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An integrated genomic, proteomic and biochemical analysis of (+)‐3‐carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil
Abstract
Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82–95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.
... In addition, traumatic resin duct formation and resin accumulation were observed, indicating not just a chemical plant response, but also a physical one. Another study conducted by Hall et al. [12] on Sitka spruce (Picea sitchensis) revealed that when under attack by the white pine weevil, resistant trees produced much higher levels of (+)-3carene than susceptible ones, in which only trace amounts were found of such a monoterpene. ...
... The lengths of the genomic sequences encompassing the ORFs of the seven genes were: A sequence alignment showed an almost perfect match among the cDNAs and the corresponding exonic regions of the genomic sequences, allowing a reliable determination of the exon/intron structure of each gene. All the seven genomic sequences were found to contain 10 exons and 9 introns (Table 1), consistent with the previously characterized genomic sequences of conifer MTPSs [12,41,42]. Moreover, the genomic structural characteristics of the seven MBOS/MTPS genes from P. laricio were found to be highly conserved, in terms of intron placements, the exon number and sizes, and the location of RR(X8)W and class-I DDxxD amino acid motifs ( Figure 4). ...
... Furthermore, the phasing of the intron insertion, identified as the location of the intron before the first, second, or third nucleotide position of the neighbouring codon and referred to as phases 0, 1, and 2, respectively [44], seemed to be equally well conserved ( Table 1). The high conserved genomic organization detected among the P. laricio MBOS/MTPS genes provided strong evidence of their common origin in conifers, confirming previous phylogenetic and protein structural studies, which demonstrated that the genes involved in the hemiterpene synthesis evolved independently in angiosperms and gymnosperms [6,12,34]. ...
In the present study, we carried out a quantitative analysis of the monoterpenes composition in different tissues of the non-model conifer Pinus nigra J.F. Arnold subsp. laricio Palib. ex Maire (P. laricio, in short). All the P. laricio tissues examined showed the presence of the same fourteen monoterpenes, among which the most abundant were β-phellandrene, α-pinene, and β-pinene, whose distribution was markedly tissue-specific. In parallel, from the same plant tissues, we isolated seven full-length cDNA transcripts coding for as many monoterpene synthases, each of which was found to be attributable to one of the seven phylogenetic groups in which the d1-clade of the canonical classification of plants' terpene synthases can be subdivided. The amino acid sequences deduced from the above cDNA transcripts allowed to predict their putative involvement in the biosynthesis of five of the monoterpenes identified. Transcripts profiling revealed a differential gene expression across the different tissues examined, and was found to be consistent with the corresponding metabolites profiles. The genomic organization of the seven isolated monoterpene synthase genes was also determined.
... According to our continuous literature review, no report has been found on the change of 3-carene in pine plant after infection of PWNs. Nonetheless, a few reports expressed the role of 3-carene against insect resistance in pine species [15][16][17][18]. The amounts of 3-carene were associated with the tolerance of Sitka spruce against white pine weevil [15,16]. ...
... Nonetheless, a few reports expressed the role of 3-carene against insect resistance in pine species [15][16][17][18]. The amounts of 3-carene were associated with the tolerance of Sitka spruce against white pine weevil [15,16]. The high amounts of 3-carene were correlated with the resistance to Douglas-fir pitch moths in Lodgepole pine (Pinus contorta) [17]. ...
... GC-MS analysis of terpenoids in resin obtained from pine wood nematode (PWN)-infected and non-infected Pinus densiflora plants: (a) Resin compounds of control P. densiflora, and (b) resin compounds of PWN-infected P. densiflora. 1. α-pinene, 2. camphene, 3. β-pinene, 4. β-myrcene, 5. 3-carene, 6. β-phellandrene, 7. terpinolene, 8. L-pinocarveol, 9. methylthymylether, 10. acetic acid, 11. α-cubebene, 12. copaene, 13. β-caryophyllene, 14. β-cubebene, 15. δ-cadinene,16. isocembrol, 17. resin acids. ...
Pinus densiflora is an important pine species in Northeast Asia due to its historical, cultural, and economic values. Pine wood nematode (PWN) seriously damages P. densiflora, causing the pine wilt disease (PWD). Changes of phyto-compounds in resin and monoterpenes in P. densiflora after infection of PWN were studied. The changes were identified by GC-MS in control and infected P. densiflora. Among the resin phytochemicals (in P. densiflora), 3-carene was distinctly enhanced after PWN inoculation. The emitted plant volatile monoterpenes were analyzed by HS-SPME/GC-MS. It was observed that the amount of 3-carene enhanced conspicuously after infection of PWNs in both P. densiflora and P. koraiensis at 9.7 and 54.7 times, respectively. 3-Carene synthase gene (Pd3-cars) of P. densiflora was isolated and functionally characterized by transgenic tobacco overexpressing Pd3-cars. Integration and expression of transgenic tobacco were confirmed by genomic and RT-PCR analysis. The Pd3-cars gene was expressed in transgenic tobacco plants. Furthermore, the production of 3-carene was identified by HS-SPME/GC-MS analysis as the volatile compounds emitted from leaves of transgenic tobacco. Treatment of 3-carene to PWNs showed a mild nematicidal activity with 45.98% mortality at the concentration of 10 mg/mL. The current findings may apply to the early diagnosis of pine wilt disease infected by PWNs through enhanced emission of 3-carene.
... The TPSs belonging to the gymnosperm-specific TPS-d subfamily produce a complex mixture of mono-, sesqui-, as well as diterpenoid specialized metabolites, which are found in volatile emissions and oleoresin secretions. These specialized metabolites are involved in the defence against pathogens and herbivores and can help to protect against abiotic stress (Zulak and Bohlman 2010;Hall et al. 2011;Tholl et al. 2015;Celedon and Bohlmann 2019). Oleoresin terpenoids are also important for the production of flavours and fragrances, therapeutics, solvents, coatings and resins, and more recently have been taken into consideration as potential precursors of biofuels (Bohlman and Keeling 2008;Zulak and Bohlman 2010;Hall et al. 2013a). ...
... In case of wounding, the resin under pressure spreads out from the ducts and reaches the wounded area, acting as a physical and chemical weapon against invading organisms (Zulak and Bohlman 2010;Celedon and Bohlmann 2019). The importance of terpenoids in the defence system of conifers against insect pests was confirmed by the study of Hall et al. (2011): the resistance to the white pine weevil (Pissodes strobi) in Picea sitchensis was found to be associated to the levels of the monoterpene ( +)-3-carene, which in turn depended on the copy number and the extent of transcriptional activation of the gene coding for its biosynthetic enzyme, as well as on the amount and catalytic efficiency of the encoded protein. ...
... Interestingly, Ab CAS is phylogenetically equidistant from Table S1. DTPSs diterpene synthases, MTPSs monoterpene synthases, STPSs sesquiterpene synthases the LAS from Ginkgo biloba (Gb LAS) and ISO and LAS enzymes from other Pinaceae, indicating that this enzymatic activity evolved before the speciation of fir, pine and spruce, and was lost in other Pinaceae (Hall et al. 2011;Zerbe et al. 2012;Fig. 5). ...
In the biosynthesis of terpenoids, the ample catalytic versatility of terpene synthases (TPS) allows the formation of thousands of different molecules. A steadily increasing number of sequenced plant genomes invariably show that the TPS gene family is medium to large in size, comprising from 30 to 100 functional members. In conifers, TPSs belonging to the gymnosperm-specific TPS-d subfamily produce a complex mixture of mono-, sesqui-, and diterpenoid specialized metabolites, which are found in volatile emissions and oleoresin secretions. Such substances are involved in the defence against pathogens and herbivores and can help to protect against abiotic stress. Oleoresin terpenoids can be also profitably used in a number of different fields, from traditional and modern medicine to fine chemicals, fragrances, and flavours, and, in the last years, in biorefinery too. In the present work, after summarizing the current views on the biosynthesis and biological functions of terpenoids, recent advances on the evolution and functional diversification of plant TPSs are reviewed, with a focus on gymnosperms. In such context, an extensive characterization and phylogeny of all the known TPSs from different Pinus species is reported, which, for such genus, can be seen as the first effort to explore the evolutionary history of the large family of TPS genes involved in specialized metabolism. Finally, an approach is described in which the phylogeny of TPSs in Pinus spp. has been exploited to isolate for the first time mono-TPS sequences from Pinus nigra subsp. laricio, an ecologically important endemic pine in the Mediterranean area.
... Spruce defences that are relevant for weevil resistance are diverse and affect multiple facets of the weevil life cycle. Much of the knowledge of these defences is based on comparative ecological, biochemical, molecular, and genomic studies with two naturally occurring Sitka spruce genotypes that represent contrasting weevil-resistant and susceptible phenotypes (Byun-McKay et al., 2003;Byun-McKay et al., 2006;Hall et al., 2011;Whitehill et al., 2019;Whitehill, Henderson, Schuetz, et al., 2016;Whitehill, Henderson, Strong, et al., 2016). The highly weevilresistant Sitka spruce genotype H898 used in those studies originates from an area in the Pacific Northwest ( Figure 1) where high weevil population densities have naturally occurred, which may have driven the evolution of natural resistance (King et al., 2011). ...
... Many of the spruce defences that affect weevils are also effective as general conifer defences against other herbivores (Franceschi, Krokene, Christiansen, & Krekling, 2005;Keeling & Bohlmann, 2006;Schiebe et al., 2012). However, certain defences may have evolved to become particularly prominent in the spruce-weevil system, such as the induced formation of traumatic resin ducts (TRDs; Byun-McKay et al., 2003;Christiansen et al., 1999;Hudgins, Christiansen, & Franceschi, 2003;Hudgins & Franceschi, 2004;Krekling, Franceschi, Krokene, & Solheim, 2004;Martin, Tholl, Gershenzon, & Bohlmann, 2002;Nagy, Franceschi, Solheim, Krekling, & Christiansen, 2000;, the terpene metabolites (+)-3-carene and dehydroabietic acid (Hall et al., 2011;Hamberger et al., 2011;Roach, Hall, Zerbe, & Bohlmann, 2014;Whitehill et al., 2019), or an abundance of stone cells (Whitehill et al., 2019;Whitehill, Henderson, Schuetz, et al., 2016;Whitehill, Henderson, Strong, et al., 2016). ...
... The physical effects of resin may not be dependent on the particular composition of resin beyond the general proportions of monoterpenes and sesquiterpenes relative to DRAs. However, individual resin chemicals, specifically the monoterpene (+)-3-carene and the DRA dehydroabietic acid, have been associated with weevil resistance in Sitka spruce (Hall et al., 2011;. Ecological roles for individual terpene metabolites remain to be explored, but evidence does point to potential roles for some compounds as juvenile hormone mimics, which could disrupt insect growth and development (Bohlmann, Crock, Jetter, & Croteau, 1998;Oh et al., 2017). ...
Insect pests are part of natural forest ecosystems contributing to forest rejuvenation, but can also cause ecological disturbance and economic losses that are expected to increase with climate change. The white pine or spruce weevil (Pissodes strobi) is a pest of conifer forests in North America. Weevil‐host interactions with various spruce (Picea) species have been explored as a genomic and molecular reference system for conifer defense against insects. Interactions occur in two major phases of the insect life cycle. In the exo‐phase, adult weevils are free‐moving and display behavior of host selection for oviposition that is affected by host traits. In the endo‐phase, insects live within the host where mobility and development from eggs to young adults is affected by a complex system of host defenses. Genetic resistance exists in several spruce species and involves synergism of constitutive and induced, chemical and physical defenses that comprise the conifer defense syndrome. Here, we review conifer defenses that disrupt the weevil life cycle and mechanisms by which trees resist weevil attack. We highlight molecular and genomic aspects and a possible role for the weevil microbiome. Knowledge of this conifer defense system is supporting forest health strategies and tree breeding for insect resistance.
... Terpenes play active roles in plant defense to multiple biotic stresses (Keeling and Bohlmann, 2006;Celedon and Bohlmann, 2019;Toffolatti et al., 2021). For example, (+)-3-carene is a monoterpene constitutively produced in many members of Pinaceae for defense against biotic agents (Fäldt et al., 2003;Hall et al., 2011;Roach et al., 2014). The correlation study of preformed terpene contents in trees and the degree of resistance will aid in a better understanding of their roles in tree defense. ...
... Variation in terpene concentrations between the samples of different susceptibility demonstrated constitutive genetic differences in resistance. Other authors have noted that the monoterpene amount increases in resistant trees after the induction of defense responses, but remains unchanged in susceptible trees (Hall et al., 2011). Thus, monoterpene contents merit to be further explored as a chemical marker for resistance or susceptibility. ...
Root and stem rot caused by Heterobasidion annosum is a severe problem in boreal Scots pine. Dissecting the features of disease resistance is generally an essential step in resistance breeding in plants and forest trees. In this study, we explored inherent resistance factors of Scots pine against H. annosum. A total of 236 families consisting of 85 full-sib (FS), 35 half-sib population mix (HSpm), and 116 half-sib (HS) families of Scots pine seedlings were inoculated with a H. annosum isolate. We sampled needle tissues before inoculation for terpene measurements and RNA sequencing. Based on the lesion area, the extremes of 12 resistant and 12 susceptible families were selected for further analyses. Necrotic lesions resulting from fungal infection were in a weak to moderate relationship with the plant height. Monoterpenes were the principal terpene compounds observed in Scots pine seedlings. Concentrations of 3-carene were significantly higher in pine genotypes inherently resistant compared with susceptible seedlings. By contrast, susceptible genotypes had significantly higher proportions of α-pinene. Gene ontology analysis of differential expressed transcripts (DETs) revealed that response to biotic factors was enriched in resistant seedlings. Functional characterization of individual DETs revealed that higher expression of transcripts involved in response to abiotic stress was common in susceptible genotypes. This observation was supported by the annotation of hub genes in a key module that was significantly correlated with the lesion trait through weighted gene co-expression network analysis (WGCNA) of 16 HS and HSpm samples. These findings contribute to our understanding of constitutive resistance factors of Scots pine against Heterobasidion root and stem rot diseases.
... There is no information on the heritability of seed resin vesicle morphology. However, the oleoresin and resin vesicles in other conifer tissues can have a strong genetic component (Hall et al. 2011;Robert et al. 2010). Western redcedar (Thuja plicata Donn ex D. Don) has seed resin vesicles, and the abundance of monoterpenes in foliage has been shown to have a genetic component and is correlated with inhibition to deer browsing (Foster et al. 2013;Russell and Yanchuk 2012;Vourc'h et al. 2002). ...
... We would expect natural variation across the population in the expression and kinetic abilities of these enzymes, as has been shown in Sitka spruce (Picea sitchensis (Bong.) Carrière) (Hall et al. 2011). These enzymes are also being identified in western redcedar (Gesell et al. 2015). ...
The seed coat of fir (Abies), hemlock (Tsuga), and cedar (Thuja) species contain terpenoid resin vesicles. Although information is limited about the morphology and allelopathy of these vesicles, their damage during seed processing can negatively impact germination success. We examined resin vesicle morphology of western redcedar (Thuja plicata Donn ex D. Don), eastern white cedar (Thuja occidentalis L.), amabilis fir (Abies amabilis Douglas ex J. Forbes), balsam fir (Abies balsamea (L.) Mill.), grand fir (Abies grandis (Douglas ex D. Don) Lindl), and subalpine fir (Abies lasiocarpa (Hook.) Nutt.) seeds by ¹H magnetic resonance imaging to characterize resin vesicle volume, shape, and number. Western redcedar genotypes with known differences in the quantity of foliar monoterpenes also had parallel differences in the resin vesicle volume of corresponding seeds. Germination assays with the cedar and fir species, eastern hemlock (Tsuga canadensis (L.) Carrière), mountain hemlock (Tsuga mertensiana (Bong.) Carrière), and western hemlock (Tsuga heterophylla (Raf.) Sarg.) confirmed that resin vesicle damage prior to stratification (moist chilling) significantly reduced germination success for most species. Extracts of these resin vesicles from the Abies and Thuja species strongly inhibited the germination of Arabidopsis Col-0 seeds but inhibited the germination of only a small percentage of Arabidopsis abscisic acid insensitive mutant abi3-6 seeds. Resin extracts from Thuja species were 10 times more effective than those from Abies species in inhibiting Arabidopsis Col-0 germination.
... Various volatile or semivolatile terpenoids, including low-molecular-weight monoterpenoids, diterpenoids, and sesquiterpenoids, have been found to have anti-phytopathogen properties, both above and below ground [77]. For example, (E)-β-caryophyllene emitted by A. thaliana was shown to be involved in plant protection against P. syringae [78], and (+)-3-carene in Picea sitchensis was associated with resistance to white pine weevil (Pissodes strobi) [79]. Some isoprenoids have also demonstrated signaling functions; for example, dehydroabietinal, which is produced in Arabidopsis leaf tissue, serves as a vascular signaling compound and a potent activator of SAR [80]. ...
Gray mold caused by Botrytis cinerea causes significant losses in tomato crops. B. cinerea infection may be halted by volatile organic compounds (VOCs), which may exhibit fungistatic activity or enhance the defense responses of plants against the pathogen. The enhanced VOC generation was observed in tomato (Solanum lycopersicum L.), with the soil-applied biocontrol agent Trichoderma virens (106 spores/1 g soil), which decreased the gray mold disease index in plant leaves at 72 hpi with B. cinerea suspension (1 × 106 spores/mL). The tomato leaves were found to emit 100 VOCs, annotated and putatively annotated, assigned to six classes by the headspace GCxGC TOF-MS method. In Trichoderma-treated plants with a decreased grey mold disease index, the increased emission or appearance of 2-hexenal, (2E,4E)-2,4-hexadienal, 2-hexyn-1-ol, 3,6,6-trimethyl-2-cyclohexen-1-one, 1-octen-3-ol, 1,5-octadien-3-ol, 2-octenal, octanal, 2-penten-1-ol, (Z)-6-nonenal, prenol, and acetophenone, and 2-hydroxyacetophenone, β-phellandrene, β-myrcene, 2-carene, δ-elemene, and isocaryophyllene, and β-ionone, 2-methyltetrahydrofuran, and 2-ethyl-, and 2-pentylfuran, ethyl, butyl, and hexyl acetate were most noticeable. This is the first report of the VOCs that were released by tomato plants treated with Trichoderma, which may be used in practice against B. cinerea, although this requires further analysis, including the complete identification of VOCs and determination of their potential as agents that are capable of the direct and indirect control of pathogens.
... Furthermore, glandular trichomes also store volatile terpenoids, which act as insect repellents, e.g., sesquiterpenes found in the trichomes of wild tomato are strong repellents to homopterans [58]. The monoterpene (+)-3-carene are connected with insecticidal activity of Picea sitchensis Carr on the Engelmann spruce weevil (Pissodes strobi) [59]. Additionally, they also interact with herbivores and their natural enemies, constitutive and prompted mixtures can play role in interspecific, intraspecific, and even via "alarm" signals that may activate the defensive mechanism in adjacent plants [60,61]. ...
The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is the most notorious insect pest of potato globally. Injudicious use of insecticides for management of this pest has resulted in resistance to all major groups of insecticides along with many human, animal health, and environmental concerns. Additionally, the input cost of insecticide development/discovery is markedly increasing because each year thousands of chemicals are produced and tested for their insecticidal properties, requiring billions of dollars. For the management of resistance in insect pests, synergists can play a pivotal role by reducing the application dose of most insecticides. These eco-friendly synergists can be classified into two types: plant-based synergists and RNAi-based synergists. The use of plant-based and RNAi-based synergists in resistance management of insect pests can give promising results with lesser environmental side effects. This review summarizes the resistance status of CPB and discusses the potential advantage of plant-based and RNAi-based synergists for CPB resistance management. It will motivate researchers to further investigate the techniques of using plant- and RNAi-based synergists in combination with insecticides.
... Several authors found that d3-CAR is associated with tree-killing bark beetles or weevil resistance in spruce (e.g. Hall et al., 2011). Lüpke et al. (2016) classified Scots pine provenances into d3-CAR dominated and non-d3-CAR (with either high alpha-/beta-pinene or beta-myrcene fraction) type. ...
Currently, large areas of Picea abies (Norway spruce) stands in Europe are increasingly affected by drought and heat waves. Moreover, early spring drought has occurred with much higher frequency. Our work focuses on physiological changes induced by drought in four-year-old spruce seedlings during shoot elongation. We investigated drought effect on photosynthetic rate, concentration of abscisic acid and its metabolites, amount and composition of monoterpenes in needles of seedlings from five different provenances (altitude range 550–1280 m above sea level) in Western Carpathians. Spruce seedlings subjected to one-month drought stress of moderate intensity (about 50% of soil water content at the end of experiment) showed significant reduction of CO2 uptake and increased concentration of ABA related to untreated controls. Induced drought affected needle monoterpene content and composition. Observed changes in drought-induced physiological parameters were influenced by seedling provenance. The provenance from 920 m above sea level showed the greatest sensitivity to drought with significantly highest ABA content and, at the same time, a clear decline of CO2 uptake and amounts of total monoterpenes. Our results indicating intra-specific provenance-related variability in physiological response of spruce seedlings to drought may provide a basis for improved reforestation strategies in drought risk areas.
... Moreover, high and low levels of d-3-carene in growing shoots define Sitka spruce (Picea sitchensis (Bong.) Carrière) genotypes resistant and susceptible to white pine weevil [50,51]. This compound seems the best candidate among monoterpenes for a defensive effect in conifer foliage. ...
Changes during leaf ontogeny affect palatability to herbivores, such that many insects, including the eastern spruce budworm (Choristoneura fumiferana (Clem.)), are specialist feeders on growing conifer leaves and buds. Developmental constraints imply lower toughness in developing foliage, and optimal defense theory predicts higher investment in chemical defense in these vulnerable yet valuable developing leaves. We summarize the literature on the time course of defensive compounds in developing white spruce (Picea glauca (Moench) Voss) needles and report original research findings on the ontogeny of white spruce needle toughness. Our results show the predicted pattern of buds decreasing in toughness followed by leaves increasing in toughness during expansion, accompanied by opposite trends in water content. Toughness of mature foliage decreased slightly during the growing season, with no significant relationship with water content. Toughness of sun-grown leaves was slightly higher than that of shade-grown leaves. However, the literature review did not support the expected pattern of higher defensive compounds in expanding leaves than in mature leaves, suggesting that white spruce might instead exhibit a fast-growth low-defense strategy.
... Tree breeding for insect resistance stands to benefit from the application of genomics-based tools, such as genetic markers identified from genome and transcriptome analyses or genomic selection to overcome challenges associated with screening for pest resistance (Beaulieu et al., 2020;Isik, 2014;Lenz et al., 2019;Neale & Kremer, 2011;Plomion et al., 2011). Previous comparisons of R and S trees elucidated defense mechanisms that contribute to weevil resistance, such as oleoresin terpenes and stone cells (Hall et al., 2011;Roach et al., 2014;Whitehill, Henderson, Schuetz, et al., 2016;Whitehill, Henderson, Strong, et al., 2016;. The present transcriptome analysis was performed using a statistical model to identify weevil specific responses and assess differences in gene expression between R (Miller et al., 2005;Ralph et al., 2006Ralph et al., , 2008. ...
Spruce weevil (Pissodes strobi) is a significant pest of regenerating spruce (Picea) and pine (Pinus) forests in North America. Weevil larvae feed in the bark, phloem, cambium, and outer xylem of apical shoots, causing stunted growth or mortality of young trees. We identified and characterized constitutive and weevil‐induced patterns of Sitka spruce (Picea sitchensis) transcriptomes in weevil‐resistant (R) and susceptible (S) trees using RNA sequencing (RNA‐seq) and differential expression (DE) analyses. We developed a statistical model for the analysis of RNA‐seq data from treatment experiments with a 2 × 3 factorial design to differentiate insect‐induced responses from the effects of mechanical damage. Across the different comparisons, we identified two major transcriptome contrasts: A large set of genes that was constitutively DE between R and S trees, and another set of genes that was DE in weevil‐induced S‐trees. The constitutive transcriptome unique to R trees appeared to be attuned to defense, while the constitutive transcriptome unique to S trees was enriched for growth‐related transcripts. Notably, a set of transcripts annotated as “fungal” was detected consistently in the transcriptomes. Fungal transcripts were identified as DE in the comparison of R and S trees and in the weevil‐affected DE transcriptome of S trees, suggesting a potential microbiome role in this conifer‐insect interaction. Overview of differentially expressed (DE) genes. Number of DE genes in R and S trees. Only two major comparisons contained DE contigs and are defined as the constitutive difference (blue circle) between R and S trees, and genes DE following weevil activity in S trees defined as weevil induced (orange circle).
... The trace/presence pattern in (+)-3carene is likely to be inflating the variation among families and genotypes, and therefore potentially driving up the heritability estimate. This pattern of very low and high concentrations of 3carene across individual trees has been observed in other studies of Sitka spruce (Hall et al., 2011) and in other conifers, resulting in similarly high estimates of heritability of this terpene (Baradat and Yazdani, 1988;Hanover, 1966). In future work, increasing the sample size within families could determine the degree of variation across individuals and help further resolve if this oscillating phenotypic variation is due to additive genetic variance or to another source like phenotypic plasticity or environmental variation. ...
Reforestation in the British Isles (UK and Ireland) has been dominated with the use of an exotic conifer tree species, Sitka spruce (Picea sitchensis [Bong.] Carr.). Sitka breeding in the UK was developed from a single provenance, the Haida Gwaii Islands (Canada), which is both well suited to the British climate and highly susceptible to the white pine weevil (Pissodes strobi L.) in its native range. We examined variation and heritability of insect resistance related traits and assessed potential trade-offs with tree growth in 50 full-sibling families and 13 clonally replicated genotypes growing in the UK. We measured bark levels of three terpenes (dehydroabietic acid, (+)-3-carene and terpinolene) shown to confer resistance to the white pine weevil in Sitka spruce’s native range, on the principle that these defence compounds may also contribute to pest resistance in the UK. We compared our results with published findings from the native range and also used individuals from a Haida Gwaii seed lot grown in the UK for comparison of terpene levels. Dehydroabietic acid content in the UK breeding population was similar to populations from resistant native populations, but (+)-3-carene and terpinolene levels were relatively low. Narrow sense heritability for dehydroabietic acid, (+)-3-carene and terpinolene was estimated as 0.20, 0.93 and 0.98, respectively from the full-sib data, and this evidence of genetic variance was supported by estimates of broad sense heritability from the smaller clonal study. Terpene content was found to be positively correlated to growth traits. The heritability estimates and genetic correlations indicate that selective breeding should be effective in raising levels in the UK breeding population of the three candidate terpenes implicated in weevil resistance. However, low levels observed indicate that other provenances from the native range may produce greater short-term improvements for two of the terpenes.
... (+)-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 . ...
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 . ...
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.
... For example, structure-guided mutagenesis of Mentha spicata limonene synthase, the key enzyme in the menthol production (Lange et al., 2011), identified two amino acids, His579 and Trp324, substitution of which led to premature neutralization of the carbocation intermediate to form both linear and cyclic monoterpenoids, including myrcene, linalool, and terpineol (Srividya et al., 2015) (Figure 6F). Similarly, reciprocal mutagenesis analyses of the mono-TPSs, Sitka spruce (Picea sitchensis) 3-carene synthase, and sabinene synthase associated with tree resistance against white pine weevil (Hall et al., 2011) revealed that two corresponding residues, 3-carene synthase Leu596, and sabinene synthase Phe596 located near the helix G break are critical for rearranging the central α-terpinyl + carbocation toward 3-carene and sabinene, respectively (Roach et al., 2014). ...
Terpenoids comprise tens of thousands of small molecule natural products that are widely distributed across all domains of life. Plants produce by far the largest array of terpenoids with various roles in development and chemical ecology. Driven by selective pressure to adapt to their specific ecological niche, individual species form only a fraction of the myriad plant terpenoids, typically representing unique metabolite blends. Terpene synthase (TPS) enzymes are the gatekeepers in generating terpenoid diversity by catalyzing complex carbocation-driven cyclization, rearrangement, and elimination reactions that enable the transformation of a few acyclic prenyl diphosphate substrates into a vast chemical library of hydrocarbon and, for a few enzymes, oxygenated terpene scaffolds. The seven currently defined clades (a-h) forming the plant TPS family evolved from ancestral triterpene synthase- and prenyl transferase–type enzymes through repeated events of gene duplication and subsequent loss, gain, or fusion of protein domains and further functional diversification. Lineage-specific expansion of these TPS clades led to variable family sizes that may range from a single TPS gene to families of more than 100 members that may further function as part of modular metabolic networks to maximize the number of possible products. Accompanying gene family expansion, the TPS family shows a profound functional plasticity, where minor active site alterations can dramatically impact product outcome, thus enabling the emergence of new functions with minimal investment in evolving new enzymes. This article reviews current knowledge on the functional diversity and molecular evolution of the plant TPS family that underlies the chemical diversity of bioactive terpenoids across the plant kingdom.
... Advances in the technologies for proteome analysis are slowly getting visibility in forest tree proteomics research (Jorrín-Novo et al., 2015). These technologies include mainly liquid chromatography (LC)-MS-based systems (Mast et al., 2010;Chen et al., 2012;Marmiroli et al., 2013;de Francisco et al., 2016;Abraham et al., 2017) and the use of selected reaction monitoring (SRM) targeted proteomics for accurate protein quantification (Zulak et al., 2009;Hall et al., 2011). ...
Research in forest tree species has advanced slowly when compared with other agricultural crops and model organisms, mainly due to the long‐life cycles, large genome sizes, and lack of genomic tools. Additionally, trees are complex matrices, and the presence of interferents (e.g., oleoresins and cellulose) challenges the analysis of tree tissues with mass spectrometry (MS)‐based analytical platforms. In this review, advances in MS‐based forest tree metabolomics are discussed. Given their economic and ecological significance, particular focus is given to Pinus, Quercus, and Eucalyptus forest tree species to better understand their metabolite responses to abiotic and biotic stresses in the current climate change scenario. Furthermore, MS‐based metabolomics technologies produce large and complex datasets that require expertize to adequately manage, process, analyze, and store the data in dedicated repositories. To ensure that the full potential of forest tree metabolomics data are translated into new knowledge, these data should comply with the FAIR principles (i.e., Findable, Accessible, Interoperable, and Re‐usable). It is essential that adequate standards are implemented to annotate metadata from forest tree metabolomics studies as is already required by many science and governmental agencies and some major scientific publishers. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 00:1–32, 2019.
... The recent development of a mass spectrometry-based targeted proteomics approach, such as Multiple Reaction Monitoring (MRM: the acquisition of multiple product ions from one precursor ion by Selected Reaction Monitoring (SRM) transitions), and its utilization for the quantitative analysis of peptides derived from proteins of specific organelles in plant cells has become a powerful, hypothesis-driven analytical tool to achieve specific, sensitive, and precise protein quantification (Zulak et al., 2009;Hall et al., 2011;Fan et al., 2012;Hooper et al., 2017). The high specificity of MRM analysis comes from the mass measurement (accurately measured to an atomic mass unit) of the whole peptide and its multiple fragments, the unique peptide ion fragmentation pattern, and the precise peptide retention time in the liquid chromatography (LC) separation (Liebler and Zimmerman, 2013). ...
Seed germination provides an excellent model to study the process of mitochondrial biogenesis. It is a complex and strictly regulated process which requires a proper biogenesis of fully active organelles from existing promitochondrial structures. We have previously reported that the lack of the inner mitochondrial membrane protease FTSH4 delayed Arabidopsis seed germination. Here, we implemented a targeted mass spectrometry-based approach, Multiple Reaction Monitoring (MRM), with stable-isotope-labeled standard peptides for increased sensitivity, to quantify mitochondrial proteins in dry and germinating wild-type and ftsh4 mutant seeds, lacking the FTSH4 protease. Using total seed protein extracts we measured the abundance of the peptide targets belonging to the OXPHOS complexes, AOX1A, transport, and inner membrane scaffold as well as mitochondrial proteins that are highly specific to dry and germinating seeds. The MRM assay showed that the abundance of these proteins in ftsh4 did not differ substantially from that observed in wild-type at the level of dry seed and after stratification, but we observed a reduction in protein abundance in most of the examined OXPHOS subunits in the later stages of germination. These changes in OXPHOS protein levels in ftsh4 mutants were accompanied by a lower cytochrome pathway activity as well as an increased AOX1A amount at the transcript and protein level and alternative pathway activity. The analyses of the steady-state transcript levels of mitochondrial and nuclear genes encoding OXPHOS subunits did not show significant difference in their amount, indicating that the observed changes in the OXPHOS occurred at the post-transcriptional level. At the time when ftsh4 seeds were fully germinated, the abundance of the OXPHOS proteins in the mutant was either slightly lowered or comparable to these amounts in wild-type seeds at the similar developmental stage. By the implementation of an integrative approach combining targeted proteomics, quantitative transcriptomics, and physiological studies we have shown that the FTSH4 protease has an important role in the biogenesis of OXPHOS and thus biogenesis of mitochondria during germination of Arabidopsis seeds.
... Biomarkers have also been identified for trees affected by insect pests and pathogens. For example, constitutive concentrations of selected monoterpenes were higher in white pine weevilresistant than -susceptible Sitka spruce (Hall et al. 2011), phloem phenolic composition was used to predict Norway spruce resistance to Ceratocystis polonica, a fungus associated with the bark beetle Ips typographus (Brignolas et al. 1998), and phenolic biomarkers were identified from mango leaf extracts that were associated with cultivars that were tolerant or susceptible to Fusarium (Augustyn et al. 2014). Our goal for the work presented here is supported by a previous study, in which chemical fingerprinting (a less analytically sensitive approach than metabolite profiling -Fiehn 2001) was sufficient to differentiate the most resistant and most susceptible CLOs in the same population analyzed herein ). ...
Sudden oak death, caused by the invasive pathogen Phytophthora ramorum Werres, de Cock & Man in't Veld, can be deadly for Quercus agrifolia Neé (coast live oak, CLO). However, resistant trees have been observed in natural populations. The objective of this study was to examine if pre-attack (constitutive) levels of phenolic compounds can be used as biomarkers to identify trees likely to be resistant. Naïve trees were selected from a natural population and phloem was sampled for analysis of constitutive phenolics. Following P. ramorum inoculation, trees were phenotyped to determine disease susceptibility and constitutive phenolic biomarkers of resistance were identified. Seasonal variation in phloem phenolics was also assessed in a subset of non-inoculated trees. Four biomarkers, including myricitrin and three incompletely characterized flavonoids, together correctly classified 80% of trees. Biomarker levels were then used to predict survival of inoculated CLO and the proportion of resistant trees within a subset of non-inoculated trees from the same population. Levels of five phenolics were significantly affected by season, but with no pronounced variation in average levels among seasons. These results suggest that pre-infection levels of specific phenolic compounds (i.e., biomarkers) can identify trees naturally resistant to this invasive forest pathogen. Knowledge of resistant trees within natural populations may be useful for conserving and breeding resistant trees and for disease management.
Canada has made significant contributions to the field of plant biochemistry, with numerous researchers actively focusing on elucidating the biosynthetic pathways of plant specialized metabolites and producing these compounds in heterologous systems, such as bacteria, yeast, or other plant species. The review aims to highlight the strengths of Canadian research in this domain over the last three decades. It will describe advances in pathway elucidation, enzyme characterization, and production of enzymes and metabolites in heterologous systems, particularly in the areas of alkaloids, terpenoids, and phenolic compounds. Canadian researchers have not only made pivotal scientific discoveries but have also ensured the continuity of scientific excellence by mentoring new generations of principal investigators in plant specialized metabolites. These advances warrant recognition and financial support to retain future talent and to maintain Canada's leadership in scientific progress on the global stage.
Pine pitch canker (PPC) emerged as a significant problem in 1945 in Southeastern USA. The causal agent, Fusarium circinatum , has spread widely and now occurs in pine forests and plantations worldwide. Fusarium circinatum causes damping off, shoot and tip die‐back, and the death of seedlings in nurseries. Infection of mature trees leads to excessive resin bleeding on branches and main stems, sunken cankers with bark remaining attached, and finally, tree death. Arguably, F. circinatum is the most important pathogen of pine seedlings in many areas of the world. At least 67 species of Pinus , 18 Pinus hybrids, and 6 other non‐pine tree species are susceptible to PPC. The selection and development of tree germplasm resistant to pathogens is considered the most robust approach to reduce losses to diseases. Genetic variation in resistance to F. circinatum certainly exists, even in generally very susceptible hosts, such as P. radiata . Exploiting genetic resistance as a tool to manage PPC requires screening large numbers of tree genotypes and fine‐tuning efficient phenotyping protocols. The greater the number of genetic lines and replications, the higher the selection differentials and accuracy of the genetic parameter estimates. Recent advances in physiological and molecular techniques exploring the plant‐pathogen interaction have expanded our understanding of genetic resistance mechanisms in pines to PPC and represent an added‐value tool to support phenotypic selection. This review provides an overview of current knowledge on the molecular, physiological, and genetic basis of resistance to F. circinatum in pines and considers possibilities to improve the resistance of pines against the pathogen through breeding and selection.
Volatile organic compounds (VOCs) and essential oils of conifers are widely used in the pharmaceutical industry. This work aimed to analyze the VOCs of 30 conifer species representing the Pinaceae and Cupressaceae families. Samples were collected from arboreta in Hungary, and their chemical composition was determined by gas chromatography (SPME-GC/MS); then, chemometric analyses were performed using multivariate methods to identify characteristic VOCs of conifers. Here, we present results for monoterpene and sesquiterpene profiles of the examined conifer samples. The most abundant compounds detected were α-pinene, bornyl acetate, limonene, β-pinene, β-caryophyllene, β-myrcene, δ-3-carene, and β-phellandrene. The results showed that the following volatiles were characteristic of the conifer groups: sabinene (RRT=6.0) for the cupressoid group (which includes the Cupressaceae species), longifolene (RRT=15.0) and β-pinene (RRT=6.1) were characteristic of the pinoid group (including Picea, Pinus, and Pseudotsuga species), and camphene (RRT=5.5) and bornyl acetate (RRT=12.6) were characteristic of the abietoid group (including Abies, Cedrus, and Tsuga species). Our results on VOCs in the Pinaceae and Cupressaceae families contribute to the elucidation of biodiversity patterns of conifer species and, in addition, may support the industrial application of terpenes.
With climate warming and economic globalization, insect–microbe assemblages are becoming increasingly responsible for various devastating forest diseases worldwide. Japanese larch (Larix kaempferi) is extensively cultivated in China because of its high survival rate, rapid maturation and robust mechanical properties. Endoconidiophora fujiensis, an ophiostomatoid fungus associated with Ips subelongatus, has been identified as a lethal pathogen of L. kaempferi in Japan. However, there is a dearth of research on the pathogenicity of E. fujiensis in larches in China. Therefore, we investigated the pathogenicity of E. fujiensis in introduced L. kaempferi and indigenous larch (Larix olgensis) trees and compared the induced resistance responses to the pathogen in both tree species in terms of physiology and gene expression. Five-year-old saplings and 25-year-old adult trees of L. olgensis and L. kaempferi were inoculated in parallel during the same growing season. Endoconidiophora fujiensis exhibited high pathogenicity in both larch species, but particularly in L. kaempferi compared with L. olgensis adult trees; adult L. olgensis was more resistant to E. fujiensis than adult L. kaempferi, which was reflected in higher accumulation of defensive monoterpenes, such as myrcene, 3-carene and limonene and the earlier induction of defense genes catalase (CAT) and pathogenesis-related protein 1 (PR1). This study contributes to our understanding of the interactions between bark beetle-associated ophiostomatoid fungi and host larches, from phenotypic responses to alterations in secondary metabolites via defense- and metabolism-related gene activation, providing a valuable foundation for the management of larch diseases and pests in the future.
Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and βglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of βglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of βglu-1 and Ugt5 genes. We observed very large copy numbers of βglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of βglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.
Purpose of Review
Forestry in northern temperate and boreal regions relies heavily on conifers. Rapid climate change and associated increases in adverse growing conditions predispose conifers to pathogens and pests. The much longer generation time and presumably, therefore, lower adaptive capacity of conifers relative to their native or non-native biotic stressors may have devastating consequences. We provide an updated overview of conifer defences underlying pathogen and pest resistance and discuss how defence traits can be used in tree breeding and forest management to improve resistance.
Recent Findings
Breeding of more resilient and stress-resistant trees will benefit from new genomic tools, such as genotyping arrays with increased genomic coverage, which will aid in genomic and relationship-based selection strategies. However, to successfully increase the resilience of conifer forests, improved genetic materials from breeding programs must be combined with more flexible and site-specific adaptive forest management.
Summary
Successful breeding programs to improve conifer resistance to pathogens and pests provide hope as well as valuable lessons: with a coordinated and sustained effort, increased resistance can be achieved. However, mechanisms underlying resistance against one stressor, even if involving many genes, may not provide any protection against other sympatric stressors. To maintain the adaptive capacity of conifer forests, it is important to keep high genetic diversity in the tree breeding programs. Choosing forest management options that include diversification of tree-species and forest structure and are coupled with the use of genetically improved plants and assisted migration is a proactive measure to increase forest resistance and resilience to foreseen and unanticipated biotic stressors in a changing climate.
Tana (Zanthoxylum ailanthoides), a perennial deciduous species in the Rutaceae family, possesses leaves with a unique fragrance that indigenous peoples incorporate into their traditional cuisine. In Kalibuan, the cultivated tana trees were pruned repeatedly to maintain a shorter height, which led to the growth of new leaves that were spicier and pricklier. Tana leaves contain a range of volatile terpenoids, and the pungent aroma may arise from the presence of monoterpenoids. To gain insight into the biosynthetic pathway, five candidate monoterpene synthase genes were cloned and characterized using a purified recombinant protein assay. The main product of Za_mTPS1, Za_mTPS2, and Za_mTPS5 is sabinene, geraniol, and (E)-β-ocimene, respectively. The main product of Za_mTPS3 and Za_mTPS4 is linalool. Real-time PCR analysis revealed that Za_mTPS1 and Za_mTPS5 are expressed at higher levels in prickly leaves of cultivated tana, suggesting that they may contribute to the distinctive aroma of this plant.
Stone cells are a specialized, highly lignified cell type found in both angiosperms and gymnosperms. In conifers, abundance of stone cells in the cortex provides a robust constitutive physical defense against stem feeding insects. Stone cells are a major insect‐resistance trait in Sitka spruce (Picea sitchensis), occurring in dense clusters in apical shoots of trees resistant (R) to spruce weevil (Pissodes strobi) but being rare in susceptible (S) trees.
To learn more about molecular mechanisms of stone cell formation in conifers, we used laser microdissection and RNA sequencing to develop cell‐type‐specific transcriptomes of developing stone cells from R and S trees. Using light, immunohistochemical, and fluorescence microscopy, we also visualized the deposition of cellulose, xylan, and lignin associated with stone cell development.
A total of 1293 genes were differentially expressed at higher levels in developing stone cells relative to cortical parenchyma. Genes with potential roles in stone cell secondary cell wall formation (SCW) were identified and their expression evaluated over a time course of stone cell formation in R and S trees.
The expression of several transcriptional regulators was associated with stone cell formation, including a NAC family transcription factor and several genes annotated as MYB transcription factors with known roles in SCW formation.
Insects and plants dominate terrestrial ecosystems in terms of both species numbers and biomass. Ecological relationships between insects and plants are ubiquitous and insect-plant interactions are important for ecosystem structuring and functioning.
In this chapter we move to the final stage of the Central Dogma and beyond. Having previously discussed the genome (Chaps. 10.1007/978-3-319-46807-5_2, 10.1007/978-3-319-46807-5_3, 10.1007/978-3-319-46807-5_4 and 10.1007/978-3-319-46807-5_5) and the transcriptome (Chap. 10.1007/978-3-319-46807-5_6), we will now present the proteome and the metabolome. It has only been in the last 20 years that technologies have been developed to capture, characterize, and quantify the complete pool of proteins and metabolites from an animal or plant tissue sample. Prior to that, individual proteins and metabolites generally had to be assayed one at a time. This work is known as protein or metabolite profiling.
Artificial light has been used to control plant growth and secondary metabolite production. Artemisia annua L. plants were illuminated with three light-emitting diode (LED) spectra to investigate proteomic and biochemical responses. After 7 days, proteomic data revealed different protein numbers in leaves, stems, and roots under particular treatments. Results demonstrated increased accumulation of several proteins, including secondary metabolite-related proteins. The red light (R) (660 nm) highly induced terpenoid proteins. Similar biochemical profiles were observed in white light (W) (445, 544 nm) and blue light (B) (445 nm) conditions, while profiles from R treatment were different. Functional proteins of W and B treatments were involved in the MVA and MEP pathways and sesquiterpene biosynthesis. By contrast, unique proteins in R treatment were mainly expressed in sesquiterpene and tetraterpene biosynthesis. Specific relationships between biosynthetic proteins and sesquiterpenes were observed, suggesting the indispensable role of the light spectrum in regulating terpenoid biosynthesis.
Forests and trees have considerable economic and ecological value as well as serve a critical role as an essential alternative bioenergy source. Forests and forest soils act as major carbon sinks and storages and are therefore a potential tool in combatting climate
change. Global nutrient cycles, and as an extension all of humankind, are dependent on forests and trees. The health of the world’s forests is currently threatened by the impending shift in growth conditions caused by climate change and invasive pathogens and pests. Invasive pathogens pose a greater threat than previously due to climate change. Finding effective ways to manage the diseases of long-lived trees is therefore a daunting task. However, a better understanding of the anatomical, chemical, molecular, and genetic basis for tree defenses will be vital for sustainable management of forest tree diseases. Breeding for disease resistance has been touted as a promising approach in addition to traditional prevention methods, like the use of biocontrol agents and fungicides, in disease disturbance prevention. Either way, a better understanding of the anatomical, constitutive, and induced defenses used by forest trees to defend themselves against biotic stressors will be useful for effective disease management. The application of marker assisted selection, genomic selection, and gene editing is a potential novel alternative in the fight against forest tree diseases.
Background
Needle rust caused by the fungus Chrysomyxa rhododendri causes significant growth decline and increased mortality of young Norway spruce trees in subalpine forests. Extremely rare trees with enhanced resistance represent promising candidates for practice-oriented reproduction approaches. They also enable the investigation of tree molecular defence and resistance mechanisms against this fungal disease. Here, we combined RNA-Seq, RT-qPCR and secondary metabolite analyses during a period of 38 days following natural infection to investigate differences in constitutive and infection-induced defence between the resistant genotype PRA-R and three susceptible genotypes.
Results
Gene expression and secondary metabolites significantly differed among genotypes from day 7 on and revealed already known, but also novel candidate genes involved in spruce molecular defence against this pathogen. Several key genes related to (here and previously identified) spruce defence pathways to needle rust were differentially expressed in PRA-R compared to susceptible genotypes, both constitutively (in non-symptomatic needles) and infection-induced (in symptomatic needles). These genes encoded both new and well-known antifungal proteins such as endochitinases and chitinases. Specific genetic characteristics concurred with varying phenolic, terpene, and hormone needle contents in the resistant genotype, among them higher accumulation of several flavonoids (mainly kaempferol and taxifolin), stilbenes, geranyl acetone, α-ionone, abscisic acid and salicylic acid.
Conclusions
Combined transcriptional and metabolic profiling of the Norway spruce defence response to infection by C. rhododendri in adult trees under subalpine conditions confirmed the results previously gained on artificially infected young clones in the greenhouse, both regarding timing and development of infection, and providing new insights into genes and metabolic pathways involved. The comparison of genotypes with different degrees of susceptibility proved that several of the identified key genes are differently regulated in PRA-R, and that the resistant genotype combines a strong constitutive defence with an induced response in infected symptomatic needles following fungal invasion. Genetic and metabolic differences between the resistant and susceptible genotypes indicated a more effective hypersensitive response (HR) in needles of PRA-R that prevents penetration and spread of the rust fungus and leads to a lower proportion of symptomatic needles as well as reduced symptom development on the few affected needles.
Success of plants largely depends on their ability to defend against herbivores. Since emergence of the first voracious consumers, plants maintained adapting their structures and chemistry to escape from extinction. The constant pressure was further accelerated by adaptation of herbivores to plant defenses, which all together sparked the rise of a chemical empire comprised of thousands of specialized metabolites currently found in plants. Metabolic diversity in the plant kingdom is truly amazing, and although many plant metabolites have already been identified, a large number of potentially useful chemicals remain unexplored in plant bio‐resources. Similarly, biosynthetic routes for plant metabolites involve many enzymes, some of which still wait for identification and biochemical characterization. Moreover, regulatory mechanisms that control gene expression and enzyme activities in specialized metabolism of plants are scarcely known. Finally, understanding of how plant defense chemicals exert their toxicity and/or repellency against herbivores remains limited to typical examples, such as proteinase inhibitors, cyanogenic compounds and nicotine. In this review, we attempt summarizing the current status quo in metabolic defense of plants that is predominantly based on the survey of ubiquitous examples of plant interactions with chewing herbivores.
Plants produce a wide variety of natural volatile organic compounds (NVOCs), many of which are unique to each species. These compounds serve many purposes, such as fending off herbivores and adapting to changes in temperature and water supply. Interestingly, although NVOCs are synthesized to deter herbivores, many of these compounds have been found to possess several therapeutic qualities, such as promoting nerve stability, enhancing sleep, and suppressing hyperresponsiveness, in addition to acting as antioxidants and anti-inflammatory agents. Therefore, many NVOCs are promising drug candidates for disease treatment and prevention. Given their volatile nature, these compounds can be administered to patients through inhalation, which is often more comfortable and convenient than other administration routes. However, the development of NVOC-based drug candidates requires a careful evaluation of the molecular mechanisms that drive their therapeutic properties to avoid potential adverse effects. Furthermore, even compounds that appear generally safe might have toxic effects depending on their dose, and therefore their toxicological assessment is also critical. In order to enhance the usage of NVOCs this short review focuses not only on the biological activities and therapeutic mode of action of representative NVOCs but also their toxic effects.
Plant monoterpenoids with structural diversities have extensive applications in food, cosmetics, pharmaceuticals, and biofuels. Due to the strong dependence on the geographical locations and seasonal annual growth of plants, agricultural production for monoterpenoids is less effective. Chemical synthesis is also uneconomic because of its high cost and pollution. Recently, emerging synthetic biology enables engineered microbes to possess great potential for the production of plant monoterpenoids. Both acyclic and cyclic monoterpenoids have been synthesized from fermentative sugars through heterologously reconstructing monoterpenoid biosynthetic pathways in microbes. Acting as catalytic templates, plant monoterpene synthases (MTPSs) take elaborate control of the monoterpenoids production. Most plant MTPSs have broad substrate or product properties, and show functional plasticity. Thus, the substrate selectivity, product outcomes, or enzymatic activities can be achieved by the active site mutations and domain swapping of plant MTPSs. This makes plasticity engineering a promising way to engineer MTPSs for efficient production of natural and non-natural monoterpenoids in microbial cell factories. Here, this review summarizes the key advances in plasticity engineering of plant MTPSs, including the fundamental aspects of functional plasticity, the utilization of natural and non-natural substrates, and the outcomes from product isomers to complexity-divergent monoterpenoids. Furthermore, the applications of plasticity engineering for improving monoterpenoids production in microbes are addressed.
Nursery-grown Norway spruce Picea abies seedlings are often heavily attacked by the pine weevil Hylobius abietis on clear-cuts the first years after planting. Because the seedlings are not resource-limited during the growing phase in the nursery they are expected to invest less in defence than naturally regenerated seedlings already present on the clear-cuts. The latter have had to cope with various environmental stressors that could make them invest more in defence. We tested if naturally regenerated plants have stronger chemical defences than nursery-grown plants. Nursery-grown plants were planted in-between naturally regenerated plants on fresh clear cuts, and phenolic and terpene compounds in the stem bark were measured after one growing season. To test both constitutive and inducible defences, plants were either wounded, painted with methyl jasmonate (MeJA) to induce defences, or given a combination of both treatments. Growth and pine weevil attacks of the plants were registered. Nursery-grown plants had higher total concentrations of phenolic compounds and lower concentrations of terpenes than naturally regenerated plants. These opposite responses were reflected in very different compound profiles in the two plant types. We suggest the differences between plant types to be results of differences in plant age, stress level, genetic origin or possibly a combination of these factors. Most compounds showed no response to wounding, MeJA-treatment or wounding and MeJA-treatment combined, but the terpenes 3-carene, euca-lyptol, limonene and para-cymene had higher concentrations in MeJA-treated nursery-grown plants than in control plants. These compounds are known to be effective in conifer resistance against weevils and bark beetles. Overall, 27% of our 400 study plants had signs of pine weevil damage after 3 ½ months in the field. However, treatment or plant type had no significant effect on whether plants were attacked or not and this might have been a result of the relatively low overall level of attacks in this study. Further studies are needed to disentangle the importance of plant age, stress level, genetic origin and resource availability for chemical defence mechanisms of young Norway spruce plants, as strengthening the natural resistance of nursery plants may be increasingly important in a future with less pesticide use.
In plants, low molecular weight terpenes produced by terpene synthases (TPS) contribute to multiple ecologically and economically important traits. The present study investigates a carrot terpene synthase gene cluster on chromosome 4 associated with volatile monoterpene production. Two carrot mutants, yellow and cola, which are contrasting in the content of low molecular weight terpenes, were crossed to develop an F 2 mapping population. The mapping analysis revealed overlapping QTLs on chromosome 4 for sabinene, α-thujene, α-terpinene, γ-terpinene, terpinen-4-ol and 4-carene. The genomic region of this locus includes a cluster of five terpene synthase genes (DcTPS04, DcTPS26, DcTPS27, DcTPS54 and DcTPS55). DcTPS04 and DcTPS54 displayed genotype-and tissue-specific variation in gene expression. Based on the QTL mapping results and the gene expression patterns, DcTPS04 and DcTPS54 were selected for functional characterization. In vitro enzyme assays showed that DcTPS54 is a single-product enzyme catalysing the formation of sabinene, whereas DcTPS04 is a multiple-product terpene synthase producing α-terpineol as a major product and four additional products including sabinene, β-limonene, β-pinene and myrcene. Furthermore, we developed a functional molecular marker that could discriminate carrot genotypes with different sabinene content in a set of 85 accessions.
p-Menthanes are among the most valuable groups of monoterpenoids produced by plants. Products such as menthol, carvone, pulegone, and limonene are well-known fragrance compounds that are also used as pesticides or medicines. In flowering plants, they are synthesized and stored in specialized anatomical structures such as glandular trichomes (GT), secretory cavities, and root resin ducts. These metabolites originate through the 2C-methyl-D-erythritol-4-phosphate precursor pathway in the plastids of GT secretory cells but also require the activity of prenyltransferases and modifying enzymes in the mitochondria and endoplasmic reticulum to complete their functionalization to biologically active forms. (−)-Menthol is the best characterized of these and is pharmacologically active due to its affinity for the Cold and Menthol Receptor 1 and the κ-Opioid Receptor. p-Menthanes are found throughout the plant kingdom, and although their essential oils are still obtained mostly through traditional agriculture, synthetic biology approaches increasingly enable large scale production of economically valuable examples in microbes and cell free systems. Limonene is the model p-menthane monoterpene, and detailed studies of (+)-limonene synthase and (−)-limonene synthase crystal structures have enabled identification of catalytic residues and determinants of stereospecificity, which has implications for the ecological properties of these volatile products in defense and interspecific signaling. This review examines the chemical diversity of the p-menthanes in the plant kingdom, their biosynthetic origins in specialized anatomical structures, and the enzymology of p-menthane biosynthetic enzymes from diverse plant families.
Terpenoids make up the biggest and most diversified class of chemical substances discovered in plants, encompassing over 40,000 individual compounds. In conifers, the production of terpenoids, either as oleoresin or emitted as volatile compounds, play an important role in the physical and chemical defence responses against pathogens and herbivores. In the present work, we examined, for the first time to the best of our knowledge, the terpenic defensive relations of Calabrian pine (Pinus nigra subsp. laricio (Poiret) Maire), facing the attack of the pine processionary moth (Thaumetopoea pityocampa (Denis and Schiffermüller, 1775)), brought about in the open on adult plant individuals growing at two distinct forest sites. Among the volatile terpenoids emitted from pine needles, bornyl acetate [(4,7,7-trimethyl-3-bicyclo[2.2.1]heptanyl) acetate] was the most frequently and selectively associated with the infestation, increasing during the period of most intense trophic activity of the caterpillars (defoliation), and decreasing thereafter. Although further work is needed to clarify whether the observed response reflects defence reactions and/or they are involved in communication among the infested plants and their biotic environment, the present results boost the currently growing interest in the isolation and characterization of plant secondary metabolites that can be used to control pests, pathogens, and weeds.
Terpenoids make up the biggest and most diversified class of chemical substances discovered in plants, encompassing over 40,000 individual compounds. In conifers, the production of terpenoids, either as oleoresin or emitted as volatile compounds, play an important role in the physical and chemical defence responses against pathogens and herbivores. In the present work, we examined, for the first time to the best of our knowledge, the terpenic defensive relations of Calabrian pine (Pinus nigra subsp. laricio (Poiret) Maire), facing the attack of the pine processionary moth (Thaumetopoea pityocampa (Denis and Schiffermüller, 1775)), brought about in the open on adult plant individuals growing at two distinct forest sites. Among the volatile terpenoids emitted from pine needles, bornyl acetate [(4,7,7-trimethyl-3-bicyclo[2.2.1]heptanyl) acetate] was the most frequently and selectively associated with the infestation, increasing during the period of most intense trophic activity of the caterpillars (defoliation), and decreasing thereafter. Although further work is needed to clarify whether the observed response reflects defence reactions and/or they are involved in communication among the infested plants and their biotic environment, the present results boost the currently growing interest in the isolation and characterization of plant secondary metabolites that can be used to control pests, pathogens, and weeds.
In contrast to flowering plants, the genomes of gymnosperms are still poorly understood. Current knowledge on the genomic architecture, gene space, and macrostructure of gymnosperm genomes mainly comes from Pinaceae species, in which spruce species (genera with most sequenced genomes to date) had played a significant role. Whereas the accumulation of nuclear genomes’ sequence information has followed a slow pace, the sequencing of chloroplast genomes skyrocketed in the last 10 years, with many spruce species sequenced so far. From the comparison of nuclear, mitochondrial, and chloroplast genomes, we can conclude that they all differ in their genomic macrostructure and rates of sequence evolution. The high structural conservation among species, genera, and families is characteristic of nuclear genomes, whereas chloroplast and mitochondrial genomes show less conserved structures due to genomic arrangements. Finally, Picea species follow trends observed in other gymnosperms and angiosperms, in which rates of sequence evolution are the highest in nuclear DNA, followed by chloroplast and then mitochondrial DNA.
Cannabis (Cannabis sativa) resin is the foundation of a multi-billion dollar medicinal and recreational plant bioproducts industry. Major components of the cannabis resin are the cannabinoids and terpenes. Variations of cannabis terpene profiles contribute much to the different flavor and fragrance phenotypes that affect consumer preferences. A major problem in the cannabis industry is the lack of proper metabolic characterization of many of the existing cultivars, combined with sometimes incorrect cultivar labeling. We characterized foliar terpene profiles of plants grown from 32 seed sources and found large variation both within and between sets of plants labeled as the same cultivar. We selected five plants representing different cultivars with contrasting terpene profiles for clonal propagation, floral metabolite profiling and trichome-specific transcriptome sequencing. Sequence analysis of these five cultivars and the reference genome of the Purple Kush (PK) cultivar revealed a total of 33 different cannabis terpene synthase (CsTPS) genes as well as variations of the CsTPS gene family and differential expression of terpenoid and cannabinoid pathway genes between cultivars. Our annotation of the PK reference genome identified 19 complete CsTPS gene models, and tandem arrays of isoprenoid and cannabinoid biosynthetic genes. An updated phylogeny of the CsTPS gene family showed three cannabis-specific clades, including a clade of sesquiterpene synthases within the TPS-b subfamily that typically contains mostly monoterpene synthases. The CsTPSs described and functionally characterized here include 13 that had not been previously characterized and collectively explain a diverse range of cannabis terpenes.
Agricultural ethanol has not been an optional fuel for petrol due to a big difference in prices. Nowadays purposefulness of developing alternative fuel production processes, including bioethanol production, is supported by both economic and environmental concerns. The role of secondary metabolites in development of plant growth and architect for enhancing the biofuel production. Besides now as days the main focus are on gene and gene products as they construct the main apparatus for the studying the plant genome. The sequencing technology and reduced cost of sequencing are accelerating the ability to discover new natural product pathways by identifying the underlying genes. In this chapter various plant products and substances are documented right from the classical biofules to gene products vis a vis gene clusters.
Conifers have evolved complex oleoresin terpene defenses against herbivores and pathogens. In co‐evolved bark beetles, conifer terpenes also serve chemo‐ecological functions as pheromone precursors, chemical barcodes for host identification, or nutrients for insect‐associated microbiomes. We highlight the genomic, molecular and biochemical underpinnings of the large chemical space of conifer oleoresin terpenes and volatiles. Conifer terpenes are predominantly the products of the conifer terpene synthase (TPS) gene family. Terpene diversity is increased by cytochromes P450 of the CYP720B class. Many conifer TPS are multiproduct enzymes. Multisubstrate CYP720B enzymes catalyse multistep oxidations. We summarise known terpenoid gene functions in various different conifer species with reference to the annotated terpenoid gene space in a spruce genome. Overall, biosynthesis of terpene diversity in conifers is achieved through a system of biochemical radiation and metabolic grids. Expression of TPS and CYP720B genes can be specific to individual cell types of constitutive or traumatic resin duct systems. Induced terpenoid transcriptomes in resin duct cells lead to dynamic changes of terpene composition and quantity to fend off herbivores and pathogens. While terpenoid defenses have contributed much to the evolutionary success of conifers, under new conditions of climate change, these defences may become inconsequential against range‐expanding forest pests.
Cannabis sativa (cannabis) produces a resin that is valued for its psychoactive and medicinal properties. Despite being the foundation of a multi-billion dollar global industry, scientific knowledge and research on cannabis is lagging behind compared to other high-value crops. This is largely due to legal restrictions that have prevented many researchers from studying cannabis, its products, and their effects in humans. Cannabis resin contains hundreds of different terpene and cannabinoid metabolites. Many of these metabolites have not been conclusively identified. Our understanding of the genomic and biosynthetic systems of these metabolites in cannabis, and the factors that affect their variability, is rudimentary. As a consequence, there is concern about lack of consistency with regard to the terpene and cannabinoid composition of different cannabis ‘strains’. Likewise, claims of some of the medicinal properties attributed to cannabis metabolites would benefit from thorough scientific validation.
Developing carrot varieties highly resistant to Alternaria dauci is a top priority for breeders. Meanwhile, consumers are increasingly demanding as regards final product quality, particularly taste. Bitterness is one of the five common taste sensations, but it is rejected by most carrot consumers. Therefore, providing tools for efficient plant breeding of resistant, low bitter carrots would be helpful. While resistance QTLs (rQTLs) have already been identified for carrot resistance to A. dauci, the genetic control and mapping of the metabolites involved in bitterness perception have not been addressed so far. We identified the metabolites most involved in bitterness by combining chemical and sensory analyses of a set of resistant and susceptible carrot genotypes grown in different environments. We evaluated their genetic control and heritability in a segregating F2:3 population over 2 years of field trials and searched for colocalizations between rQTLs and metabolite QTLs (mQTLs) to evaluate the link between bitterness and resistance traits. Our results suggest that it is possible to increase resistance while favoring low bitter varieties by selecting genomic regions involved in the expression of one or the other trait and counter-selecting others when r- and mQTL colocalization is unfavorable.
Conifers depend on complex defense systems against herbivores. Stone cells (SC) and oleoresin are physical and chemical defenses of Sitka spruce that have been separately studied in previous work.
Weevil oviposit at the tip of the previous year's apical shoot (PYAS). We investigated interactions between weevil larvae and trees in controlled oviposition experiments with resistant (R) and susceptible (S) Sitka spruce. R trees have an abundance of SC in the PYAS cortex. SC are mostly absent in S trees. R trees and S trees also differ in the composition of oleoresin terpenes. Transcriptomes of R and S trees revealed differences in long‐term weevil‐induced responses.
Performance of larvae was significantly reduced on R trees compared with S trees under experimental conditions that mimicked natural oviposition behavior at apical shoot tips and may be attributed to the effects of SC. In oviposition experiments designed for larvae to feed below the area of highest SC abundance, larvae showed an unusual feeding behavior and oleoresin appeared to function as the major defense.
The results support a role for both SC and oleoresin terpenes and possible synergies between these traits in the defense syndrome of weevil‐resistant Sitka spruce.
Sabinene is an important naturally occurring bicyclic monoterpene which can be used as flavorings, perfume additives, fine chemicals, and advanced biofuels. Up to now, this valuable terpene is commercially unavailable since there is no applicable manufacturing process. Microbial synthesis can be a promising route for sabinene production. In this review, we summarize knowledge about the metabolic pathway and key enzymes for sabinene biosynthesis. Recent advances that have been made in production of sabinene by microbial fermentation are highlighted. In these studies, researchers have identified the general synthetic pathway of sabinene from simple intermediate metabolites. Sabinene synthases of different origins were also cloned and characterized. Additionally, heterologous systems of the model microbes Escherichia coli and Saccharomyces cerevisiae were constructed to produce sabinene. This review also suggests new directions and attempts to gain some insights for achieving an industrial level production of sabinene. The combination of traditional molecular biology with new genome and proteome analysis tools will provide a better view of sabinene biosynthesis and a greater potential of microbial production.
Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells, and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue-complex. Overall, ~3,700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell-type, revealing cell specialization at the transcriptome level. MeJa caused cell-type specific transcriptome responses and changed the overall patterns of cell-type specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue-complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type specific transcriptome signatures. Characterization of cell-type specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell-types. This article is protected by copyright. All rights reserved.
The mountain pine beetle (Dendroctonus ponderosae; MPB) is an eruptive bark beetle species affecting pine forests of western North America. MPB are exposed to volatile monoterpenes, which are important host defense chemicals. We assessed the toxicity of the ten most abundant monoterpenes of lodgepole pine (Pinus contorta), a major host in the current MPB epidemic, against adult MPB from two locations in British Columbia, Canada. Monoterpenes were tested as individual volatiles and included (−)-β-phellandrene, (+)-3-carene, myrcene, terpinolene, and both enantiomers of α-pinene, β-pinene and limonene. Dose-mortality experiments identified (−)-limonene as the most toxic (LC50: 32 μL/L), and (−)-α-pinene (LC50: 290 μL/L) and terpinolene (LC50: >500 μL/L) as the least toxic. MPB body weight had a significant positive effect on the ability to survive most monoterpene volatiles, while sex did not have a significant effect with most monoterpenes. This study helps to quantitatively define the effects of individual monoterpenes towards MPB mortality, which is critical when assessing the variable monoterpene chemical defense profiles of its host species.
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.
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.
The white pine weevil (Pissodes strobi Peck) is an important insect pest in the Pacific Northwest that attacks the apical stem leaders of spruce (Picea spp.) causing damage to tree form, growth, and stand development. Because of attacks by weevils, Sitka spruce (P.sitchensis Bong.) is not commonly replanted as a commercial species in coastal British Columbia, despite its economic and ecological importance. In the last decade, the focus of research on Sitka spruce resistance against weevils has moved from silvicultural approaches to breeding for resistance. The British Columbia Ministry of Forests and Range, in collaboration with the Canadian Forest Service, has developed a successful program to screen populations and select tree genotypes for resistance to weevil attack. Part of this effort has been the establishment of clonebanks that contain genotypes from throughout the range of Sitka spruce. For metabolite profiling, using gas chromatography coupled with flame ionization detection or mass spectrometry, we analysed 111 different genotypes to determine the relationship of mono- and di-terpenoid oleoresin compounds with the resistance rating. Dehydroabietic acid, a diterpene, was identified as a strong indicator of resistance. Two monoterpenes, (+)-3-carene and terpinolene, were also associated with resistance in genotypes originating from one of the areas (Haney) in which resistance has been noted.
Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express
sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified
a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers
and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product
in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species.
Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed
multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide
similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism
in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce
and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified
product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably
lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps
are offset by their lower polymorphism.
White pine weevil (Pissodes strobi, Peck.) is a native forest insect pest in the Pacific Northwest of North America that attacks species of spruce (Picea spp.) and pine (Pinus spp.). Young Sitka spruce [Picea sitchensis (Bong.) Carr.] trees are particularly susceptible to weevil attack. Pockets of naturally occurring Sitka spruce resistance have been identified in high weevil hazard areas in coastal British Columbia. In this study, we characterize behavioral, physiological and reproductive responses of weevils to an extremely resistant Sitka spruce genotype (H898) in comparison to a highly susceptible genotype (Q903). The experiments relied on a large number of three-year-old clonally propagated trees and were therefore restricted to two contrasting Sitka spruce genotypes. When exposed to resistant trees, both male and female weevils were deterred during host selection and mating, females showed delayed or reduced ovary development, and successful reproduction of weevils was prevented on resistant trees.
Arabidopsis thaliana is the model plant and is grown worldwide by plant biologists seeking to dissect the molecular underpinning of plant growth
and development. Gene copy number variation (CNV) is a common form of genome natural diversity that is currently poorly studied
in plants and may have broad implications for model organism research, evolutionary biology, and crop science. Herein, comparative
genomic hybridization (CGH) was used to identify and interrogate regions of gene CNV across the A. thaliana genome. A common temperature condition used for growth of A. thaliana in our laboratory and many around the globe is 22 °C. The current study sought to test whether A. thaliana, grown under different temperature (16 and 28 °C) and stress regimes (salicylic acid spray) for five generations, selecting
for fecundity at each generation, displayed any differences in CNV relative to a plant lineage growing under normal conditions.
Three siblings from each alternative temperature or stress lineage were also compared with the reference genome (22 °C) by
CGH to determine repetitive and nonrepetitive CNVs. Findings document exceptional rates of CNV in the genome of A. thaliana over immediate family generational scales. A propensity for duplication and nonrepetitive CNVs was documented in 28 °C CGH,
which was correlated with the greatest plant stress and infers a potential CNV–environmental interaction. A broad diversity
of gene species were observed within CNVs, but transposable elements and biotic stress response genes were notably overrepresented
as a proportion of total genes and genes initiating CNVs. Results support a model whereby segmental CNV and the genes encoded
within these regions contribute to adaptive capacity of plants through natural genome variation.
Following the domestication of maize over the past approximately 10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.
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.
Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood.
Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA.
Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes.
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.
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.
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.
Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl jasmonate (MeJA) (Martin et al., 2002). Increase of enzyme activities of terpenoid biosynthesis and accumulation of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts. The formation of defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after infestation with fungal pathogens. In the present study, we analyzed the traumatic resin response in Norway spruce further at the molecular genetic level. Treatment of trees with MeJA induced transient transcript accumulation of monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce. In screening for defense-related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF67, was isolated and the recombinant enzyme expressed in E. coli and functionally characterized in vitro. The cloned PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene synthase. The enzyme encoded by PaJF67 forms stereospecifically (+)-3-carene (78% of total product) together with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total product). (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway spruce and other members of the Pinaceae.
Terpenoids are characteristic constitutive and inducible defense chemicals of conifers. The biochemical regulation of terpene formation, accumulation, and release from conifer needles was studied in Norway spruce [Picea abies L. (Karst)] saplings using methyl jasmonate (MeJA) to induce defensive responses without inflicting physical damage to terpene storage structures. MeJA treatment caused a 2-fold increase in monoterpene and sesquiterpene accumulation in needles without changes in terpene composition, much less than the 10- and 40-fold increases in monoterpenes and diterpenes, respectively, observed in wood tissue after MeJA treatment (D. Martin, D. Tholl, J. Gershenzon, J. Bohlmann [2002] Plant Physiol 129: 1003-1018). At the same time, MeJA triggered a 5-fold increase in total terpene emission from foliage, with a shift in composition to a blend dominated by oxygenated monoterpenes (e.g. linalool) and sesquiterpenes [e.g. (E)-beta-farnesene] that also included methyl salicylate. The rate of linalool emission increased more than 100-fold and that of sesquiterpenes increased more than 30-fold. Emission of these compounds followed a pronounced diurnal rhythm with the maximum amount released during the light period. The major MeJA-induced volatile terpenes appear to be synthesized de novo after treatment, rather than being released from stored terpene pools, because they are almost completely absent from needle oleoresin and are the major products of terpene synthase activity measured after MeJA treatment. Based on precedents in other species, the induced emission of terpenes from Norway spruce foliage may have ecological and physiological significance.
An improved protocol was developed for efficient and reliable extraction of high-quality total RNA and mRNA from various tissues of spruce (Picea spp.) and poplar (Populus spp.) trees, as well as other plant species. This method was specifically optimized for tissues with high content of polysaccharides, oleoresin terpenoids, and phenolic secondary metabolites, which often co-precipitate with RNA and inhibit subsequent reverse transcription. The improved protocol yielded up to 600 micrograms of total RNA per gram of tissue suitable for standard expressed sequence tags (ESTs), full-length cDNA library construction, and for microarray applications.
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.
Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of RNA transcript abundance. Useful data from this method depend on fitting data to theoretical curves that allow computation of mRNA levels. Calculating accurate mRNA levels requires important parameters such as reaction efficiency and the fractional cycle number at threshold (CT) to be used; however, many algorithms currently in use estimate these important parameters. Here we describe an objective method for quantifying qRT-PCR results using calculations based on the kinetics of individual PCR reactions without the need of the standard curve, independent of any assumptions or subjective judgments which allow direct calculation of efficiency and CT. We use a four-parameter logistic model to fit the raw fluorescence data as a function of PCR cycles to identify the exponential phase of the reaction. Next, we use a three-parameter simple exponent model to fit the exponential phase using an iterative nonlinear regression algorithm. Within the exponential portion of the curve, our technique automatically identifies candidate regression values using the P-value of regression and then uses a weighted average to compute a final efficiency for quantification. For CT determination, we chose the first positive second derivative maximum from the logistic model. This algorithm provides an objective and noise-resistant method for quantification of qRT-PCR results that is independent of the specific equipment used to perform PCR reactions.
We analyzed the expression pattern of various terpene synthase (TPS) genes in response to a wounding injury applied to the apical leader of Sitka spruce (Picea sitchensis Bong. Carr.) genotypes known to be resistant (R) or susceptible (S) to white pine weevil (Pissodes strobi Peck.) attack. The purpose was to test if differences in constitutive or wound-induced TPS expression can be associated with established weevil resistance. All wounding treatments were conducted on 9-year-old R and S trees growing under natural field conditions within the range of variation for weevil R and S genotypes. Representative cDNAs of the monoterpene synthase (mono-TPS), sesquiterpene synthase (sesqui-TPS), and diterpene synthase (di-TPS) classes were isolated from Sitka spruce to assess TPS transcript levels. Based on amino acid sequence similarity, the cDNAs resemble Norway spruce (Picea abies) (-)-linalool synthase (mono-TPS; PsTPS-Linl) and levopimaradiene/abietadiene synthase (di-TPS; PsTPS-LASl), and grand fir (Abies grandis) delta-selinene synthase (sesqui-TPS; PsTPS-Sell). One other mono-TPS was functionally identified as (-)-limonene synthase (PsTPS-Lim). No significant difference in constitutive expression levels for these TPSs was detected between R and S trees. However, over a postwounding period of 16 d, only R trees exhibited significant transcript accumulation for the mono- and sesqui-TPS tested. Both R and S trees exhibited a significant accumulation of PsTPS-LASl transcripts. An assessment of traumatic resin duct formation in wounded leaders showed that both R and S trees responded by forming traumatic resin ducts; however, the magnitude of this response was significantly greater in R trees. Collectively, our data imply that the induced resinosis response is an important aspect of defense in weevil R Sitka spruce trees growing under natural conditions.
It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity). The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues). To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase gamma-humulene synthase. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.
Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.
Forty-one 2-year-old clones of Picea sitchensis (Bong.) Carr. from three full-sib families (14 clones from each of two families and 13 clones from a third family) were either wounded and inoculated with an isolate of Heterobasidion annosum (Fr.) Bref. or wounded without inoculation. Lesion lengths on the inner bark from the point of inoculation varied among clones 35 days after treatment. There was no relationship between lesion length and relatedness within families. Two clones (21342 and 25202) with the shortest lesions, tentatively designated as less susceptible to H. annosum, and two clones (21176 and 27166) with the longest lesions, designated more susceptible, were selected for comparison of host anatomical and chemical responses to infection. The position and structure of the ligno-suberized boundary zone (LSZ) in the bark of the clones suggested that the less susceptible clones formed thicker layers of suberized cells in the LSZ following wounding plus inoculation. No LSZ was observed in two ramets of the more susceptible Clone 27166 following wounding and inoculation with H. annosum. Compared with more susceptible genotypes, clones of P. sitchensis with low susceptibility to H. annosum had high relative proportions of (+)-alpha-pinene, (-)-beta-pinene and one unidentified terpene constituent (Unknown-15) in cortical resin sampled 25 cm from the lesions. In contrast, more susceptible clones had higher relative proportions of (-)-limonene, Unknown-16, Unknown-18 and Unknown-19. In the secondary resin produced in bark tissues surrounding the lesions, proportions of several monoterpene constituents varied; these changes included a decrease in the relative amount of beta-phellandrene and corresponding small increases in some minor constituents. The concentrations of the monoterpenes, except a few minor constituents, increased in the infected tissues. Wounding plus inoculation with H. annosum resulted in varied monoterpene responses, with distinct differences between less susceptible and more susceptible clones. In less susceptible clones, Unknown-19 increased following wounding plus inoculation, whereas in more susceptible clones, concentrations of delta-3-carene and Unknown-13 and Unknown-16 increased. Differences in both constitutive and induced resin monoterpene profiles may provide useful markers for resistance to H. annosum in selection and breeding programs.
The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant–herbivore, plant–pollinator, and plant–pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin acids found in the defensive oleoresin of conifers. Although these paralogous enzymes are 91% identical at the amino acid level, one is a single-product enzyme, whereas the other is a multiproduct enzyme that forms completely different products. We used a rational approach of homology modeling, protein sequence comparison, domain swapping, and a series of reciprocal site-directed mutagenesis to identify the specific residues that direct the different product outcomes. A one-amino acid mutation switched the levopimaradiene/abietadiene synthase into producing isopimaradiene and sandaracopimaradiene and none of its normal products. Four mutations were sufficient to reciprocally reverse the product profiles for both of these paralogous enzymes while maintaining catalytic efficiencies similar to the wild-type enzymes. This study illustrates how neofunctionalization can result from relatively minor changes in protein sequence, increasing the diversity of secondary metabolites important for conifer defense.
• biochemical evolution
• natural product biosynthesis
• plant defense
• terpene diversity
• site-directed mutagenesis
Conifers possess inducible terpenoid defense systems. These systems are associated with the formation of traumatic resin ducts (TRD) and are underpinned by enhanced gene expression and activity of terpene synthases (TPS), enzymes responsible for oleoresin formation. We first determined that Sitka spruce (Picea sitchensis [Bong.] Carriere) had the capacity for TRD formation by mechanically wounding representative trees. We then proceeded to investigate whether the white pine weevil (Pissodes strobi Peck.), a stem-boring insect, can influence the expression of genes encoding monoterpene synthases (mono-tps) in Sitka spruce. We went on to compare this response with the effects of a simulated insect attack by drill wounding. A significant increase in mono-tps transcript level was observed in the leaders of lateral branches of weevil-attacked and mechanically wounded trees. In this study, weevils induced a more rapid enhancement of mono-tps gene expression. A full-length Sitka spruce mono-tps cDNA (PsTPS2) was isolated, expressed in Escherichia coli, and functionally identified as (-)-pinene synthase. The recombinant (-)-pinene synthase catalyzes the formation of (-)-alpha-pinene and (-)-beta-pinene, both of which are known constituents of stem oleoresin in Sitka spruce and increase in abundance after weevil attack. These data suggest that increased (-)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.
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.
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.
Some plant terpenes such as sterols and carotenes are part of primary metabolism and found essentially in all plants. However, the majority of the terpenes found in plants are classified as 'secondary' compounds, those chemicals whose synthesis has evolved in plants as a result of selection for increased fitness via better adaptation to the local ecological niche of each species. Thousands of such terpenes have been found in the plant kingdom, but each species is capable of synthesizing only a small fraction of this total. In plants, a family of terpene synthases (TPSs) is responsible for the synthesis of the various terpene molecules from two isomeric 5-carbon precursor 'building blocks', leading to 5-carbon isoprene, 10-carbon monoterpenes, 15-carbon sesquiterpenes and 20-carbon diterpenes. The bryophyte Physcomitrella patens has a single TPS gene, copalyl synthase/kaurene synthase (CPS/KS), encoding a bifunctional enzyme producing ent-kaurene, which is a precursor of gibberellins. The genome of the lycophyte Selaginella moellendorffii contains 18 TPS genes, and the genomes of some model angiosperms and gymnosperms contain 40-152 TPS genes, not all of them functional and most of the functional ones having lost activity in either the CPS- or KS-type domains. TPS genes are generally divided into seven clades, with some plant lineages having a majority of their TPS genes in one or two clades, indicating lineage-specific expansion of specific types of genes. Evolutionary plasticity is evident in the TPS family, with closely related enzymes differing in their product profiles, subcellular localization, or the in planta substrates they use.
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.
Induction of terpene synthase (TPS) gene expression and enzyme activity is known to occur in response to various chemical and biological stimuli in several species of spruce (genus Picea). However, high sequence identity between TPS family members has made it difficult to determine the induction patterns of individual TPS at the protein and transcript levels and whether specific TPS enzymes respond differentially to treatment. In the present study we used a multi-level approach to measure the induction and activity of TPS enzymes in protein extracts of Norway spruce (Picea abies) bark tissue following treatment with methyl jasmonate (MeJA). Measurements were made on the transcript, protein, enzyme activity and metabolite levels. Using a relatively new proteomics application, selective reaction monitoring (SRM), it was possible to differentiate and quantitatively measure the abundance of several known TPS proteins and three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) isoforms in Norway spruce. Protein levels of individual TPS and DXS enzymes were differentially induced upon MeJA treatment and good correlation was generally observed between induction of transcripts, proteins, and enzyme activities. Most of the mono- and diterpenoid metabolites accumulated with similar temporal patterns of induction as part of the coordinated multi-compound chemical defense response. Protein and enzyme activity levels of the monoTPS (+)-3-carene synthase and the corresponding accumulation of (+)-3-carene was induced to a higher fold change than any other TPS or metabolite measured, indicating an important role in the induced terpenoid defense response in Norway spruce.
Enzymologists have never had it so good. Thanks to advances in gene discovery, driven by high-throughput sequencing of genomes and transcriptomes, and improved heterologous expression of proteins, the functional characterization of new enzymes is generally straightforward: clone the gene, express the protein, assay for activity, and publish the results. However, without knowing the correct substrate to use in the assay such efforts can easily go astray. A paper by Schilmiller et al. (1) published in the latest issue of PNAS shows how one can elegantly avoid this pitfall by integrating genomic, genetic, enzymological, and metabolite-profiling approaches. In this important contribution, the authors report the discovery of a new substrate for enzymes of plant terpenoid biosynthesis.
Cellulose synthase (CesA) is a central catalyst in the generation of the plant cell wall biomass and is, therefore, the focus of intense research. Characterization of individual CesA genes from Populus species has led to the publication of several different naming conventions for CesA gene family members in this model tree. To help reduce the resulting confusion, we propose here a new phylogeny-based CesA nomenclature that aligns the Populus CesA gene family with the established Arabidopsis thaliana CesA family structure.
The monoterpene cyclase limonene synthase transforms geranyl diphosphate to a monocyclic olefin and constitutes the simplest model for terpenoid cyclase catalysis. (-)-4S-Limonene synthase preprotein from spearmint bears a long plastidial targeting sequence. Difficulty expressing the full-length preprotein in Escherichia coli is encountered because of host codon usage, inclusion body formation, and the tight association of bacterial chaperones with the transit peptide. The purified preprotein is also kinetically impaired relative to the mixture of N-blocked native proteins produced in vivo by proteolytic processing in plastids. Therefore, the targeting sequence, that precedes a tandem pair of arginines (R58R59) which is highly conserved in the monoterpene synthases, was removed. Expression of this truncated protein, from a vector that encodes a tRNA for two rare arginine codons (pSBET), affords a soluble, tractable 'pseudomature' form of the enzyme that is catalytically more efficient than the native species. Truncation up to and including R58, or substitution of R59, yields enzymes that are incapable of converting the natural substrate geranyl diphosphate, via the enzymatically formed tertiary allylic isomer 3S-linalyl diphosphate, to (-)-limonene. However, these enzymes are able to cyclize exogenously supplied 3S-linalyl diphosphate to the olefinic product. This result indicates a role for the tandem arginines in the unique diphosphate migration step accompanying formation of the intermediate 3S-linalyl diphosphate and preceding the final cyclization reaction catalyzed by the monoterpene synthases. The structural basis for this coupled isomerization-cyclization reaction sequence can be inferred by homology modeling of (-)-4S-limonene synthase based on the three-dimensional structure of the sesquiterpene cyclase epi-aristolochene synthase [Starks, C. M., Back, K., Chappell, J., and Noel, J. P. (1997) Science 277, 1815-1820].
Bark beetle infestation and associated fungal infection are a serious disease problem in conifer species. Conifers have evolved elaborate, constitutive and inducible, terpene-based defense mechanisms to deter insect pests and their symbiotic fungal pathogens. This process involves the secretion of oleoresin, a complex mixture of monoterpenes, sesquiterpenes and diterpenoid acids. Induced oleoresinosis in grand fir (Abies grandis) provides a model system for studying the regulation of defensive terpene biosynthesis and for identifying relevant genes. The ecological relationships between conifers, beetle pests, beetle predators and fungal pathogens present several possible avenues for manipulating oleoresin composition to improve tree resistance. Possible examples include chemically disguising the host, adding toxins and altering the levels of pheromone precursors, attractants for predators or hormone mimics to disrupt insect development. Strategies and prospects for generating transgenic conifers with increased defense capability are discussed.
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.
(-)-(4S)-limonene synthase (LS) and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase (LPS) from grand fir (Abies grandis) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.
A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.
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.
Numerous terpenoid compounds are present in copious amounts in the oleoresin produced by conifers, especially following exposure to insect or fungal pests. CDNA clones for many terpene synthases responsible for the biosynthesis of these defense compounds have been recovered from several conifer species. Here, the use of three terpene synthase sequences as heterologous probes for the discovery of related terpene synthase genes in Douglas-fir, Pseudotsuga menziesii (Mirbel) Franco (Pinaceae), is reported. Four full-length terpene synthase cDNAs were recovered from a methyl jasmonate-induced Douglas-fir bark and shoot cDNA library. These clones encode two multi-product monoterpene synthases [a (-)-alpha-pinene/(-)-camphene synthase and a terpinolene synthase] and two single-product sesquiterpene synthases [an (E)-beta-farnesene synthase and a (E)-gamma-bisabolene synthase].
Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.
Summary 657
Acknowledgements 671
References 671
Insects select their hosts, but trees cannot select which herbivores will feed upon them. Thus, as long-lived stationary organisms, conifers must resist the onslaught of varying and multiple attackers over their lifetime. Arguably, the greatest threats to conifers are herbivorous insects and their associated pathogens. Insects such as bark beetles, stem- and wood-boring insects, shoot-feeding weevils, and foliage-feeding budworms and sawflies are among the most devastating pests of conifer forests. Conifer trees produce a great diversity of compounds, such as an enormous array of terpenoids and phenolics, that may impart resistance to a variety of herbivores and microorganisms. Insects have evolved to specialize in resistance to these chemicals – choosing, feeding upon, and colonizing hosts they perceive to be best suited to reproduction. This review focuses on the plant–insect interactions mediated by conifer-produced terpenoids. To understand the role of terpenoids in conifer–insect interactions, we must understand how conifers produce the wide diversity of terpenoids, as well as understand how these specific compounds affect insect behaviour and physiology. This review examines what chemicals are produced, the genes and proteins involved in their biosynthesis, how they work, and how they are regulated. It also examines how insects and their associated pathogens interact with, elicit, and are affected by conifer-produced terpenoids.
The chemical defence of pine against herbivorous insects has been intensively studied with respect to its effects on the performance and behaviour of the herbivores as well as on the natural enemies of pine herbivores. The huge variety of terpenoid pine components play a major role in mediating numerous specific food web interactions. The constitutive terpenoid pattern can be adjusted to herbivore attack by changes induced by insect feeding or oviposition activity. Recent studies on folivorous pine sawflies have highlighted the role of induced pine responses in herbivore attack and have demonstrated the importance of analysing the variability of pine defence and its finely tuned specificity with respect to the herbivore attacker in a multitrophic context.
Diterpene resin acids are a significant component of conifer oleoresin, which is a viscous mixture of terpenoids present constitutively or inducibly upon herbivore or pathogen attack and comprises one form of chemical resistance to such attacks. This review focuses on the recent discoveries in the chemistry, biosynthesis, molecular biology, regulation, and biology of these compounds in conifers.
Identification and validation of COS markers in Pinaceae
- C Liewlaksaneeyanawin
- J Zhuang
- M Tang
- G Lueng
- C Oddy
- S Findlay
- N Farzaneh
- C E Ritland
- J Bohlmann
- K Ritland
Liewlaksaneeyanawin, C., Zhuang, J., Tang, M., Lueng, G., Oddy, C., Findlay,
S., Farzaneh, N., Ritland, C.E., Bohlmann, J. and Ritland, K. (2009) Identification and validation of COS markers in Pinaceae. Tree Genet. Genom. 5,
247-255.
phloem caused by mechanical wounding
- Karst
Karst., phloem caused by mechanical wounding. J. Chem. Ecol. 22, 559-573.