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Keywords:

  • conifer defense;
  • monoterpene synthase;
  • TPS gene family;
  • gene copy number variation;
  • biomarker

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Sitka spruce (Picea sitchensis [Bong.] Carr.) is a conifer species native to the temperate rainforests of the Pacific coast of Canada and the United States that contributes to the large biodiversity of the Pacific Northwest coastal ecosystem. Sitka spruce is also planted commercially in North America and Europe. In North America, the major pest of young Sitka spruce trees is the white pine weevil (or spruce shoot weevil; Pissodes strobi Peck). Weevil infestation has substantially reduced both the natural regeneration and commercial re-forestation of Sitka spruce. Female weevils oviposit near the top of young trees, and the emergent larvae feed on the phloem and cambium, resulting in death of the apical stem section, loss of apical dominance, and reduced growth. Individual trees are often attacked repeatedly over several years when weevil populations are high, resulting in death of the tree or out-competition for light by surrounding vegetation (King et al., 2004; King and Alfaro, 2009). Naturally occurring resistance has been identified in native Sitka spruce populations (King et al., 2004; King and Alfaro, 2009). However, little is known about the genomic and molecular mechanisms of this resistance, except that multiple components including chemical, physical and anatomical traits of the bark tissue as well as phenology may be involved (King et al., 2004; King and Alfaro, 2009).

One component of the chemical defense system of conifers is the production of terpenoid oleoresin and terpenoid volatile emissions (Phillips and Croteau, 1999; Mumm and Hilker, 2006; Keeling and Bohlmann, 2006a,b; Zulak and Bohlmann, 2010). The oleoresin of any individual Sitka spruce tree comprises dozens of cyclic and acyclic monoterpenes, sesquiterpenes and diterpenes (Miller et al., 2005; Robert et al., 2010). These terpenoids are produced enzymatically by terpene synthases (TPSs), and their diversity reflects the size of the TPS gene family as well as the single- and multi-product profiles of these enzymes (Martin et al., 2004; Keeling and Bohlmann, 2006b; Feng et al., 2011). The large TPS gene family of conifers probably resulted from gene duplication and functional separation events such as neo-functionalization and/or sub-functionalization (Martin et al., 2004; Keeling et al., 2008). TPS genes for many oleoresin terpenoids have been cloned and functionally characterized, for example in grand fir (Abies grandis) (Bohlmann et al., 1999), Norway spruce (Picea abies) (Martin et al., 2004) and Sitka spruce (Byun-McKay et al., 2003). The terpenoid defenses of Sitka spruce are constitutive, and can be induced following insect attack (Byun-McKay et al., 2003; Miller et al., 2005), wounding (Byun-McKay et al., 2006), fungal colonization (Woodward et al., 2007) or treatment with the octadecanoid defense elicitor methyl jasmonate (MeJa) (Miller et al., 2005). Previous work in Norway spruce showed that induced monoterpene accumulation positively correlates with increased monoterpene synthase enzyme activity, transcript and protein abundance (Zulak et al., 2009).

As part of a long-term program on weevil resistance in Sitka spruce, genotypes from across its native geographic range have been evaluated for insect resistance in replicated clonal trials (King et al., 2004; King and Alfaro, 2009), and the terpenoid metabolite composition of more than 100 genotypes was determined (Robert et al., 2010). The monoterpenes (+)-3-carene and terpinolene, as well as the diterpenoid dehydroabietic acid, were associated with resistance in trees originating from the Haney region of British Columbia, Canada (Robert et al., 2010). One particular genotype (H898) from this region has extreme resistance to white pine weevil (King and Alfaro, 2009), and has higher levels of (+)-3-carene (Robert and Bohlmann, 2010) than a representative susceptible Sitka spruce genotype (Q903) from the Haida Gwaii Islands in British Columbia, Canada, which accumulates only trace amounts of (+)-3-carene (Robert and Bohlmann, 2010). In behavioral and physiological studies using H898 as a resistant genotype and Q903 as a susceptible genotype, weevils were found to be deterred from feeding on resistant trees compared to susceptible trees, and, when forced to feed on resistant trees, female weevils had delayed ovary development and reduced reproductive success (Robert and Bohlmann, 2010). Additionally, increased levels of (+)-3-carene in Norway spruce trees are correlated with decreased larval survival of the great spruce bark beetle (Storer and Speight, 1996), and lodgepole pine clones that are resistant to the Douglas fir pitch moth had higher relative levels of (+)-3-carene than susceptible clones (Rocchini et al., 2000). A TPS gene responsible for (+)-3-carene biosynthesis has previously been cloned and functionally characterized from Norway spruce (Fäldt et al., 2003). The recombinant PaTPS-3car protein produced (+)-3-carene and terpinolene as major products, and expression of the gene and protein was induced in Norway spruce by treatment with MeJa (Fäldt et al., 2003; Zulak et al., 2009). Closely related sequences from Sitka spruce have been identified by mining EST and full-length cDNA resources (Ralph et al., 2008; C.I. Keeling and J. Bohlmann, unpublished data).

The contrasting (+)-3-carene phenotypes of weevil-resistant (H898) and weevil-susceptible (Q903) genotypes of Sitka spruce provide a unique opportunity to study the genomic, molecular and biochemical basis of monoterpene variation associated with conifer resistance. Using comparative genomics, proteomics and biochemical approaches, we tested the following possibilities to explain the variation in (+)-3-carene phenotypes: (i) a difference of the number of (+)-3-carene synthase gene copies, (ii) a difference in (+)-3-carene synthase gene expression, (iii) a difference in (+)-3-carene synthase protein expression, or (iv) a difference in (+)-3-carene synthase enzyme activities. We describe identification and molecular and enzyme kinetic analyses of a small family of (+)-3-carene synthase genes from resistant and susceptible Sitka spruce trees.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This study extends the previously reported terpenoid metabolite profiling of 111 Sitka spruce genotypes that identified an association between constitutive levels of (+)-3-carene and weevil resistance in trees originating from the Haney area of coastal British Columbia (Robert et al., 2010). The present analysis of the molecular and biochemical control of constitutive and induced (+)-3-carene biosynthesis required a large number of clonally propagated sapling trees, and was therefore performed using a contrasting pair of representative genotypes, for which weevil resistance and susceptibility had been previously established (Robert and Bohlmann, 2010). Resistant (H898) and susceptible (Q903) Sitka spruce trees were treated with MeJa to induce a defense response, and the basis of differential (+)-3-carene accumulation in these trees was investigated by monitoring the monoterpene profile, enzyme activity and protein and transcript abundance over time. All experiments were performed with four biological replicates per genotype, treatment and time point, using apical stem sections, which are the natural target of weevils for oviposition and feeding.

Constitutive monoterpene profiles of resistant and susceptible Sitka spruce

Monoterpene compounds were extracted from apical stem sections of resistant and susceptible plants and were analyzed by GC/MS. Resistant trees had a significantly (F1,6 = 8.40, P = 0.027) higher amount of total monoterpenes than susceptible trees (Figure 1a). Analysis of individual monoterpenes in untreated trees revealed that resistant trees accumulated significantly higher amounts of (+)-α-pinene (F1,6 = 11.484, P = 0.015), α-thujene (F1,6 = 13.51, P = 0.010), (−)-β-pinene (F1,6 = 16.22, P = 0.007), (+)-3-carene (F1,6 = 44.96, P = 0.001), α-terpinene (F1,6 = 39.66, P = 0.001) and terpinolene (F1,6 = 28.34, P = 0.002) than susceptible trees (Figure 1b). Resistant trees accumulated 120-fold more (+)-3-carene than susceptible trees (1.28 versus 0.011 μg g−1 dry weight [DW], respectively). This result and the previously established association between (+)-3-carene and weevil resistance identified (+)-3-carene as a relevant target for further investigation into the genomic, molecular and biochemical basis of differential monoterpene biosynthesis in Sitka spruce.

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Figure 1.  Resistant and susceptible Sitka spruce trees have different constitutive resin monoterpene composition. (a) Constitutive amount of total monoterpenes extracted. (b) Amount of individual monoterpenes extracted. All values are the mean (μg g−1 DW) ± 1 SE of four biological replicates (three technical replicates per sample). In (a), asterisks indicate significant difference between resistant and susceptible trees at < 0.05. In (b), asterisks indicate significant difference between resistant and susceptible trees at < 0.05.

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cDNA cloning and functional characterization of (+)-3-carene synthase-like genes in resistant and susceptible Sitka spruce

Primers based on the previously characterized Norway spruce (+)-3-carene synthase (PaTPS-3car; Fäldt et al., 2003) and on Sitka spruce EST sequences with high homology to PaTPS-3car (Ralph et al., 2008) were used to amplify candidate TPS cDNAs from resistant and susceptible trees. PCR screening and sequencing of the ORF and UTR regions of candidate cDNAs in both resistant (R) and susceptible (S) trees identified four distinct (+)-3-carene synthase-like genes (Figure 2a). Full-length cDNAs of two of the genes (PsTPS-3car1 and PsTPS-sab) were cloned from both R- and S-trees, with the allelic variants from R- and S-trees having 99% sequence identity (Figure 2 and Table S1). PsTPS-3car1 and PsTPS-sab share 82% predicted protein sequence identity. A third gene, PsTPS-3car2(R), was found only in the resistant genotype, and had 95% protein sequence identity with PaTPS-3car from Norway spruce (Fäldt et al., 2003) and with a fourth gene, PsTPS-3car3(S), which was amplified from cDNA only in the susceptible genotype. In total, six different full-length cDNAs were cloned for functional characterization, corresponding to the four different (+)-3-carene synthase-like genes and including the two allelic variants (R and S) of PsTPS-3car1 and PsTPS-sab: PsTPS-3car1(R), PsTPS-3car2(R), and PsTPS-sab(R) from resistant trees; and PsTPS-3car1(S), PsTPS-3car3(S), and PsTPS-sab(S) from susceptible trees (Figure 2a).

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Figure 2.  cDNA cloning and functional characterization of PsTPS-3car and PsTPS-sab genes. (a) Neighbor-joining phylogenetic tree and (b) background-subtracted GC/MS product profiles of (+)-3-carene and (+)-sabinene synthases cloned from cDNA of resistant (R) and susceptible (S) Sitka spruce trees. The scale bar in (a) corresponds to 0.06 amino acid substitutions per site. Bootstrap values for distances greater than 50% (1000 runs) are shown at nodes, calculated using the neighbor-joining method in CLC bio main workbench (http://www.clcbio.com/main/). Allelic variants of the same gene are labelled in the same colour; genes labelled in different colors have less than 99% amino acid sequence identity.

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Full-length cDNAs were expressed in Escherichia coli, and the recombinant proteins were Ni-affinity purified and assayed for TPS activity. All of the recombinant proteins were active with geranyl diphosphate (GPP), and were identified as multi-product monoterpene synthases. PsTPS-3car1(R), PsTPS-3car1(S), PsTPS-3car2(R) and PsTPS-3car3(S) produced predominantly (+)-3-carene, while PsTPS-sab(R) and PsTPS-sab(S) produced predominantly (+)-sabinene (Figure 2c and Table S2). All of the recombinant proteins produced terpinolene as the second most abundant monoterpene, regardless of whether (+)-3-carene or (+)-sabinene was the primary product. Assays of the recombinant proteins using neryl diphosphate (NDP) as a possible alternative monoterpene synthase substrate (Schilmiller et al., 2009; Bohlmann and Gershenzon, 2009) produced small amounts of the identical products, but assays using farnesyl diphosphate (FPP) and geranyl geranyl diphosphate (GGPP) did not yield products.

Genomic sequences for PsTPS-3car2 and PsTPS-3car3

PsTPS-3car2 was not found in the transcriptome (i.e. cDNA) of susceptible trees, and, conversely, PsTPS-3car3 was not found in the transcriptome of resistant trees, despite extensive efforts to PCR-amplify full-length or small representative cDNA fragments of these genes, suggesting that they were not expressed in the tissues tested. To assess the presence of these genes within the genomes of resistant and susceptible trees, we designed primers for PsTPS-3car2 and PsTPS-3car3 and used genomic DNA from each tree genotype as a template. PCR using primers targeting PsTPS-3car3 yielded an amplicon of approximately 3 kb from both resistant and susceptible trees. Sequencing of the PCR product from susceptible trees identified a gene with 100% nucleotide sequence identity to the PsTPS-3car3(S) transcript; and sequencing of the PCR product from resistant trees identified a gene with 99% nucleotide sequence identity to the genomic sequence of susceptible trees, confirming the presence of the PsTPS-3car3 gene in the genome of resistant and susceptible trees. The genomic PsTPS-3car3 clone contained ten exons and nine introns, consistent with the previously characterized genomic sequences of conifer monoterpene synthases (Trapp and Croteau, 2001; Hamberger et al., 2009).

Amplification using primers for PsTPS-3car2 and genomic DNA from resistant trees resulted in a sequence of approximately 3 kb in length, and a deduced ORF with 100% sequence identity to the PsTPS-3car2(R) transcript. This sequence had 97% nucleotide sequence identity to the genomic DNA sequences of PsTPS-3car3(R) and PsTPS-3car3(S) across ten exons and nine introns. Several primer combinations were used in attempts to amplify PsTPS-3car2(S) from the genomic DNA of susceptible trees. No bands were generated using any of the primer combinations, suggesting that the PsTPS-3car2 gene is not present within the genome of susceptible trees.

A clade of (+)-3-carene synthase-like genes within the conifer TPS-d1 sub-family

A neighbor-joining phylogeny of the deduced protein sequences of previously published conifer TPS genes (Martin et al., 2004; Keeling and Bohlmann, 2006b) and the genes from this study places the Sitka spruce cDNAs and genomic DNAs encoding (+)-3-carene synthases and (+)-sabinene synthase with other conifer monoterpene synthases in the TPS-d1 subfamily (Figure S1 and Table S3). Within the TPS-d1 subfamily, the PsTPS-3car and PsTPS-sab genes form a distinct clade of functionally related enzymes that also includes the previously characterized Norway spruce PaTPS-3car genes (Fäldt et al., 2003) and the Douglas fir (Pseudotsuga menziesii) terpinolene synthase (Huber et al., 2005). The formation of the three major terpene products of these multi-product TPS, (+)-3-carene, (+)-sabinene and terpinolene, can be explained on the basis of a common reaction mechanism (Fäldt et al., 2003).

Genotype-specific patterns of constitutive and MeJa-induced (+)-3-carene accumulation, PsTPS-3car transcript accumulation, and PsTPS-3car protein abundance

To investigate the role of members of the PsTPS-3car/PsTPS-sab clade in the (+)-3-carene phenotypes observed in resistant and susceptible Sitka spruce, we profiled metabolites, transcripts, proteins and enzyme activities in the apical stem section of trees treated with MeJa or with Tween-20 (control) at day 0 (untreated) and at 2, 7, 14, 21 and 28 days following treatment. Samples were analyzed for (i) monoterpene accumulation, (ii) monoterpene synthase enzyme activity, and (iii) transcript abundance of monoterpene synthases. The abundance of monoterpene synthase proteins was monitored using selected reaction monitoring (SRM) at 0 and 7 days after treatment. In addition to monitoring genes and proteins of the PsTPS-3car/PsTPS-sab clade, we included a Sitka spruce (−)-α-pinene synthase (PsTPS-αpin; C.I. Keeling and J. Bohlmann, unpublished data) for comparison and reference, as untreated resistant trees accumulate similar levels of (−)-α-pinene and (+)-3-carene (Figure 1b). However (−)-α-pinene is not associated with resistance (Robert et al., 2010).

Monoterpene accumulation and volatilization in resistant and susceptible Sitka spruce

Untreated (day 0) resistant trees accumulated significantly (F1,6 = 8.40, P = 0.027) higher levels of monoterpenes (10.87 ± 2.08 μg g−1 DW) than susceptible trees (4.70 ± 0.48 μg g−1 DW) (Figure 3), and, throughout the time course, resistant trees had consistently significantly higher total monoterpene levels than susceptible trees following treatment with MeJa (Table S4). Significant increases in total monoterpene accumulation in response to MeJa compared to controls were observed in resistant trees at 21 days after treatment (F1,6 = 9.41, P = 0.022) and in susceptible trees at 7 (F1,6 = 14.93, P = 0.008), 14 (F1,6 = 23.71, P = 0.003) and 28 (F1,6 = 8.97, P = 0.024) days after treatment (Figure 3). The accumulation profiles of individual monoterpenes were also investigated. Untreated resistant trees accumulated significantly (F1,6 = 44.96, P = 0.001) higher levels (1.28 ± 0.19 μg g−1 DW) of (+)-3-carene than susceptible trees (0.011 ± 0.002 μg g−1 DW), but treatment with MeJa did not increase the level of (+)-3-carene in resistant trees, although accumulation of (+)-sabinene (day 7: F1,6 = 6.65, P = 0.042; day 14: F1,6 = 11.38, = 0.015; day 21: F1,6 = 18.81, = 0.005; day 28: F1,6 = 14.08, = 0.009) and (−)-α-pinene (day 7: F1,6 = 8.61, = 0.026; day 21: F1,6 = 13.62, = 0.010; day 28: F1,6 = 6.18, = 0.047) was significantly increased. In susceptible trees, MeJa significantly induced a weak increase in (+)-3-carene (day 7: F1,6 = 7.88, = 0.031; day 14: F1,6 = 8.90, = 0.025; day 21: F1,6 = 6.28, = 0.046; day 28: F1,6 = 21.99, = 0.003) and (−)-α-pinene (day 14: F1,6 = 6.76, = 0.041; day 28: F1,6 = 6.73, = 0.041) levels, but not accumulation of (+)-sabinene (Figure 3). Throughout the time course, regardless of treatment with MeJa or Tween-20, resistant trees had significantly higher levels of (+)-3-carene and (+)-sabinene, but not (−)-α-pinene, compared with susceptible trees (Table S4).

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Figure 3.  Amount of total monoterpenes, (+)-3-carene, (+)-sabinene and (−)-α-pinene extracted from resistant and susceptible Sitka spruce trees at 0, 2, 7, 14, 21 and 28 days after treatment with 0.1% methyl jasmonate (MeJa) or 0.1% Tween-20 (Tween). Each data point represents the mean of four biological replicates (three technical replicates per sample) ± 1 SE. Asterisks indicate a significant difference (< 0.05) between MeJa and Tween. The results of statistical analysis discriminating between resistant and susceptible trees (F and P values) are given in Table S4A.

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In addition to extraction of monoterpenes from stem tissues, emission of volatile monoterpenes was monitored in MeJa-treated trees. Prior to treatment (day 0), and throughout the time course, resistant trees emitted higher levels of total monoterpenes, (+)-3-carene, (+)-sabinene and (−)-α-pinene than susceptible trees (Figure S2). Emission of (+)-3-carene from susceptible trees was not detected, consistent with the results from the analysis of monoterpene extracts of these trees (Figure 3).

Monoterpene synthase activity in protein extracts from resistant and susceptible Sitka spruce

Protein was extracted from the apical stem sections of resistant and susceptible trees and assayed for monoterpene synthase activity by measuring the formation of monoterpenes with GPP as substrate. Untreated (day 0) resistant and susceptible trees had the same amount of total monoterpene synthase and (−)-α-pinene synthase activities (Figure 4). However, untreated resistant trees had significantly higher levels of (+)-3-carene and (+)-sabinene synthase activities than untreated susceptible trees (Table S4).

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Figure 4.  Total monoterpene synthase activity and (+)-3-carene, (+)-sabinene and (−)-α-pinene production from protein extracts of resistant and susceptible Sitka spruce trees at 0, 2, 7, 14, 21 and 28 days after treatment with 0.1% methyl jasmonate (MeJa) or 0.1% Tween-20 (Tween). Each data point represents the mean of four biological replicates (three technical replicates per sample) ± 1 SE. Asterisks indicate a significant difference (< 0.05) between MeJa and Tween. The results of statistical analysis discriminating between resistant and susceptible trees (F and P values) are given in Table S4B.

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After MeJa treatment, (+)-sabinene synthase activity was significantly (day 7: F1,6 = 32.38, = 0.001; day 21: F1,6 = 10.89, = 0.016) induced in resistant trees, whereas non-significant increases in enzyme activity were detected for (+)-3-carene and (−)-α-pinene production (Figure 4). In susceptible trees, (+)-3-carene (day 7: F1,6 = 12.87, = 0.012; day 14: F1,6 = 11.69, = 0.014), (+)-sabinene (day 7: F1,6 = 79.41, = 0.000; day 14: F1,6 = 18.45, = 0.005) and (−)-α-pinene (day 7: F1,6 = 7.98, = 0.030) synthase activities were significantly induced after treatment with MeJa. There was no significant difference in induced (+)-3-carene formation between resistant and susceptible trees throughout the time course following treatment with MeJa. Significant differences in (+)-sabinene formation at days 2, 7, 21 and 28, and in (−)-α-pinene formation at days 2, 21 and 28 after MeJa treatment were seen between these two genotypes (Table S4).

Targeted proteome analysis using SRM identifies differential expression of PsTPS-3car proteins in resistant and susceptible Sitka spruce

We used SRM for target-specific TPS proteome analysis (Zulak et al., 2009) to measure the abundance of PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, PsTPS-sab and PsTPS-αpin in resistant and susceptible trees at day 0 (untreated) and day 7 after treatment (Figure 5). PsTPS-3car1 was detected at low abundance at day 0 in resistant and susceptible trees. Following MeJa treatment, PsTPS-3car1 was significantly (F2,9 = 10.64, = 0.004) induced in susceptible but not in resistant trees. PsTPS-3car2(R) was detected in resistant trees at day 0, and the level of abundance was maintained at day 7 following treatment with MeJa or Tween-20. PsTPS-3car2 was not detected in protein extracts from susceptible trees in samples taken at day 0 or day 7 after treatment, consistent with several unsuccessful attempts to amplify this gene from cDNA and genomic DNA of these trees. Conversely, PsTPS-3car3(S) was detected and induced in susceptible but not resistant trees, consistent with failed attempts to clone this gene from cDNA of resistant trees. PsTPS-sab and PsTPS-αpin were expressed at day 0 in both genotypes, and were significantly induced at day 7 following MeJa treatment in resistant (PsTPS-sab: F2,9 = 100.99, = 0.000; PsTPS-αpin: F2,9 = 52.16, = 0.000) and susceptible (PsTPS-sab: F2,9 = 53.08, = 0.000; PsTPS-αpin: F2,9 = 28.60, = 0.000) trees. PsTPS-3car1 protein was detected at significantly higher levels in susceptible trees compared to resistant trees at day 7 after MeJa treatment, and the levels of both PsTPS-sab and PsTPS-αpin were significantly higher in resistant trees at this time point (Figure 5 and Table S4).

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Figure 5.  Abundance of (+)-3-carene, (+)-sabinene and (−)-α-pinene synthase proteins per μg of total extracted protein from resistant and susceptible Sitka spruce trees detected by SRM at day 0 (uninduced) and 7 days after treatment with Tween-20 (7 Tween) or methyl jasmonate (7 MeJa). Each value represents the mean of four biological replicates ± 1 SE. Bars with different letters are significantly different at < 0.05 (Table S4C). ND, protein not detected.

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Quantitative RT-PCR confirms patterns of differential abundance of PsTPS-3car transcripts in resistant and susceptible Sitka spruce

Gene-specific primers were used to monitor the transcript abundance of PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, PsTPS-sab and PsTPS-αpin at day 0 and over the 28-day time course following treatment with MeJa or Tween-20. PsTPS-3car1 transcripts were detected at low levels in untreated (day 0) resistant and susceptible trees, and were significantly induced in both resistant (day 2: F2,9 = 781.73, = 0.000; day 7: F2,9 = 31.12, = 0.003) and susceptible (day 2: F2,9 = 36.07, = 0.001; day 7: F2,9 = 11.41, = 0.015) genotypes after MeJa treatment (Figure 6). Consistent with the results of SRM proteome analysis (Figure 5), PsTPS-3car2 transcripts were detected in resistant but not in susceptible trees, and PsTPS-3car3 transcripts were detected only in susceptible trees. Transcript levels of PsTPS-3car2(R) and PsTPS-3car3(S) were induced following MeJa treatment. PsTPS-sab transcripts were detected in both tree genotypes and were significantly induced in resistant (day 2: F2,9 = 26.84, = 0.002) and susceptible (day 2: F2,9 = 13.58, = 0.010; day 7: F2,9 = 30.73 = 0.001; day 14: F2,9 = 23.50, = 0.003) trees following MeJa treatment. Similarly, PsTPS-αpin transcripts were detected in both tree genotypes and were significantly induced in resistant (day 2: F2,9 = 40.99, = 0.001; day 14: F2,9 = 6.67, = 0.042) and susceptible (day 2: F2,9 = 22.92, = 0.003; day 7: F2,9 = 12.03, = 0.013; day 14: F2,9 = 9.44, = 0.022) trees following MeJa treatment. At various time points after treatment with MeJa, PsTPS-3car1 (days 7, 21 and 28), PsTPS-sab (days 2 and 14) and PsTPS-αpin (day 7) transcripts were detected at significantly higher abundance in resistant trees compared to susceptible trees (Table S4).

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Figure 6.  Transcript abundance of (+)-3-carene, (+)-sabinene and (−)-α-pinene synthases relative to abundance of the reference gene (eIF-5A) from resistant and susceptible Sitka spruce trees at 0, 2, 7, 14, 21 and 28 days following treatment with 0.1% methyl jasmonate (MeJa) or 0.1% Tween-20 (Tween). Each value represents the mean of four biological replicates (and three technical replicates per sample) ± 1SE. Asterisks indicate a significant difference (< 0.05) between MeJa and Tween. ND, transcript not detected. The results of statistical analysis discriminating between resistant and susceptible trees (F and P values) are given in Table S4D.

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Kinetic properties of the recombinant PsTPS-3car1, PsTPS-3car2, PsTPS-3car3 and PsTPS-sab enzymes from resistant and susceptible Sitka spruce

Kinetic parameters were determined for the recombinant (+)-3-carene and (+)-sabinene synthases from resistant and susceptible Sitka spruce (Table 1). The recombinant PsTPS-3car1 proteins from both genotypes have similar kinetic parameters, with Km values of 6.88 ± 0.22 μm and 8.09 ± 0.21 μm, and kcat values of 2.21 ± 0.31 sec−1 and 2.25 ± 0.30 sec−1, respectively. The recombinant PsTPS-sab proteins from resistant and susceptible trees also had similar characteristics. PsTPS-sab enzymes had higher affinity (i.e. lower Km) for GPP but lower turnover than PsTPS-3car1 enzymes. Overall, the four recombinant PsTPS-3car1 and PsTPS-sab proteins [PsTPS-3car1(R), PsTPS-3car1(S), PsTPS-sab(R) and PsTPS-sab(S)] had similar catalytic efficiencies (kcat/Km) and these properties were similar to those of previously characterized conifer TPS (Keeling et al., 2008). The PsTPS-3car2(R) protein from resistant trees had a higher affinity for GPP but a lower turnover than PsTPS-3car1 or PsTPS-sab, resulting in a lower catalytic efficiency value. In contrast to all of the above proteins, PsTPS-3car3(S) from susceptible trees had extremely low activity, such that enzyme assays for product detection required 400–900 nm of recombinant protein, compared with 2–15 nm for PsTPS-3car1, PsTPS-3car2 and PsTPS-sab. The very low activity of PsTPS-3car3(S) hindered kinetic characterization of this protein (Table 1). A value for Vmax was estimated for PsTPS-3car3(S) on the assumption that substrate concentration in the PsTPS-3car3(S) assays was nearing saturation and that the corresponding activity was nearing Vmax, which allowed kcat to be estimated at less than 10−5 sec−1.

Table 1.   Kinetic parameters of recombinant (+)-3-carene synthase (PsTPS-3car1, PsTPS-3car2 and PsTPS-3car3) and (+)-sabinene synthase (PsTPS-sab) enzymes from resistant (R) and susceptible (S) Sitka spruce using GPP as substrate
 Kmm)Vmax (pmol sec−1 μg−1)kcat (sec−1)kcat/Km (sec−1 μm−1)
  1. All values represent the mean of at least seven replicates ± standard error. The substrate affinity (Km) could not be assessed (NA) for PsTPS-3car3(S) due to the low reaction rate of the recombinant protein. Full-length PsTPS-3car2(S) and PsTPS-3car3(R) were not cloned from cDNA (NC), thus their kinetic parameters could not be determined.

PsTPS_3car1(R)6.88 ± 0.2232.1 ± 4.72.21 ± 0.310.31
PsTPS_3car1(S)8.09 ± 0.2134.1 ± 4.52.25 ± 0.300.28
PsTPS_3car2(R)1.47 ± 0.130.91 ± 0.080.060 ± 0.0050.041
PsTPS_3car2(S)NCNCNCNC
PsTPS_3car3(R)NCNCNCNC
PsTPS_3car3(S)NA<10−3<10−5NA
PsTPS_sab(R)3.59 ± 0.06113.5 ± 0.450.89 ± 0.030.25
PsTPS_sab(S)3.67 ± 0.2014.5 ± 1.80.96 ± 0.120.26

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Genomic, molecular and biochemical basis of (+)-3-carene biosynthesis in Sitka spruce

Although conifers are large, long-lived (up to hundreds of years) and immobile targets on the battleground of plant–herbivore interactions, they tolerate, deter or resist attack from many highly mobile and potentially faster evolving herbivores and pathogens. As part of their chemical and physical defenses, conifers deploy a diverse and dynamic system of terpenoid chemicals (Phillips and Croteau, 1999; Zulak and Bohlmann, 2010). The evolution of a family of TPS genes for specialized metabolism contributes much to the diversity and plasticity of this defense system (Keeling and Bohlmann, 2006b). The goal of this study was to characterize the mechanisms that control the fine-tuning of terpenoid chemical diversity and plasticity of terpenoid profiles observed in conifers, using the biosynthesis of monoterpene (+)-3-carene as a model. We investigated the differential accumulation of (+)-3-carene in a contrasting pair of resistant and susceptible Sitka spruce genotypes through analysis of the (+)-3-carene synthase TPS family at the genomic, transcriptomic, proteomic and biochemical levels. We identified three distinct (+)-3-carene synthase genes (PsTPS-3car1, -2 and -3) and a closely related (+)-sabinene synthase (PsTPS-sab) from Sitka spruce. The three distinct PsTPS-3car genes and the PsTPS-sab gene are probably the result of gene duplications, with either retention of gene function, or sub- or neo-functionalization in the case of PsTPS-sab. The PsTPS-3car/PsTPS-sab clade represents a previously undetected level of genetic diversity for conifer monoterpene synthases. Previously, of all conifers, only a single (+)-3-carene synthase gene had been identified in Norway spruce (Fäldt et al., 2003). A detailed analysis of the abundance of transcripts and proteins of the (+)-3-carene synthase family in untreated and MeJa-induced trees suggested that the (+)-3-carene accumulation phenotype in resistant and susceptible Sitka spruce genotypes is the result of differential expression of genes and proteins with different enzyme kinetic properties.

Both the resistant and the susceptible genotype carry a copy of the PsTPS-3car1 gene. This gene is expressed at low constitutive transcript levels in both genotypes, and has low constitutive protein abundance. The catalytic efficiency is the same for PsTPS-3car1 from the resistant and susceptible trees. Thus, there is no evidence that this gene is responsible for the difference in the constitutive levels of (+)-3-carene in these resistant and susceptible trees. This gene and its protein may contribute to the low levels of (+)-3-carene found in the susceptible trees. The PsTPS-3car2 gene, as well as its transcript and protein, were detected only in the resistant genotype. Although the PsTPS-3car2(R) enzyme had a lower catalytic efficiency than the PsTPS-3car1 enzyme, it is likely that PsTPS-3car2(R) is a major contributor to formation of (+)-3-carene in the resistant genotype due to its abundance. The apparent lack of PsTPS-3car2 in the genome, transcriptome and proteome of the susceptible trees may explain the much lower levels of (+)-3-carene in this genotype. Although present in both genomes, transcripts and proteins of PsTPS-3car3 were detected only in the susceptible genotype. The encoded protein appears to be a vestigial (+)-3-carene synthase with extremely low catalytic activity. Thus, PsTPS-3car3 is probably not a considerable contributor to (+)-3-carene formation, consistent with the low levels of (+)-3-carene found in the susceptible trees.

The monoterpene (+)-3-carene has been identified as associated with resistance to weevils in Sitka spruce from a geographic region of British Columbia in which extreme and durable weevil resistance has been demonstrated, exemplified by the H898 genotype (King et al., 2004; King and Alfaro, 2009; Robert et al., 2010). The possibility of using this metabolite and individual members of the PsTPS-3car gene and protein family as biomarkers (measured in the form of proteins, transcripts or metabolites) or genetic markers to screen for weevil resistance should now be explored.

The PsTPS-3car clade as an example of copy number variation

Conifers have extremely large genomes, with an estimated size of 20–40 Gb, that are thought to be of high complexity (Ahuja and Neale, 2005; Morse et al., 2009). One mechanism contributing to the increasing size of genomes and gene families is gene duplication, which can lead to copy number variation (CNV) between the genomes of two individuals (Springer et al., 2009; DeBolt, 2010). CNV is common in the genomes of Arabidopsis (DeBolt, 2010) and maize (Springer et al., 2009), and screening of Arabidopsis for genes with CNV suggested that transposons and defense-related genes have an increased likelihood of having CNV (DeBolt, 2010). The TPS-d multi-gene family of conifer specialized metabolism represents a gene family in which CNV is likely to have occurred. The PsTPS-3car/PsTPS-sab clade within the conifer TPS-d1 sub-family is probably the result of repeated gene duplication (Figure 2a and Figure S1). The presence of three PsTPS-3car genes in the resistant H898 genotype, but only two PsTPS-3car genes in the susceptible Q903 genotype, may represent a case of CNV for a conifer defense-related gene family. Presence/absence variation (PAV) is a specific type of CNV in which one genotype possesses a gene but a second genotype does not (Springer et al., 2009). In the system described here, the presence of PsTPS-3car2 in the genome of the resistant trees and its apparent absence in the susceptible genotype represent a possible example of PAV and associated phenotypic variation in a conifer.

The PsTPS-sab and PsTPS-3car1 gene as a case of sub- or neo-functionalization of conifer monoterpene synthases

The presence of a (+)-sabinene synthase function within the PsTPS-3car/PsTPS-sab clade (Figure 2 and Figures S1 and S2) suggests a pattern of divergent functional evolution of TPSs within this clade. As all members of the this clade are multi-product enzymes (this study, and Fäldt et al., 2003), and as (+)-sabinene is a minor product of the Sitka spruce PsTPS-3car and Norway spruce PaTPS-3car enzymes, it is possible that PsTPS-3car1 and PsTPS-sab originated from a common ancestor via gene duplication, followed by loss of the capacity to produce (+)-3-carene in one of the gene copies. This copy may have evolved from having the ancestral function of forming (+)-sabinene as a minor product to producing (+)-sabinene as the major product, thus representing a case of sub-functionalization and/or neo-functionalization. Previous in vitro mutagenesis studies of multi-product conifer TPSs showed that substituting just one or a few amino acids resulted in altered product profiles (Katoh et al., 2004; Hyatt and Croteau, 2005; Yoshikuni et al., 2006; Keeling et al., 2008). In the case of 1,8-cineole synthase from Salvia fruticosa, just two amino acid changes converted this enzyme into a sabinene synthase in vitro (Kampranis et al., 2007). Work is now underway to investigate the critical mutations that may have occurred in nature during the divergent functional evolution of the PsTPS-3car and PsTPS-sab genes.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials

The origins and growing conditions of 3-year-old clonally propagated Sitka spruce of the highly resistant (H898) genotype and the highly susceptible (Q903) genotype have been described previously (Robert and Bohlmann, 2010). One month prior to experiments, plants were moved into the ambient temperature and light zone of the greenhouse at the University of British Columbia (Miller et al., 2005). All experiments were performed using four biological replicates. Procedures for MeJa treatment have been described previously (Miller et al., 2005). Four trees per treatment per genotype were sampled at day 0 (no treatment) and at days 2, 7, 14, 21 and 28 after treatment with either 50 ml of 0.1% v/v MeJa in 0.1% v/v Tween-20 or 0.1% Tween-20 alone (control). Apical stem sections were clipped and needles removed, and the sections were flash-frozen in liquid nitrogen and stored at −80°C. All analyses were performed on tissue extracts from the apical stem, including both phloem and xylem.

Terpenoid extraction

Monoterpenes were extracted as described previously (Lewinsohn et al., 1993; Martin et al., 2002) with minor modifications. Tissue (0.2 g DW) was immersed in 1.5 ml methyl tert-butyl ether (MTBE), containing 745 μm isobutylbenzene, and shaken overnight at room temperature. Extracts were washed using 0.3 ml of 0.1 m (NH4)2CO3 (pH 8.0), and processed for GC/MS analysis (see Appendix S1).

Volatile collection

An automated volatile collection system (Analytical Research Systems, http://www.ars-fla.com/) was used to trap monoterpenes emitted from the above-ground parts of plants in a controlled-environment chamber (PGW36; Conviron, http://www.conviron.com/) as described by Martin et al. (2003). Plants of 30–40 cm in height were placed inside the collection system 1 day prior to volatile collections. Two 4 h sample collections were made per day between 8 am and 4 pm. Volatiles were collected on 30 mg SuperQ (Alltech Inc., http://www.discoverysciences.com/), eluted using 0.5 ml pentane containing 338 μM isobutylbenzene as an internal standard, and analyzed by GC/MS (see Appendix S1).

cDNA cloning and expression of recombinant TPS proteins

RNA was isolated from the apical stem 2 days after MeJa treatment as described previously (Kolosova et al., 2004), except that RNA was further purified using an RNeasy mini kit with on-column DNase treatment (Qiagen, http://www.qiagen.com/). Total RNA (3 μg) was used to synthesize cDNA using a Sprint RT Complete-Double Pre-Primed first-strand cDNA synthesis kit (Clontech, http://www.clontech.com/). RNA quality was assessed using an Agilent 2100 bioanalyser (http://www.agilent.com), yielding a mean RNA integrity number of 7.4. For cDNA identification, PCR primers Ps_3CarFL_F1 (5′-GTAGTCCATAAGGAGCAGAAATG-3′) and Ps_Car_R1 (5′-TTACATAGGCACAGGTTCAAGAACGG-3′) were designed based on the ORF of the previously characterized Norway spruce (+)-3-carene synthase (Fäldt et al., 2003). A second set of primers (Ps_Car_F2, 5′-CACCATGTCTGTTATTTCCATTGTGCCG-3′; Ps_Car_R2, 5′-CCTAATAGGCTGAAAAGTACAATAATAAACCACATAACCTG-3′) was based on Sitka spruce EST sequences (Ralph et al., 2008). Amplified cDNA products of the expected size (1.9 kb) were cloned into either pDRIVE (Qiagen) or pJet1.2 (Fermentas, http://www.fermentas.com/) vectors and sequenced. Full-length cDNAs were cloned into a pET28b(+) expression vector (EMD Chemicals, http://www.emd-chemicals.ca/) in-frame with an N-terminal 6 x His tag. Recombinant protein expression and nickel affinity purification were performed as previously described (Keeling et al., 2008), except that, after elution, the protein buffer used was 25 mm HEPES (pH 7.4), 100 mm KCl and 10% glycerol. Protein concentrations were determined using the BCA assay (Thermo Scientific, http://www.thermoscientific.com). For kinetic analysis, pseudomature TPSs were produced by truncation of the full-length cDNAs at residue 58, six residues upstream of the RRX8W motif, thus removing the plastid targeting sequence (Williams et al., 1998).

Cloning of genomic DNA

Genomic DNA was extracted from apical stems as previously described (Liewlaksaneeyanawin et al., 2009) with minor modifications. Primers Ps_3CarFL_F1 and Ps_Car_R2 were used to amplify full-length genomic clones of PsTPS-3car3 (Table S5). Primers PsTPS_3car2_F2, PsTPS_3car2_R2, PsTPS_3car2_F4 and PsTPS_3car2 _R4 were used to amplify PsTPS-3car2 from resistant trees. The above primers and additional PsTPS-3car2 target primers were used in the attempted cloning of representative sequences of PsTPS-3car2 from susceptible trees (Table S5).

Protein extraction from combined bark and wood tissues

Protein extraction from apical stems was performed as described previously (Martin et al., 2002) with minor modifications. Protein extracts were desalted on Sephadex PD minitrap G-25 columns (GE Healthcare, http://www.gehealthcare.com) pre-equilibrated with 25 mm HEPES, pH 7.2 (Sigma-Aldrich, http://www.sigmaaldrich.com/), 100 mm KCl and 10% glycerol for use in TPS enzyme assays. Desalted protein extracts were transferred to a 2 ml GC vial, inverted several times, and transferred to a fresh GC vial for enzyme assays.

TPS enzyme assays and kinetics

Single vial assays were performed as described previously (O’Maille et al., 2004; Keeling et al., 2008). Total protein (377–1168 μg) extracted from stem tissue or Ni affinity-purified recombinant protein (0.5 μg for PsTPS-sab; 1.0 μg for PsTPS-3car1; 4 μg for PsTPS-3car2; 50–94 μg for PsTPS-3car3) was incubated in buffer (25 mm HEPES, 100 mm KCl, 10 mm MnCl2, 10% glycerol, 5 mm dithiothreitol) with 50 μm of either GPP (Echelon Biosciences Inc., http://www.echelon-inc.com), NPP (Echelon Biosciences Inc.), FPP (Sigma-Aldrich) or GGPP (Sigma-Aldrich). Aqueous assays were overlaid with 500 μl pentane containing 2.5 μm isobutylbenzene as an internal standard and incubated at 30°C for 1 h. Products were extracted into the organic overlay by vortexing for 20 s and centrifugation at 1000 g for 30 min at 4°C prior to GC/MS analysis (see Appendix S1).

To determine kinetic parameters, Ni affinity-purified recombinant protein was assayed using varying amounts of GPP (1–90 μm for PsTPS-3car1; 1–30 μm for PsTPS-sab; 0.1–30 μm for PsTPS-3car2R). Assay times of 10 min for PsTPS-3car1 or 15 min for PsTPS-sab and PsTPS-3car2(R) maintained the linearity of product formation. Recombinant TPS in the Ni affinity-purified protein preparations was quantified by SDS–PAGE and measurement of protein band intensity using Scion Image (Scion Corporation, http://www.scioncorp.com/). The concentrations of TPS protein per assay were 3.0–6.6 nm for PsTPS-3car1, 10.1–15.7 nm for PsTPS-3car2(R) and 2.0–3.7 nm for PsTPS-sab. Enzyme assays with 400–900 nm of PsTPS-3car3S and 20 μm GPP were incubated for 60 min, and yielded only small amounts of product, precluding accurate kinetic characterization of this protein. Vmax for PsTPS-3car3S was estimated based on the assumption that the substrate concentration is nearing saturation and that the corresponding activity is nearing maximal velocity.

RNA extraction and quantitative RT-PCR

RNA was isolated from apical stems and cDNA was synthesized as described above. Gene-specific primers (Table S5) were designed for each of the target TPS genes and for the reference gene (translation initiation factor EIF-5A) (Udvardi et al., 2008). The plate set-up allowed comparison of one tree from each treatment, time point and genotype (complete block design) on one plate (Rieu and Powers, 2009). Each well was replicated twice per plate, and appropriate ‘no reverse transcription’ and ‘no template’ controls were performed for each sample and primer set. The thermocycler (DNA Engine Opticon 2, http://www.gmi-inc.com/DNA-Engine-Opticon-2-Real-Time-Cycler.html) program was 95°C for 15 min, 40 cycles of 94°C for 1 min, 60°C for 30 s and 72°C for 30 s, with fluorescence reading between cycles. Data were compiled using real-time PCR Miner software (Zhao and Fernald, 2005), and the relative quantity of each transcript was calculated and normalized to the reference gene expression as described previously (Zhao and Fernald, 2005; Rieu and Powers, 2009).

SRM protein analysis

Protein was extracted from apical stem sections of plants treated with MeJa or Tween-20 (0 and 7 days after treatment), digested with trypsin and analysed using MultiQuant 1.1 (Applied Biosystems, http://www.appliedbiosystems.com/) as described previously (Zulak et al., 2009). Isotopically labeled tryptic peptides for the five TPS targets were selected, synthesized and purified as described previously (Zulak et al., 2009), except that [13C6]Lys and [13C6 and 15N4]Arg (98% isotopic enrichment; Cambridge Isotope Laboratories, http://www.isotope.com) were used for biosynthesis of the isotope-labeled peptides (added at 0.25–0.75 pmol per gel slice), and the SRM acquisition methods were constructed using six SRM Q1/Q3 ion pairs per target. The labeled peptides for each protein were TYIDILR (PsTPS-3car1), DSGFPDLNFIR (PsTPS-3car2(R)), KPAFDISR (PsTPS-3car3(S)), ESGSPELTFIR (PsTPS-sab) and YLEESLQK (PsTPS-αpin) (isotopically labeled amino acid in bold). The amount of the peptide of interest in 20 μg of total protein extract was calculated by multiplying the light:heavy peak area ratios by the purity (fmol per gel slice) of the labeled peptide.

Statistical analyses

Statistical analyses were performed using systat 11.0 (Systat Software Inc., http://www.systat.com/), and, where data violated the assumptions of normality and homogeneity of variance (in the quantitative RT-PCR analysis), they were log-transformed to satisfy the assumptions of anova. For quantitative RT-PCR, anova was performed to identify differences between MeJa-treated trees and control trees on each sampling day for each genotype. Because the treated and control trees were paired as a consequence of the quantitative RT-PCR plate design, we calculated the ratio of MeJa-treated expression to control expression for each sample pair to determine whether this ratio was significantly (< 0.05) different than 1 (no effect). For SRM, anova was performed for each peptide to discriminate between the six categories (resistant trees – day 0, day 7 tween, day 7 MeJa; susceptible trees – day 0, day 7 tween, day 7 MeJa). For extracted metabolites and enzyme activities, t tests were performed to compare treatment and control at each time point. For volatile emissions, extracted metabolites, enzyme activity and quantitative RT-PCR, anova was performed to discriminate between resistant and susceptible trees for each treatment and time point.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Lina L. Madilao for excellent technical assistance and Karen Reid for excellent laboratory management. This research was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant to J.B., postdoctoral fellowship to D.E.H.; Canada graduate scholarship to J.A.R.) and by Genome British Columbia and Genome Canada in support of the Treenomix Conifer Forest Health Project (to J.B., http://www.treenomix.ca) and the Proteomics Innovation Centre (to C.H.B, http://www.proteincentre.com). Salary support for J.B. was provided in part by a Natural Sciences and Engineering Research Council of Canada Steacie Memorial Fellowship and the University of British Columbia Distinguished University Scholar program.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Neighbor joining phylogenetic analysis of functionally characterized conifer terpene synthases with the Physcomitrella patens ent-kaurene synthase (PpTPS-entKS) as an outgroup. The TPS-d subfamilies 1, 2 and 3 are delineated by boxes, and the Picea sitchensis (+)-3-carene and (+)-sabinene synthase gene family (3CAR) is indicated within clade TPS-d1. ORF deduced from gDNA sequence is indicated with an asterisk (*). Scale bar corresponds to 0.7 amino acid substitutions per site. NCBI accession numbers available in Table S3 in Supporting Information. Bootstrap values for distances greater than 50% (1000 runs) are shown at nodes, calculated with the neighbor joining method in CLC bio main workbench.

Figure S2. Amount of total monoterpenes, (+)-3-carene, (+)-sabinene and (−)-α-pinene emitted from resistant and susceptible Sitka spruce trees at 0, 2, 7, 14, 21 and 28 days after induction with 0.1% MeJa. Each data point represents the mean of 3 biological replicates ± 1SE. Asterisk (*) denotes a significant difference (P < 0.05) between resistant and susceptible trees. Statistics discriminating between resistant and susceptible trees (F and P values) can be found in Table S4E. † indicates that volatile emissions were not detected for susceptible trees, precluding statistical analysis.

Table S1. Amino acid sequence identity of the Norway spruce (+)-3-carene synthase (PaTPS-3car) and the Sitka spruce PsTPS-3car and PsTPS-sab proteins from this study.

Table S2. Percent total of individual monoterpenes produced by the recombinant (+)-3-carene and (+)-sabinene synthase proteins from Sitka spruce. Each value represents at least 5 replicates.

Table S3. NCBI Accession numbers of the amino acid sequences used for phylogenetic analysis of functionally characterized conifer terpene synthases. NA – no accession pending publication.

Table S4. Output of statistical analyses discriminating between resistant and susceptible trees for (A) metabolite extractions, (B) native enzyme activity assays, (C) SRM, D) RT-qPCR, (E) volatile emissions.

Table S5. Gene specific primers used in the cloning and expression profiling of Sitka spruce (+)-3-carene and (+)-sabinene synthases described in this study. (A) Gene specific primers used to amplify PsTPS-3car2 from the genomic DNA of resistant and susceptible trees. (B) Gene specific quantitative RT-PCR primers (5′–3′)

Appendix S1. GC/MS analysis.

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