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

Abstract

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.

Figure 1.

Major Norway spruce TPS products showing monoterpenes, sesquiterpenes, and diterpenes produced by these enzymes.

Figure 2.

Amino acid alignments of Norway spruce mono-TPS, sesqui-TPS, and di-TPS generated by ClustalX and GeneDoc. The RRX₈W, common to nearly all mono-TPS and positionally preserved in some sesqui-TPS and di-TPS. Asp rich motifs, DxDD for di-TPS, and DDxxD for mono-TPS, sesqui-TPS, and di-TPS—necessary for the binding of cationic cofactors—are shown. The ancestral conifer di-TPS motif of 200 to 215 amino acids is delineated by a dashed and dotted line. Conserved similarity shading is based on 100% (black), 60% (dark gray), and 30% (light gray).

Figure 3.

GC-MS chiral analysis of products formed by PaTPS-Lin, (−)-linalool synthase. Total ion chromatogram of assay products (A) showing a major peak, R_t = 7.78 along with (B) chiral terpene standards showing (−)-linalool, R_t = 7.79 (1), and (+)-linaool, R_t = 7.87 (2). Mass spectra of major peak (C) identifies this peak as (−)-linalool.

Figure 4.

GC-MS analysis of the multiple-product forming PaTPS-Lon, longifolene synthase. Total ion chromatogram of the assay showing 19 sesquiterpene products (A). Mass spectra of the major peak (B) and six additional products according to decreasing abundance including α-longicyclene (C), α-longipinene (D), E-β-farnesene (E), cyclostativene (F), β-longipinene (G), and longiborneol (H).

Figure 5.

GC-MS analysis of products formed by PaTPS-LAS and PaTPS-Iso. Total ion chromatogram of multiple assay products of PaTPS-LAS (A). Mass spectra are shown of peak one with insert of levopimaradiene standard (B), peak two with insert of authentic abietadiene (C) and of peak three with insert of neoabietadiene standard (D). Total ion chromatogram of the single product produced by PaTPS-Iso (E). Mass spectra of this product are shown with an insert of authentic isopimara-7,15-diene (F).

Figure 6.

Proposed reaction mechanisms for diterpene synthases PaTPS-LAS and PaTPS-Iso. The substrate GGDP undergoes initial cyclization reaction yielding (+)-copalyl diphosphate. In a second cyclization reaction, the intermediate sandaracopimarenyl carbocation is formed. The pathway to the left (a) reflects the reaction mechanism of PaTPS-LAS whereby a 1,2 methyl migration results in the abietane skeleton. Final deprotonations from this skeleton yield the four detected products, levopimaradiene, abietadiene, neoabietadiene, and palustradiene, each with different double bond configurations. The pathway toward the right (b) indicates the reaction mechanism of PaTPS-Iso whereby the sandaracodimarenyl carbocation is deprotonated to yield isopimara-7,15-diene.

Figure 7.

Phylogenetic tree of gymnosperm TPS amino acid sequences (Supplemental Table I) showing the TPS-d subfamily branching into three distinct groups of TPS involved in secondary metabolism. Branching patterns seen in this analysis are cause for the further separation into TPS-d1 (primarily mono-TPS), TPS-d2 (sesqui-TPS), and TPS-d3 (primarily di-TPS). Kaurene synthase from Cucurbita maxima shown as outgroup in this analysis. Bootstrap values over 50% for maximum likelihood (upper) and distance (lower) analyses are shown at nodes. Maximum likelihood values represent percentages of 100 γ-corrected (log L = −52520.29) replicates analyzed using Phyml.

Figure 8.

Phylogenetic tree illustrating the relationship of TPS involved in primary and secondary metabolism from angiosperms and gymnosperms (Supplemental Table I). Amino acids sequences of 67 TPS were analyzed by maximum likelihood using Phyml. Bootstrap values over 50% present at nodes for maximum likelihood (upper) and distance (lower) analyses. Maximum likelihood values represent percentages of 100 gamma-corrected (log L = −20452.16) replicates.

Figure 9.

Comparative modeling of Norway spruce mono-TPS based on the crystal structure of bornyl diphosphate synthase from S. officialis. A, Models of Norway spruce myrcene synthase (orange) and grand fir myrcene synthase (red) show identical amino acids present in the active site of these two homologous enzymes. B, (−)-pinene synthase models from Norway spruce (orange), Sitka spruce (pink), and grand fir (red) show that these enzymes differ in the proximity of several amino acids with respect to the active site. Fewer amino acids seen in the Norway spruce model may help explain why this enzyme produces many products as opposed to only two products formed from the other (−)-pinene synthases.

Figure 10.

Comparative modeling of Norway spruce and grand fir sesqui-TPS and di-TPS. A, E-α-bisabolene synthases from Norway spruce (orange) and grand fir (red) demonstrate the same amino acids model in the active site of these homologs. B, Models of PaTPS-Iso (orange) and PaTPS-LAS (yellow) from Norway spruce as compared to abietadiene synthase AgAS (red) from grand fir demonstrate two main amino acid differences: H964_PaTPS-Iso versus Y686_PaTPS-LAS and Y696_AgAS; and S721_PaTPS-Iso versus A713_PaTPS-LAS (not shown) and A721_AgAS (not shown) between these three related proteins. Differences are unique to the functionally distinct PaTPS-Iso.