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Terpenoid profiling and biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are susceptible or resistant to attack by the white pine weevil (Pissodes strobi)
... As part of our continuing investigation of essential oils of North American gymnosperms, we have obtained the foliar essential oils from three individual P. sitchensis trees growing in the Van Duzer Forest, Oregon Coast Range. There has been a previous report on P. sitchensis essential oils from coastal British Columbia [16], as well as a report on volatiles identified in P. sitchensis bark extracts [17]. ...
... That is, the major components in both collections were α-pinene, β-pinene, myrcene, β-phellandrene, isoamyl isovalerate, 3-methyl-3- butenyl isovalerate, and piperitone. Furthermore, the major monoterpenoids, α-pinene, β-pinene, δ-3carene, myrcene, and β-phellandrene, were also observed to be major components in the bark extracts from Vancouver Island, British Columbia [17]. A comparison of the main foliar essential oil components is summarized in Table 3. α-Pinene is a relatively abundant constituent in the foliar essential oils of Picea species [25][26][27]. ...
... Camphene, linalool, terpinen-4-ol, and αterpineol were virtually racemic. Robert [17] also found (-)-α-pinene, (-)-β-pinene, (+)-δ-3-carene, and (-)-β-phellandrene to dominate the bark extract of P. sitchensis. However, (+)-limonene rather than (-)limonene was identified in the bark extract. ...
... (+)-3-Carene and terpinolene were found to be associated with resistance in Sitka spruce (Picea sitchensis) genotypes originating from an area that may have been subject to high weevil pressure (Hall et al., 2011;Robert, 2010;. In a feeding bioassay, an extremely resistant clone with a high (+)-3-carene content reduced white pine weevil (Pissodes strobi) feeding by 50% compared with an extremely susceptible clone with trace (+)-3-carene . ...
... Several diterpene acids, including abietic and dehydroabietic acids, are known to have a significant impact on the growth of Hp (Kusumoto et al., 2014) and bark beetle-associated fungal pathogens (Kopper et al., 2005). Dehydroabietic acid has also been identified as a strong indicator of resistance of Sitka spruce against white spruce weevil (Robert, 2010;. Here, we note that the two less susceptible clones (393 and 457) had higher constitutive diterpene levels in the intact bark, and higher induced levels of thunbergene, thunbergol and a few other diterpenes after fungal infection compared with the two susceptible clones. ...
Constitutive and inducible terpene production is involved in conifer resistance against insects and fungal infestations. To gain knowledge about local defence responses of Norway spruce bark against pathogens and to find potential chemical markers for resistance breeding, we inoculated the stem of 8‐year‐old Norway spruce (Picea abies) clonal trees with both Endoconidiophora polonica (Ep, a common fungal pathogen associated with the spruce bark beetle Ips typographus) and Heterobasidion parviporum (Hp, a severe pathogen causing root and stem rot disease). Three weeks after inoculation, the fungal‐inoculated and intact bark from each tree was sampled. The terpenes in tree bark were extracted with hexane and characterized by gas chromatography–mass spectrometry (GC‐MS). The two fungi induced varied terpene responses in the four spruce clones used. Three of the clones showed a 2.3‐fold to 5.7‐fold stronger terpene response to Hp relative to Ep inoculation, while one clone responded similarly to inoculation with the two fungal pathogens. The amount of the diterpenes thunbergol and geranyllinalool varied between the clones. The level of thunbergol was higher in both intact and fungal‐inoculated bark from the less susceptible clones compared with the more susceptible clones. Geranyllinalool was present in higher amounts in the susceptible clones and is thus a possible marker for susceptibility. Our observations show that Norway spruce employs a similar chemical mechanism against the two fungal pathogens. Based on the present and earlier published data, we suggest that certain Norway spruce genotypes have a strong defence reaction against these two pathogens. The diterpenes thunbergol and geranyllinalool might be useful markers of susceptibility in tree‐breeding programmes and should be the focus of further detailed investigations.
... (+)-3-Carene and terpinolene were found to be associated with resistance in Sitka spruce (Picea sitchensis) genotypes originating from an area that may have been subject to high weevil pressure (Hall et al., 2011;Robert, 2010;. In a feeding bioassay, an extremely resistant clone with a high (+)-3-carene content reduced white pine weevil (Pissodes strobi) feeding by 50% compared with an extremely susceptible clone with trace (+)-3-carene . ...
... Several diterpene acids, including abietic and dehydroabietic acids, are known to have a significant impact on the growth of Hp (Kusumoto et al., 2014) and bark beetle-associated fungal pathogens (Kopper et al., 2005). Dehydroabietic acid has also been identified as a strong indicator of resistance of Sitka spruce against white spruce weevil (Robert, 2010;. Here, we note that the two less susceptible clones (393 and 457) had higher constitutive diterpene levels in the intact bark, and higher induced levels of thunbergene, thunbergol and a few other diterpenes after fungal infection compared with the two susceptible clones. ...
Constitutive and inducible terpene production is involved in conifer resistance against insects and fungal infestations. To gain knowledge about local defence responses of Norway spruce bark against pathogens and to find potential chemical markers for re-sistance breeding, we inoculated the stem of 8-year-old Norway spruce (Picea abies) clonal trees with both Endoconidiophora polonica (Ep, a common fungal pathogen asso-ciated with the spruce bark beetle Ips typographus) and Heterobasidion parviporum (Hp, a severe pathogen causing root and stem rot disease). Three weeks after inoculation, the fungal-inoculated and intact bark from each tree was sampled. The terpenes in tree bark were extracted with hexane and characterized by gas chromatography–mass response to Hp relative to Ep inoculation, while one clone responded similarly to inocu-lation with the two fungal pathogens. The amount of the diterpenes thunbergol and geranyllinalool varied between the clones. The level of thunbergol was higher in both intact and fungal-inoculated bark from the less susceptible clones compared with the -tible clones and is thus a possible marker for susceptibility. Our observations show that Norway spruce employs a similar chemical mechanism against the two fungal patho-gens. Based on the present and earlier published data, we suggest that certain Norway spruce genotypes have a strong defence reaction against these two pathogens. The diterpenes thunbergol and geranyllinalool might be useful markers of susceptibility in tree-breeding programmes and should be the focus of further detailed investigations.
Conifers are the most widely distributed group of gymnosperms in the world. They have large genome size (1C-value) compared with most animal and plant species. The genome size ranges from similar to 6,500 Mb to similar to 37,000 Mb in conifers. How and why conifers have evolved such large genomes is not understood. The conifer genome contains similar to 75% highly repetitive DNA. Most of the repetitive DNA is composed of non-coding DNA, including ubiquitous transposable elements. Conifers have relatively larger rDNA repeat units, larger gene families generated by gene duplications, larger nuclear volume, and perhaps larger genes, as compared to angiosperm plants. These genomic components may partially account for the large genome size, as well as variation in genome size, in conifers. One of the major mechanisms for genome size expansion and evolution of species is polyploidy, which is widespread in angiosperms, but it is rare in conifers. There are only a few natural polyploids in one family of conifers, Cupressaceae. Other conifers, including well-studied pines, are nearly all diploids. Whether ancient polyploidy has played a role in the evolution of genome size in conifers still remains an open question. The mechanisms that account for the variation and evolution of genome size in conifers are addressed in this review.
A synthesis of white pine weevil (Pissodes strobi Peck) mortality caused by predation (insects, birds, and mammalian predators), crowding of pupae, and abiotic factors, acting under different stand conditions given by the type of reforestation, depth of the duff, distance from woody debris, and presence of competing vegetation is presented, based on a four-year study conducted in northeastern Ontario. The type of reforestation (planted vs. aerial seeding) did not influence mortality to immature weevils in the leaders from either bird predation, insect predators and parasitoids or crowding of pupae, but 16% more weevils died during winter in a planted than in a seeded stand suggesting that there was an effect of site condition or small mammal predation on overwintering adults in the duff. Over all conditions, overwintering mortality averaged 76-92% with small mammal predation representing about 5-13%. Site conditions which augmented small mammal populations and predation, such as proximity to woody debris and presence of competing vegetation, had no effect on overwintering mortality of the weevils. The most important factor influencing overwintering mortality was likely the depth of duff where an inverse relationship was observed between mortality and depth. Forest managers should address conditions of duff depth and small mammal predation to increase the already significant overwintering mortality of adult weevils in young jack pine stands.
Sitka spruce (Picea sitchensis) is highly susceptible to leader damage by the white pine weevil (Pissodes strobi). Population differences for resistance have been previously noted from older IUFRO provenance trials both from hybrid sources (the natural hybrids of Sitka and white spruce), but also in populations of pure Sitka from dryer ecological zones. It was postulated that this resistance might be common within this high hazard ‘dry’ zone because of potentially higher selection pressures from increased weevil density. Results from a population transect of this zone, established as an openpollinated progeny/provenance trial, show that resistance is not broadly based but is centred about one of the original IUFRO resistant sources. Although hazard-zone differences were significant, sources within zone, had the strongest effect in our model; particularly evident was a strongly defined population effect around the original IUFRO source. Heritability estimates for resistance were also affected by this strong source population effect.
This publication reports on the results of over two decades of research in the Sitka Spruce Breeding Program. The objective of the Sitka spruce (Picea sitchensis [Bong.] Carr.) breeding program is to develop, propagate, and deploy genotypes with robust resistance to the white pine weevil (Pissodes strobi Peck). The program is based on research that has been conducted on the extent and nature of genetic resistance in Sitka spruce populations in British Columbia. This research has international stature and provides a successful model for incorporating the results of research on natural genetic resistance to insect pests into applied breeding programs and proactive forest management.
Variation in tree height and numbers of attacks by the Sitka spruce weevil (= white pine weevil), Pissodes strohi (Peck), were studied among families of a resistant provenance of Picea sitchensis (Bong.) Carr. at two Vancouver Island sites. At Sayward, after 14 years, the number of trees attacked varied by family from 0 to 80%. A significant association was found between the percentage of trees attacked in a family and the mean height of the family. Tall families were generally attacked more. At Fair Harbour (a clonal test), only 12% of the trees from the resistant provenance have been attacked after seven years, with all but one of the attacks concentrated on one of the two families tested. A multigenic or multicomponent basis for resistance is proposed and discussed.
The effects of attack by the Sitka spruce weevil, Pissodes strobi (Peck.) on Sitka spruce trees, Picea sitchensis (Bong.) Carr, are described. The study was based on records of attackcollected for 10 years (between 1959-1968), in a stand located near Nitinat Lake, British Columbia. The stand was re-visited in 1984 and tree quality was rated based on stem form. Also, the consequences of each attack in the period 1959-1968 for the quality of the stem in 1984 was determined. Twenty six percent of the trees in this severely attacked area were rated as having good form; 51% had a medium form and 23% were so deformed that their condition was rated as poor (no good for lumber) The trees sustained an average 2.4 attacks per tree in the 1959-1968 period. These attacks resulted in 1.6 defects per tree. It was concluded that the Sitka sppruce weevil caused a severe productivity depletion of the stand surveyed.
Isoprenoid biosynthesis in plants proceeds via two independent pathways: 1) the cytosolic classical acetate/mevalonate pathway (biosynthesis of sterols, sesquiterpenes, triterpenoids) and 2) via the non-mevalonate 1-deoxy-D-xylulose-5-phosphate (DOX-P) pathway for the biosynthesis of plastidic isoprenoids such as carotenoids, phytol (side-chain of chlorophylls), plastoquinone-9, isoprene, mono- and diterpenes. Both pathways form isopentenyl-diphosphate (IPP) as precursors, from which all other isoprenoids are formed via head-to-tail addition. The present knowledge of the novel 1-deoxy-D-xylulose-5-phosphate (DOX-P) pathways for isopentenyl-diphosphate biosynthesis, which is apparently located in plastids, is reviewed in this contribution. It provides a new insight into chloroplast metabolism.
Der pflanzliche 1-Desoxy-D-xylulose-5-phosphat-Weg in der Biosynthese von Isoprenoiden
Die Isoprenoidbiosynthese der Pflanzen verläuft über zwei voneinander unabhängige Biosynthesewege: 1. über den cytosolischen Acetat/Mevalonat-Weg (Biosynthese von Sterolen, Sesquiterpenen und Triterpenoiden) und 2. über den Mevalonatunabhängigen 1-Desoxy-D-xylulose-5-phosphat (DOX-P)-Weg für die Bildung der plastidären Isoprenoide, z.B. Carotinoide, Phytol (Seitenkette von Chlorophyll), Plastochinon-9, Isopren, Mono- und Diterpene. Beide Biosynthesewege bilden Isopentenyldiphosphat (IPP) als aktive Isoprenoidstufe, aus der sich durch Kopf-Schwanz-Addition alle Isoprenoide ableiten. Der gegenwärtige Stand unserer Kenntnis des 1-Desoxy-D-xylulose-5-phosphat-Biosynthesewegs für die Bildung von IPP, der offensichtlich in Plastiden lokalisiert ist, wird in diesem Beitrag zusammengefaßt. Der IPP-Biosyntheseweg gibt neue Einblicke in den Chloroplasten-Stoffwechsel.
Tree diameter, height, form and number of stem defects were recorded in 26-yr-old plantations of Picea sitchensis established at 3 initial spacings (2.74 × 2.74, 3.66 × 3.66, and 4.57 × 4.57 m). These plantations were heavily attacked by Pissodes strobi from an early age. Denser plantations sustained a lower intensity of attack than the more open plantations. Although the 3 spacings had similar average number of stem defects per tree, trees in the close spacing had a significantly higher frequency of trees of good form relative to the more open plantations. As a management regime, it is recommended that Sitka spruce plantations be started at close spacing and precommercially thinned at age 25. By this age trees will average about 19 cm in dbh and 12 m in height, thus ensuring a first log of good quality. -from Authors
The feeding stimulatory activity on the white pine weevil, Pissodes strobi Peck, of various types of extracts from Sitka spruce, Picea sitchensis (Bong.) Carr., was tested using a paired agar disc bioassay. Chemicals that induce feeding apparently constitute a complex mixture of non-volatile compounds in the phloem and on the surface of the bark and needles. Volatile chemicals from the bark and foliage, captured in Porapak Q, did not attract walking P. strobi in two olfactometers, nor did they trigger a feeding response when tested on plain agar. However, the monoterpenes α-pinene, β-pinene, and β-myrcene acted as synergists to the nonvolatile chemicals in the bark, while piperitone had a marked feeding deterrent effect. (+)-Camphor and limonene stimulated the feeding at low concentrations but caused feeding inhibition when the concentration rose above a particular threshold.
A feeding bioassay forPissodes strobi Peck was developed that consisted of plastic petri dishes containing paired agar disks immersed in paraffin wax. Lens paper covering the top surface of the disks provided 1, 2, or 3 weevils in each replicate with a surface through which they made regular feeding punctures which could be easily counted. The number of feeding punctures was correlated with the amount of agar ingested. Candidate feeding stimulants or deterrents were applied to the paper covering one of the disks in the dishes, while the other served as a solvent control. Feeding stimulants were tested using disks of pure agar, and deterrents were assayed on agar disks that contained 2% ground, dried Sitka spruce bark,Picea sitchensis Carr. The weevils exhibited a concentration-dependent response to the amount of spruce bark in the agar disk. Feeding stimulation by Sitka spruce bark extracts, and deterrency by leaf oil of western red cedar,Thuja plicata Donn, was demonstrated. The bioassay would be useful in chemical isolation programs aimed at identification of feeding stimulants and deterrents.
In book: Mechanisms and Deployment of Resistance in Trees to Insects."M.R. Wagner, K.M. Clancy, F. Lieutier, and T.D. Paine (eds.). 2002. Kluwer Academic Press, Dordnecht, the Netherlands.
Conifers have evolved elaborate inducible, terpenoid-based defense mechanisms to deter attacks from bark beetles and other herbivore species. Herbivore damage triggers the production of oleoresin containing monoterpene, sesquiterpene and diterpene components that serve as toxins and physical barriers to herbivore invasion. Induced terpene formation appears to be regulated by specific enzymes of terpene metabolism whose activity increases on herbivore damage. Among the best studied of these are terpene synthases, enzymes which convert acyclic prenyl diphosphates to the parent terpene skeletons. Terpene synthase activity in turn is regulated by the transcription of terpene synthase genes. Induced terpene biosynthesis is also often accompanied by extensive cellular differentiation, including the formation of new resin ducts. The signal transduction cascades that initiate these shifts in conifer metabolism and cell differentiation are poorly understood due to the lack of well-developed model systems and appropriate genetic mutants. However, there are strong indications that octadecanoid pathway metabolites and ethylene have roles in this signaling, as they do in defense signaling in angiosperms. There are still large gaps in our knowledge of the signal transduction networks leading to herbivore-induced terpenoid accumulation in conifers. However, the development of new genomic, proteomic and metabolomic tools, as well as the establishment of convenient in vitro systems should facilitate more rapid advances in this field in the near future. The results will have important implications for understanding the evolution of conifer defense mechanisms as well as for the management of commercially important forest tree species, such as spruce, pine, and fir.
Species distribution models predict a wholesale redistribution of trees in the next century, yet migratory responses necessary to spatially track climates far exceed maximum post-glacial rates. The extent to which populations will adapt will depend upon phenotypic variation, strength of selection, fecundity, interspecific competition, and biotic interactions. Populations of temperate and boreal trees show moderate to strong clines in phenology and growth along temperature gradients, indicating substantial local adaptation. Traits involved in local adaptation appear to be the product of small effects of many genes, and the resulting genotypic redundancy combined with high fecundity may facilitate rapid local adaptation despite high gene flow. Gene flow with preadapted alleles from warmer climates may promote adaptation and migration at the leading edge, while populations at the rear will likely face extirpation. Widespread species with large populations and high fecundity are likely to persist and adapt, but will likely suffer adaptational lag for a few generations. As all tree species will be suffering lags, interspecific competition may weaken, facilitating persistence under suboptimal conditions. Species with small populations, fragmented ranges, low fecundity, or suffering declines due to introduced insects or diseases should be candidates for facilitated migration.
Sitka spruce (Picea sitchensis (Bong.) Carr), white spruce (P. glauca (Moench) Voss) and Engelmann spruce (P. engelmanni (Parry)) plantations in British Columbia (B.C.), Canada, have come under serious attack from the white pine weevil, Pissodes strobi (Peck) (Coleoptera: Curculionidae). This pest attacks and destroys the terminal leader of the tree, causing serious growth losses and stem deformities. Since 1993, we have conducted a series of screening trials to search for spruce with resistance to the white pine weevil. Spruce tree selections from across the range of the species have been planted in several replicated trials. At 12 of these trial sites, in order to accelerate the screening process, local insect populations were augmented by adding reared weevils to the site. Results from our two oldest trials, at Jordan River and Cowichan Lake (planted in 1994), both on Vancouver Island, B.C., indicate that screening for weevil resistance can be effectively accomplished by using the weevil population augmentation method. Consistent selections could be obtained in trials with as few as 10 replicates per family. If weevil attack rates of 50% cumulative attack are obtained, then consistent selections may be obtained in as little as 4 years, which is a fairly quick turn-around time for studying resistance in trees.
A survey of genome size variation in 117 gymnosperms has found that the systematic coverage of the measurements is uneven,
some families have been well sampled but others have not. There is a 14·4-fold variation in genome size within the group;
the largest genome being that of Pinus lambertiana with 63·5 pg DNA/2C nucleus and the smallest, 12·96 pg DNA/2C nucleus in Metasequoia glyptostroboides. The different methods that have been used to measure genome size in gymnosperms, the choice of standards, and problems associated
with the different methods are reviewed. The conflicting reports of intraspecific variation in genome size in gymnosperm species
are problematic. Some appear real whereas others can be attributed to problems associated with methodology. Some studies of
species show considerable uniformity of genome size over a wide geographical range whereas others find high levels of variation
which may, in some cases, be correlated with geographical parameters. Possible correlations between genome size and adaptive
features of gymnosperms are examined and a number of correlations are reported between genome size and growth-related parameters.
Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer.
We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing.
We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.
We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.
Two recent letters to the editor of The Plant Cell (Gutierrez et al., 2008; Udvardi et al., 2008) highlighted the importance of following correct experimental protocol in quantitative RT-PCR (qRT-PCR). In these letters, the authors outlined measures to allow precise estimation of gene expression by ensuring the quality of material, refining laboratory practice, and using a normalization of relative quantities of transcripts of genes of interest (GOI; also called target genes) where multiple reference genes have been analyzed appropriately. In this letter, we build on the issues raised by considering the statistical design of qRT-PCR experiments, the calculation of normalized gene expression, and the statistical analysis of the subsequent data. This letter comprises advice for taking account of, in particular, the first and the last of these three vital issues. We concentrate on the situation of comparing transcript levels in different sample types (treatments) using relative quantification, but many of the concerns, particularly those with respect to design, are equally applicable to absolute quantification.
Members of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests. These conifer forests are of critical importance for global ecosystem stability and biodiversity. They also provide the majority of the world's wood and fiber supply and serve as a renewable resource for other industrial biomaterials. In contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. Sequence-finished full-length (FL)cDNAs and large collections of expressed sequence tags (ESTs) are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation.
As part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed 20 standard, normalized or full-length enriched cDNA libraries from Sitka spruce (P. sitchensis), white spruce (P. glauca), and interior spruce (P. glauca-engelmannii complex). We sequenced and analyzed 206,875 3'- or 5'-end ESTs from these libraries, and developed a resource of 6,464 high-quality sequence-finished FLcDNAs from Sitka spruce. Clustering and assembly of 147,146 3'-end ESTs resulted in 19,941 contigs and 26,804 singletons, representing 46,745 putative unique transcripts (PUTs). The 6,464 FLcDNAs were all obtained from a single Sitka spruce genotype and represent 5,718 PUTs.
This paper provides detailed annotation and quality assessment of a large EST and FLcDNA resource for spruce. The 6,464 Sitka spruce FLcDNAs represent the third largest sequence-verified FLcDNA resource for any plant species, behind only rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the only substantial FLcDNA resource for a gymnosperm. Our emphasis on capturing FLcDNAs and ESTs from cDNA libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. Sequence comparisons against five plant genomes and the non-redundant GenBank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. Opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed.
The isolation of intact, functional RNA from conifer spp. is not easy, especially from those tissues that are heavily lignified and characterized by a low number of living cells. An efficient procedure for isolating RNA from combined wood and bark tissues of conifers was developed based on a protocol optimized for the extraction of RNA from pollen and one for the isolation of RNA from woody stems. This protocol does not involve the use of phenol, and no ultracentrifugation was required. In addition, the protocol overcame the problems of RNA degradation and low yield due to oxidation by polyphenolics and co-precipitation with polysaccharides, both of which are abundant components in conifer bark tissues. The isolated RNA was of high quality and undegraded as gauged by spectrophotometric readings and electrophoresis in denaturing agarose gels. Quality was further assessed through the subsequent use of the RNA in reverse transcription and RT-PCR, indicating that it could be used for a number of downstream purposes including Northern blot hybridization and cDNA library construction. Using this modified protocol, 80-150 micrograms of RNA was routinely obtained from 1 g of fresh material. This protocol was also used for the isolation of RNA from needles of spruce spp., from which 750-950 micrograms RNA per gram of starting material could routinely be obtained.
Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.
Available information on the interaction between Sitka spruce weevil, Pissodes strobi (Peck), and its host Sitka spruce, Picea sitchensis (Bong) Carr., is examined. Based on a theoretical consideration of this information, a new mechanism is proposed to explain the resistance exhibited by some of the genetically distinct individuals of some provenances of this species. Experimental evidence is presented to show that resistant trees can sufficiently affect P. strobi reproduction and progeny development and survival to reduce this pest’s ability to kill the leaders of these trees when challenged with weevils containing already mature eggs. Our mechanism also provides an explanation as to how weevils are able to avoid attacking and reproducing on resistant trees in which their progeny would fail.
The bark of Norway spruce trees,Picea abies (L.) Karst., was wounded to produce areas of bark with differing biochemical characteristics. Adults and larvae ofDendroctonus micans Kug. were inserted into these trees at points around the wounds. Larval survival was higher, larval dry weights were higher, and adults were more likely to oviposit at sites around the wound that had higher phloem moisture contents. Larvae showed higher survival and dry weights in phloem with low terpene contents. Adults oviposited in phloem with lower stilbene glucoside contents. Phloem moisture differed at sites around the wound compared with that at a control site away from the wound, and the nitrogen content of entire bark cores was higher above wounds than at control sites away from the wounds. Phloem moisture correlated with the nitrogen content of entire bark. Phloem terpene contents correlated with each other, as did the stilbene contents. Associations between the host exploitation pattern ofD. micans and the biochemistry of the host phloem are discussed in relation to preselection by adults of sites suitable for larval development.
In the last five years, much progress has been made in the characterization of conifer defenses using an integrated biochemical, molecular, and genomics approach. With a focus on the fascinating processes of traumatic resin duct development in direct defense and on induced volatile emissions in possible tritrophic defense, we have developed species of spruce, Sitka spruce, and Norway spruce, as two of the best characterized systems of conifer defense research at the molecular and biochemical levels. The economic importance of spruce for the forest industry and the enormous economic, environmental, and social impacts of recent, unprecedented outbreaks of conifer insect pests has mandated substantial new investment into conifer genomics research with an emphasis on the genomics of defense and resistance of conifers against insect pests.
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Variation in tree height, number of attacks by the Sitka spruce weevil (= white pine weevil) (Pissodes strobi [Peck]), tree form, and stem defect were studied in a 15-year-old Sitka spruce (Picea sitchensis [Bong.] Carr.) provenance test near Sayward, on Vancouver Island, B.C. An aggregated spatial distribution of the attacks was found in the plantation. Average number of attacks, tree form, and total tree height varied significantly among provenances and among families within provenances in both light and severe infestation patches. The number of unattacked trees varied by provenance from 5 to 51%, but in patches of severe infestation it varied from 0 to 64%. The number of attacks per tree had a significant negative effect on tree height. However, at the same level of attack, some provenances grew significantly taller than others. Thirty, 19, and 51% of all trees were classified as having a good, medium, or poor form, respectively. Among provenances located in severe infestation patches, the percentages of trees having good form varied from 4 to 64%. The type of defect that formed after an attack (minor crook, major crook, or fork) varied by provenance. Aggregated weevil attack and genetic differences among provenances may have accounted for this variation.
The 50 known species of North American Clubiona Latreille, 1804 are rearranged in seven species-groups as follows: trivialis group (1 Holarctic, 4 Nearctic), obesa group (11 Nearctic), reclusa group (2 Holarctic, 3 Nearctic), pallidula group (1 Holarctic), abboti group (25 Nearctic), lutescens group (1 Holarctic, 1 Nearctic), maritima group (1 Nearctic). Clubiona quebecana and C. angulata are described as new species from eastern North America. C. kuratai Roddy, 1966, originally described from the female only, is synonymized under C. chippewa Gertsch, 1941, which was originally described from the male only. C. opeongo Edwards, 1958 and C. bishopi Edwards, 1958 are redescribed, the male of the former and the female of the latter not having been previously described.
The objective of this investigation was to determine if external factors such as phototaxis and geotaxis determine oviposition site location by reproductively active female white pine weevils, Pissodes strobi (Peck), on intact trees or cut treetops. In prior studies, light and gravity, both external tree factors, were consistently linked with oviposition location. In this study, the external factors that were varied in whole trees or cut treetops, included two weevil populations and two tree species in upright, inverted and horizontal orientations with thick or thin stems, indoor and outdoor locations, warm and cool, wet and dry, and light and dark conditions. The results show that 100% of the oviposition occurred at or near treetops, regardless of external factors. The results are unequivocal; they are without variance. These findings suggest that the reproductively active female weevil's feeding preference is for primary cortex tissue, an internal tree factor. The primary cortex thickness gradient could account for these weevils’ capability to locate treetops, where the thickest primary cortex tissue occurs, even when treetops are not at the highest or brightest locations.
Rapid development of plantation forestry is necessary to meet an increasing demand of fiber and wood for the forest products industry. In addition to delivering high productivity and high quality of cellulose and lignin formation, trees for plantation forestry must resist increasing pressure from insect pests and pathogens. Unlike in agriculture, as practiced in much of the 20th century, forestry cannot rely on massive application of pesticides for crop protection, because forests, including plantation forests, are complex ecosystems providing essential habitats for soil living and above ground organisms. At the current stage of genetic tree improvement, there are unique opportunities to retain or recapture much of the genetic material that determines natural defense and resistance for plantation forestry. Terpenoids and terpenoid synthase (TPS) genes are major components of defense and resistance in conifers and appear to be important for defense biology of poplars as well. Terpenoid defense in forest trees present multigenic traits that are highly variable within species and natural populations. Much of the chemical diversity of terpenoids in trees is determined by variable constitutive and inducible expression of members of large TPS gene families and by multi-product reaction mechanisms of TPS enzymes. Detailed knowledge of the organization and evolution of TPS gene families, TPS gene expression and TPS enzyme biochemistry is critical to maintain natural genetic and chemical diversity of terpenoids in trees selected and improved for plantation forestry. TPS genes can be targeted for development of markers for pest resistance and also as genetic markers for formation of terpenoid extractives that affect mills and toxicity of efflux water in the pulp and paper industries. In addition, the biochemical machinery of resin terpenoid formation and resin secretion in conifers can conceivably be harnessed for biotechnological production of high value compounds in renewable plantation forest resources. Conifers and poplars also emit massive amounts of reactive hemiterpenes and monoterpenes into the atmosphere. Another system of plantation forestry, Eucalyptus (not discussed in this chapter due to lack of published studies of TPS genes in this system) also emits monoterpenes in large quantitities and is protected against many herbivores by terpenoids.
A method was devised for determining the sex of live white-pine weevils, Pissodes strobi (Peck), without harming the insect. The weevils were held venter up and observed under a binocular microscope. The last abdominal tergite was pulled down and the anogenital vestibule opened with a mounted minuten insect pin bent at a right angle. Key characters used in sex determination were: (1) the pygidium (8th tergite of the male, 7th tergite of the female), (2) the posterior view of the opened anogenital vestibule, which exposed the displaced eighth abdominal tergite and the apodeme fork of the 8th sternite in the female or a small projection of the 8th abdominal tergite in the male.
Larvae of Lonchaea corticis Taylor killed and consumed pupae and to a lesser extent prepupal larvae of Pissodes strobi (Peck) at temperatures ranging from 5 to l4°C during a 3-wk experiment in glass vials that were monitored weekly. Marginal predation of pupae but not prepupal larvae was observed at O°c. In simulated pupal cells of P. strobi stored outdoors in a Victoria, British Columbia, winter, L. corticis larvae killed and consumed pupae and prepupal larvae of P. strobi in a 3-wk experiment that left most cells devoid of obvious prey remains. Tests in glass vials in March with L. corticis larvae collected during March and during the previous August showed that L. corticis larvae remain predacious at the end of winter. When the insects were left in situ in severed leaders stored outdoors in a Victoria, British Columbia, winter, the proportion of pupal cells with live P. strobi decreased significantly and the proportion of pupal cells emptied without insect emergence increased significantly; the numbers of L. corticis larvae and total P. strobi pupal cells did not change significantly. These experiments show that P. strobi pupae and prepupal larvae can be vulnerable prey for L. corticis larvae and that predation may be common in the winter conditions of coastal British Columbia.
flight and dispersal of the white-pine weevil, Pissodes strobi (Peck), through a white pine plantation was studied by releasing 409 marked weevils at a central point within a 1431-tree plantation and checking every tree at 5-day intervals. 'Weevils flew readily at the time of release. Marked weevils were recorded throughout the plantation, up to 330 feet from the release point. A few weevils were observed on the scattered white pines 200-300 yards from the release point beyond a hardwood barrier. Total numbers of weevils present on leaders increased from April 25 to May 4, after which the number steadily decreased. Males were slightly more abundant on leaders early in the season and females were slightly more abundant later in the season, but there were no sex-related differences in dispersal. A concentration of weevils within 30 feet of the release point was attributed to short-range flight with the wind direction. Taller trees and longer and thicker leaders were related to higher percentages of tree visitation by weevils.
Adult Pissodes strobi activity and brood production were compared at spruce plantations with high and low weevil infestation levels. During 2 years of observation, weevil activity started ca. 30 days earlier at the heavily infested plantation. Caging adults on terminals at both sites showed that the subsequent brood mortality was significantly higher at the plantation with low weevil damage. Poor brood survival was attributed mainly to resinosis.
Leaders of Picea sitchensis (Bong.)Carrière freshly attacked by Pissodes strobi (Peck) were covered with sleeve cages to exclude Lonchaea corticis Taylor. Unsuccessful empty pupal cells of P. strobi formed during the summer were associated with leaders without cages where L. corticis larvae were present. Unsuccessful empty pupal cells were generated during the winter in the presenee of L. corticis. Examination of all leaders in a plantation attacked by P. strobi in each of 2 yr showed predation by L. corticis on P. strobi brood that had formed pupal cells but little if any predation of P. strobi eggs or mining larvae. When the mean percentage of successful P. strobi emergence from pupal cells was 7%, no P. strobi emerged from leaders with more than two L. corticis per pupal cell formed by P. strobi; the following year mean emergence was 25% and the threshold for no emergence was near four L. corticis per pupal cell. With mean L. corticis numbers near one per pupal cell formed by P. strobi, the success of mature P. strobi brood may have been largely regulated by weather and resultant L. corticis predation.
The genetic structure of 41 Canadian populations (27 populations from British Columbia, two of which were from east of the Continental Divide, and the remaining 14 representing localities east of the Continental Divide) of the white pine weevil, Pissodes strobi (Peck), was investigated using isozyme markers. Differentiation among populations (FST) was estimated to be 0.084 and inbreeding within populations was low with estimated mean FIS of 0.011. Although overall genetic uniformity among the studied 41 populations was high (mean Nei’s genetic distance = 0.027), differences among groups of populations were revealed in cluster analyses. The 16 weevil populations obtained from areas east of the Continental Divide formed a significant cluster with the 11 populations collected from the Interior British Columbia. The 11 weevil populations examined from the South Coast–Vancouver Island of British Columbia were distinct from this cluster, as were the three weevil populations collected from the North–Central Coast of British Columbia. Populations within the South Coast–Vancouver Island group were the most genetically dissimiliar to each other, with Nei’s genetic distance ranging from 0.004 to 0.108. This was caused, in part, by differences in allele frequencies in the Bella Coola, Eve River, and Nanaimo populations relative to the other eight populations within this group. Genetic diversity estimates, in terms of the mean number of alleles per locus, the percentage of polymorphic loci and the mean expected and observed heterozygosity, were higher in eastern than western populations.
Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack.
The turpentine fraction of the oleoresin was shown to contain at least 38 sesquiterpenes that represent 12.5% of the turpentine,
with the monoterpenes comprising the remainder. Assays of cell-free extracts from grand fir stem with farnesyl diphosphate
as substrate indicated that the constitutive sesquiterpene synthases produced the same sesquiterpenes found in the oleoresin
and that, in response to wounding, only two new products were synthesized, δ-cadinene and (E)-α-bisabolene. A similarity based cloning strategy yielded two new cDNA species from a stem cDNA library that, when expressed
in Escherichia coli and the gene products subsequently assayed, yielded a remarkable number of sesquiterpene products. The encoded enzymes have
been named δ-selinene synthase and γ-humulene synthase based on the principal products formed; however, each enzyme synthesizes
three major products and produces 34 and 52 total sesquiterpenes, respectively, thereby accounting for many of the sesquiterpenes
of the oleoresin. The deduced amino acid sequence of the δ-selinene synthase cDNA open reading frame encodes a protein of
581 residues (at 67.6 kDa), whereas that of the γ-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa).
The two amino acid sequences are 83% similar and 65% identical to each other and range in similarity from 65 to 67% and in
identity from 43 to 46% when compared with the known sequences of monoterpene and diterpene synthases from grand fir. Although
the two sesquiterpene synthases from this gymnosperm do not very closely resemble terpene synthases from angiosperm species
(52–56% similarity and 26–30% identity), there are clustered regions of significant apparent homology between the enzymes
of these two plant classes. The multi-step, multi-product reactions catalyzed by the sesquiterpene synthases from grand fir
are among the most complex of any terpenoid cyclase thus far described.
A simple and rapid procedure for the simultaneous determination of monoterpenes and diterpenoid resin acids of conifer stem oleoresin is described; it involves tissue extraction with methyl tert-butyl ether and methylation of the resin acids with CH2N2. The resin acid methyl esters and the monoterpenes are then analysed in a single gas chromatographic step. Enantiomer composition of the major monoterpene constituents is determined, after preliminary purification by thin layer chromatography, by capillary gas chromatography on a cyclodextrin column. The method is applicable to conifer species of high resin content (Iodgepole pine) and low resin content (grand fir).
Resin flow differed significantly among three of six clones of lodgepole pine seed orchard trees, but did not differ among the clones categorized as susceptible or resistant to attack by the Douglas-fir pitch moth. A stepwise regression analysis identified δ-3-carene, cyclohexene, and -terpinolene as significant compounds, explaining 49.9% of the variation in the number of attacks per tree. δ-3-Carene alone explained 41.8% of the variation in the regression, and analysis of variance showed that resistant clones consistently had high relative amounts (>17.9%) of this compound, whereas susceptible clones had low amounts (<10%). The significant effect by cyclohexene and -terpinolene in the stepwise regression appeared to be due to a correlation between -terpinolene and δ-3-carene in several clones, and that cyclohexene was only present in one clone, rather than any discernable biological relationship. Limonene co-eluted with β-phellandrene, so its role must be determined by additional study.
Controlled-cross progeny of interior spruce (Picea glauca (Moench) Voss × Picea engelmanni Parry ex Engelm.) parents ranked as resistant or susceptible to the white pine weevil, Pissodes strobi (Peck) were screened for resistance to the same insect by augmentation of the trial site with weevils. Progeny from two resistant parents (R × R progeny) sustained significantly fewer weevil attacks (13% of the trees were attacked) in the year following the augmentation, than progeny from susceptible parents (S × S progeny) (68% of the trees were attacked). Progeny obtained by crossing one resistant and one susceptible parent (R × S progeny) sustained intermediate attack levels (47% were attacked). Characteristics of the bark resin canals of the crosses were explored using microscopy techniques. Bark resin canal density was highest in R × R progeny, lowest in S × S progeny and intermediate in R × S progeny. There was a negative correlation between the percentage of trees attacked in each cross and the average density of the outer resin canals for each cross. A discriminant function was developed that distinguished between resistant and susceptible progeny using bark characteristics. The function was characterized by positive coefficients for outer resin canal density and inner resin canal size, and a negative coefficient for bark thickness. Thus, trees with thin bark, large inner resin canals and dense outer resin canals are more likely to be resistant to P. strobi.
The braconid parasitoids Glyptapanteles indiensis (Marsh) and G. liparidis (Bouché) occur in sympatry and allopatry, respectively, with their congener G. flavicoxis (Marsh). We tested the hypothesis that all three parasitoids, but particularly sympatric G. indiensis and G. flavicoxis, use species-specific sex pheromone blends for close-range sexual communication. In coupled gas chromatographic–electroantennographic detection (GC–EAD) analyses of body extracts of conspecific females, male G. indiensis antennae responded to five components, one of which is specific to G. indiensis, and four are in common with G. flavicoxis. Male G. liparidis antennae responded to six components, two of which are specific to G. liparidis, and four are in common with G. flavicoxis. In Y-tube olfactometer experiments, body extracts of females elicited close-range attraction and wing-fanning responses only by conspecific but not by heterospecific males, supporting the hypothesis of close-range species-specific sex pheromone blends.
The circum-polar boreal forest has played an important role in the wealth of northern nations since the 15th century. Its natural resources spurred strategic geopolitical developments beginning in the 16th century but intense development of the boreal forest is largely limited to the 20th century. Insects cause considerable loss of wood that has an adverse effect on the balance of carbon sequestered by forests. Current understanding of processes that lead to stand-replacing outbreaks in three insect species is reviewed in this paper. Many of these processes depend on climate either directly, such as reduced survival with extreme weather events, or indirectly, mainly through effects on the host trees. In the boreal zone of Canada, pest-caused timber losses may be as much as 1.3–2.0 times the mean annual depletions due to fires. Pests are thus major, but consistently overlooked forest ecosystem components that have manifold consequences to the structure and functions of future forests. Global change will have demonstrable changes in the frequency and intensity of pest outbreaks, particularly at the margins of host ranges. The consequent shunting of carbon back to the atmosphere rather than to sequestration in forests as biomass is thought to have positive feedback to global warming. Whereas significant progress has been made in developing carbon budget models for the boreal forests of Canada, enormous problems remain in incorporating pest effects in these models. These problems have their origins in the nature of interactions among pests with forest productivity, and problems with scaling. The common problems of verification and validation of model results are particularly troublesome in projecting future forest productivity. The interaction of insects with fires must be accounted for if realistic carbon sequestration forecasts in a warming climate are to be made. These problems make assessments of mitigation and adaptation of pest management alternatives difficult to evaluate at present. Nevertheless, the impacts of stand-replacing insect population outbreaks is important in formulating future resource management policy.
Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis experiments. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, we have developed an environment for comparative protein modeling that consists of SWISS-MODEL, a server for automated comparative protein modeling and of the SWISS-PdbViewer, a sequence to structure workbench. The Swiss-PdbViewer not only acts as a client for SWISS-MODEL, but also provides a large selection of structure analysis and display tools. In addition, we provide the SWISS-MODEL Repository, a database containing more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, we hope to increase the use of protein structures and models in the process of experiment design.
Defense-related terpenoid biosynthesis in conifers is a dynamic process closely associated with specialized anatomical structures that allows conifers to cope with attack from many potential pests and pathogens. The constitutive and inducible terpenoid defense of conifers involves several hundred different monoterpenes, sesquiterpenes and diterpenes. Changing arrays of these many compounds are formed from the general isoprenoid pathway by activities of large gene families for two classes of enzymes, the terpene synthases and the cytochrome P450-dependent monooxygenases of the CYP720B group. Extensive studies have been conducted on the genomics, proteomics and molecular biochemical characterization of these enzymes. Many of the conifer terpene synthases are multi-product enzymes, and the P450 enzymes of the CYP720B group are promiscuous in catalyzing multiple oxidations, along homologous series of diterpenoids, from a broad spectrum of substrates. The terpene synthases and CYP720B genes respond to authentic or simulated insect attack with increased transcript levels, protein abundance and enzyme activity. The constitutive and induced oleoresin terpenoids for conifer defense accumulate in preformed cortical resin ducts and in xylem trauma-associated resin ducts. Formation of these resin ducts de novo in the cambium zone and developing xylem, following insect attack or treatment of trees with methyl jasmonate, is a unique feature of the induced defense of long-lived conifer trees.
Abietic acid is a major component of the rosin fraction of oleoresin synthesized by conifer species, such as grand fir (Abies grandis) and lodgepole pine (Pinus contorta), as a defensive secretion against insect and pathogen attack. This diterpenoid resin acid is derived from geranylgeranyl pyrophosphate by conversion to abietadiene and sequential oxidation of the C18-methyl group of the precursor olefin to a carboxyl function. Resin acid biosynthesis is constitutively expressed at high levels in lodgepole pine stem and is induced to these levels by stem wounding in grand fir. Soluble enzyme extracts of lodgepole pine stem and of mechanically wounded grand fir stem catalyzed the divalent metal ion-dependent cyclization of [1-3H]geranylgeranyl pyrophosphate to (-)-abieta-7(8),13(14)-diene. The wound-inducible enzyme was partially purified and identified as an 80-kDa protein with general characteristics typical of those of other terpenoid cyclases. Although the enzymatic cyclization sequence almost certainly involves the formation of copalyl pyrophosphate and a pimaradiene as stable intermediates, no evidence for the separation of the corresponding partial activities was obtained. A pathway involving the production of various pimaradiene and abietadiene isomers is proposed to account for the origin of several common resin acids.
Reproductive maturation and oviposition of the spruce terminal weevil (Pissodes strobi, Peck) are inhibited by resistant Sitka spruce (Picea sitchensis (Bong) Carr.). Vitellogenin is an egg yolk protein precursor which accumulates during the maturation of eggs. We describe the molecular cloning and characterization of a fragment of the vitellogenin gene from the spruce terminal weevil. The DNA sequence of this fragment has high identity to vitellogenin sequences from other insects and less to nematode and vertebrate vitellogenins. It hybridizes on Northern blots to a single 6.0 kb mRNA that is expressed only in females, and only after they have commenced reproductive development. Vitellogenin gene expression is induced by treatment with juvenile hormone, and is differentially regulated in insects feeding on resistant or susceptible trees. The effects of resistance on vitellogenin gene expression were studied to confirm our earlier findings and to provide a sensitive bioassay for identifying resistance factors.
Recent studies have uncovered the existence of an alternative, non-mevalonate pathway for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, the two building blocks of terpene biosynthesis.