of the structural diversity of terpenoid defense compounds: The terpenoid synthases (TPS) and cytochrome P450 monooxgenases (P450). Using a single substrate, geranylgeranyl diphosphate, families of single-product and multi-product diterpene synthases generate an array of cyclic diterpene olefins. These diterpenes are converted to diterpene resin acids by activity of one or more P450 enzymes. A few conifer diterpene synthases have previously been cloned and characterized in grand fir and in Norway spruce. We have also previously shown that the loblolly pine P450 abietadienol/abietadienal oxidase (PtAO) catalyzes multiple oxidations of several diterpene alcohols and aldehydes. Conifer diterpene synthases are thought to function in plastids while P450s can also be localized to plastids or to the endoplasmic reticulum (ER). Here, we show that a loblolly pine cDNA (PtTPS-LAS) encodes a typical multi-product conifer diterpene synthase that forms levopimaradiene, abietadiene, palustradiene, and neoabietadiene similar to the grand fir abietadiene synthase and Norway spruce levopimaradiene/abietadiene synthase. Subcellular targeting of PtTPS-LAS and PtAO to plastids and ER, respectively, was shown with green fluorescent fusion protein expression in tobacco cells. These data suggest that enzymes for conifer diterpene resin acid biosynthesis are localized to at least two different subcellular compartments, plastids and ER, requiring efficient transport of intermediates and secretion of diterpene resin acids into the extracelluar space.     

White pine weevil performances in relation to budburst phenology and traumatic resin duct formation in Norway spruce

1 As the phenological window hypothesis was reported to be significant in influencing the fitness of many herbivores feeding on tree foliage, could it also explain the performance of an insect such as the white pine weevil Pissodes strobi mainly attacking the bark phloem of conifers? 2 Under field conditions, adult weevils were caged on Norway spruce trees presenting a natural variation in their shoot growth phenology. 3 We evaluated white pine weevil biological performances, including oviposition, the number of emerged insects, survival, adult mean weight and tree defense responses as reflected by the production of induced resin canals. 4 None of the white pine weevil biological parameters was significantly affected by Norway spruce phenology. 5 The number of eggs per hole, the number of oviposition holes per leader, the number of emerged adults and their mean weight were not affected by host phenology. 6 The intensity of the traumatic response observed was variable and not correlated with budburst phenology. 7 Trees with higher traumatic responses, forming two or more layers of traumatic ducts, had lower adult emergence and estimated survival. 8 The distance between the first layer of traumatic resin ducts and the start of the annual ring was not correlated with the number of emerged weevils. 9 Norway spruce, which is an exotic tree in North America and a relatively recent host for the white pine weevil, might not possess the defense mechanisms necessary to fight off the white pine weevil.     

cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis).

Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction.     

茉莉酸甲酯对马尾松萜类合成酶活性及其细胞化学定位的影响

马尾松(Pinus massoniana)是我国南方生态建设与造林用材的主要树种,为了揭示马尾松抗虫机理尤其是诱导抗虫性的分子机制,该研究以马尾松幼苗为材料,通过外源喷施茉利酸甲酯(Me JA),分析了处理与对照间植株针叶显微结构、萜类合成酶活性及其细胞化学定位的变化。结果表明:在0.2 mmol·L~(-1)Me JA处理下马尾松植株松针中萜类物质,尤其是单萜、二萜的相对含量增加,马尾松毛虫拒食性明显,诱导抗性增强。显微观测中,针叶叶肉细胞内树脂道分泌物增加,叶绿体数目减少,但叶绿体体积增大,叶绿体片层结构增加。Me JA处理4周后,针叶中萜类合成酶活性增加,通过电镜酶细胞化学观察,膜系统尤其是叶绿体膜上萜类合成酶活性定位明显增强。这说明Me JA诱导的马尾松诱导抗性可能与改变的叶绿体结构及绿色质体萜类合成酶活性密切相关。     

Exogenous application of methyl jasmonate elicits defenses in Norway spruce (Picea abies) and reduces host colonization by the bark beetle Ips typographus

The terpenoid and phenolic constituents of conifers have been implicated in protecting trees from infestation by bark beetles and phytopathogenic fungi, but it has been difficult to prove these defensive roles under natural conditions. We used methyl jasmonate, a well-known inducer of plant defense responses, to manipulate the biochemistry and anatomy of mature Picea abies (Norway spruce) trees and to test their resistance to attack by Ips typographus (the spruce bark beetle). Bark sections of P. abies treated with methyl jasmonate had significantly less I. typographus colonization than bark sections in the controls and exhibited shorter parental galleries and fewer eggs had been deposited. The numbers of beetles that emerged and mean dry weight per beetle were also significantly lower in methyl jasmonate-treated bark. In addition, fewer beetles were attracted to conspecifics tunneling in methyl jasmonate-treated bark. Stem sections of P. abies treated with methyl jasmonate had an increased number of traumatic resin ducts and a higher concentration of terpenes than untreated sections, whereas the concentration of soluble phenolics did not differ between treatments. The increased amount of terpenoid resin present in methyl jasmonate-treated bark could be directly responsible for the observed decrease in I. typographus colonization and reproduction.     

Monoterpene synthase activities in leaves of Picea abies (L.) Karst. and Quercus ilex L

In addition to direct ecological functions in the interaction of plants with the environment, the emission of monoterpenes, especially from the foliage of evergreen trees, is of great importance for the production of ozone and photochemical oxidants in the troposphere. In the present work, we established a reproducible non-radioactive standard enzyme assay and characterized monoterpene synthase activities in needles of Norway spruce (Picea abies (L.) Karst.) and in leaves of holm oak (Quercus ilex L.). In Norway spruce, the dominant monoterpenes formed were alpha-pinene, camphene, and to a lesser extent beta-pinene and limonene. In holm oak, alpha-pinene, sabinene, and beta-pinene were the main products, while limonene was a minor component. Under optimum conditions, in both Norway spruce and holm oak, monoterpene formation remained constant up to 180 min and 90 min, respectively, and varied with the buffer and Mg2+ and Mn2+ concentrations used. Optimum temperature for monoterpene synthase activity was 40 degrees C in both species; optimal pH ranged between 6.5 and 7.5 in both species. Apparent Michaelis-constants for the substrate GDP were ca. 17.9 +/- 5.1 microM for Norway spruce and ca. 69.4 +/- 22.1 microM for holm oak. Molecular weight determination by FPLC indicated that the monoterpene synthases in Norway spruce and holm oak have native molecular weights of ca. 59 and 50 kDa, respectively.