Functional Characterization of PmDXR, a Critical Rate-Limiting Enzyme, for Turpentine Biosynthesis in Masson Pine (Pinus massoniana Lamb.).

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

Transcriptomic and Coexpression Network Analyses Revealed Pine Chalcone Synthase Genes Associated with Pine Wood Nematode Infection.

Pine wood nematode (PWN) causes serious diseases in conifers, especially pine species. To investigate the transcriptomic profiles of genes involved in pine-PWN interactions, two different pine species, namely, Pinus thunbergii and P. massoniana, were selected for this study. Weighted gene coexpression network analysis (WGCNA) was used to determine the relationship between changes in gene expression and the PWN population after PWN infection. PWN infection negatively affects the expression of most genes in pine trees, including plant defense-related genes such as genes related to plant hormone signal transduction, plant-pathogen interactions, and the MAPK signaling pathway in plants. However, the expression of chalcone synthase genes and their related genes were proportional to the changes in nematode populations, and chalcone synthase genes were dominant within the coexpression module enriched by genes highly correlated with the nematode population. Many genes that were closely related to chalcone synthase genes in the module were related to flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Pine trees could actively adjust their defense strategies in response to changes in the number of invasive PWNs, but the sustained expression of chalcone synthase genes should play an important role in the inhibition of PWN infection.

Characterization and Function of the 1-Deoxy-D-xylose-5-Phosphate Synthase (DXS) Gene Related to Terpenoid Synthesis in Pinus massoniana.

In the methyl-D-erythritol-4-phosphate (MEP) pathway, 1-deoxy-D-xylose-5-phosphate synthase (DXS) is considered the key enzyme for the biosynthesis of terpenoids. In this study, PmDXS (MK970590) was isolated from Pinus massoniana. Bioinformatics analysis revealed homology of MK970590 with DXS proteins from other species. Relative expression analysis suggested that PmDXS expression was higher in roots than in other plant parts, and the treatment of P. massoniana seedlings with mechanical injury via 15% polyethylene glycol 6000, 10 mM H2O2, 50 µM ethephon (ETH), 10 mM methyl jasmonate (MeJA), and 1 mM salicylic acid (SA) resulted in an increased expression of PmDXS. pET28a-PmDXS was expressed in Escherichia coli TransB (DE3) cells, and stress analysis showed that the recombinant protein was involved in resistance to NaCl and drought stresses. The subcellular localization of PmDXS was in the chloroplast. We also cloned a full-length 1024 bp PmDXS promoter. GUS expression was observed in Nicotiana benthamiana roots, stems, and leaves. PmDXS overexpression significantly increased carotenoid, chlorophyll a, and chlorophyll b contents and DXS enzyme activity, suggesting that DXS is important in isoprenoid biosynthesis. This study provides a theoretical basis for molecular breeding for terpene synthesis regulation and resistance.

On the Evolution and Functional Diversity of Terpene Synthases in the Pinus Species: A Review.

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.

Cyanobacterial production of plant essential oils.

MAIN CONCLUSION: Synechocystis (a cyanobacterium) was employed as an alternative host for the production of plant essential oil constituents. ß-Phellandrene synthase (PHLS) genes from different plants, when expressed in Synechocystis, enabled synthesis of variable monoterpene hydrocarbon blends, converting Synechocystis into a cell factory that photosynthesized and released useful products. Monoterpene synthases are secondary metabolism enzymes that catalyze the generation of essential oil constituents in terrestrial plants. Essential oils, including monoterpene hydrocarbons, are of interest for their commercial application and value. Therefore, heterologous expression of monoterpene synthases for high-capacity essential oil production in photosynthetic microorganism transformants is of current interest. In the present work, the cyanobacterium Synechocystsis PCC 6803 was employed as an alternative host for the production of plant essential oil constituents. As a case study, ß-phellandrene synthase (PHLS) genes from different plants were heterologously expressed in Synechocystis. Genomic integration of individual PHLS-encoding sequences endowed Synechocystis with constitutive monoterpene hydrocarbons generation, occurring concomitant with photosynthesis and cell growth. Specifically, the ß-phellandrene synthase from Lavandula angustifolia (lavender), Solanum lycopersicum (tomato), Pinus banksiana (pine), Picea sitchensis (Sitka spruce) and Abies grandis (grand fir) were active in Synechocystis transformants but, instead of a single product, they generated a blend of terpene hydrocarbons comprising ß-phellandrene, α-phellandrene, ß-myrcene, ß-pinene, and δ-carene with variable percentage ratios ranging from < 10 to > 90% in different product combinations and proportions. Our results suggested that PHLS enzyme conformation and function depends on the cytosolic environment in which they reside, with the biochemical properties of the latter causing catalytic deviations from the products naturally observed in the corresponding gene-encoding plants, giving rise to the terpene hydrocarbon blends described in this work. These findings may have commercial application in the generation of designer essential oil blends and will further assist the development of heterologous cyanobacterial platforms for the generation of desired monoterpene hydrocarbon products.

Identification of a small protein domain present in all plant lineages that confers high prephenate dehydratase activity.

l-Phenylalanine serves as a building block for the biosynthesis of proteins, but also as a precursor for a wide range of plant-derived compounds essential for plants and animals. Plants can synthesize Phe within the plastids using arogenate as a precursor; however, an alternative pathway using phenylpyruvate as an intermediate, described for most microorganisms, has recently been proposed. The functionality of this pathway requires the existence of enzymes with prephenate dehydratase (PDT) activity (EC 4.2.1.51) in plants. Using phylogenetic studies, functional complementation assays in yeast and biochemical analysis, we have identified the enzymes displaying PDT activity in Pinus pinaster. Through sequence alignment comparisons and site-directed mutagenesis we have identified a 22-amino acid region conferring PDT activity (PAC domain) and a single Ala314 residue critical to trigger this activity. Our results demonstrate that all plant clades include PAC domain-containing ADTs, suggesting that the PDT activity, and thus the ability to synthesize Phe using phenylpyruvate as an intermediate, has been preserved throughout the evolution of plants. Moreover, this pathway together with the arogenate pathway gives plants a broad and versatile capacity to synthesize Phe and its derived compounds. PAC domain-containing enzymes are also present in green and red algae, and glaucophytes, the three emerging clades following the primary endosymbiont event resulting in the acquisition of plastids in eukaryotes. The evolutionary prokaryotic origin of this domain is discussed.

Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.

Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I-IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes.

[GENETIC VARIABILITY OF MATERNAL PLANTS AND SEED EMBRYOS OF KOCH PINE POPULATIONS (PINUS KOCHIANA KLOTZSCH EX KOCH) IN CRIMEA].

Comparative studies of genetic variability were undertaken for 12 allozyme loci selections of trees and embryos of seed, and also for the crossing systems in five populations of Koch pine of (Pinus kochiana Klotzsch ex Koch) in Crimea. It was shown that in seed embryos the allelic variety peculiar to the maternal plants was restored, however the level of the available (H0) heterozygosity was considerably lower, 0.286 and 0.189 respectively. For the embryos unlike the trees, in the majority of the analyzed loci the considerable divergence was specific in the actual distribution of genotypes from the theoretically expected according to Hardy- Weinberg law. The proportion of cross pollination at the unilocal (t(s)) estimation varied from 0.384 to 0.673 in the populations, while at the multilocal ones (t(m)) it was 0.639-0.841.

The rhizosphere and hyphosphere differ in their impacts on carbon and nitrogen cycling in forests exposed to elevated CO2.

While multiple experiments have demonstrated that trees exposed to elevated CO2 can stimulate microbes to release nutrients from soil organic matter, the importance of root- versus mycorrhizal-induced changes in soil processes are presently unknown. We analyzed the contribution of roots and mycorrhizal activities to carbon (C) and nitrogen (N) turnover in a loblolly pine (Pinus taeda) forest exposed to elevated CO2 by measuring extracellular enzyme activities at soil microsites accessed via root windows. Specifically, we quantified enzyme activity from soil adjacent to root tips (rhizosphere), soil adjacent to hyphal tips (hyphosphere), and bulk soil. During the peak growing season, CO2 enrichment induced a greater increase of N-releasing enzymes in the rhizosphere (215% increase) than in the hyphosphere (36% increase), but a greater increase of recalcitrant C-degrading enzymes in the hyphosphere (118%) than in the rhizosphere (19%). Nitrogen fertilization influenced the magnitude of CO2 effects on enzyme activities in the rhizosphere only. At the ecosystem scale, the rhizosphere accounted for c. 50% and 40% of the total activity of N- and C-releasing enzymes, respectively. Collectively, our results suggest that root exudates may contribute more to accelerated N cycling under elevated CO2 at this site, while mycorrhizal fungi may contribute more to soil C degradation.

[Genetic diversity and mating system Pinus brutia var. Stankewiczii sukacz. in small localities of Sudak (Crimea)].

A comparative analysis of genetic variation at 12 polymorphic isozyme loci, and the mating system has been carried out in mature trees and their seed progeny in three small localities of Pinus brutia var. stankewiczii Sukacz. near the town of Sudak--settlement of Novyi Svet in the Crimea. We found that embryos maintain the same allelic diversity as mother plants but their observed heterozygosity is lower on the average by 37.4%. The significant deviation of genotype distribution from the theoretically expected ratios caused by the deficiency of heterozygotes was observed at 8 out of 12 loci. Multilocus estimate of outcrossing rate (t(m)) in populations varied from 68.9 to 94.9% making on the average 80.7%.

Structural and functional evolution of positively selected sites in pine glutathione S-transferase enzyme family.

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.

Ectopic expression of a loblolly pine class II 4-coumarate:CoA ligase alters soluble phenylpropanoid metabolism but not lignin biosynthesis in Populus.

4-Coumarate:CoA ligase (4CL) catalyzes the formation of hydroxycinnamoyl-CoA esters for phenylpropanoid biosynthesis. Phylogenetically distinct Class I and Class II 4CL isoforms occur in angiosperms, and support lignin and non-lignin phenylpropanoid biosynthesis, respectively. In contrast, the few experimentally characterized gymnosperm 4CLs are associated with lignin biosynthesis and belong to the conifer-specific Class III. Here we report a new Pinus taeda isoform Pinta4CL3 that is phylogenetically more closely related to Class II angiosperm 4CLs than to Class III Pinta4CL1. Like angiosperm Class II 4CLs, Pinta4CL3 transcript levels were detected in foliar and root tissues but were absent in xylem, and recombinant Pinta4CL3 exhibited a substrate preference for 4-coumaric acid. Constitutive expression of Pinta4CL3 in transgenic Populus led to significant increases of hydroxycinnamoyl-quinate esters at the expense of hydroxycinnamoyl-glucose esters in green tissues. In particular, large increases of cinnamoyl-quinate in transgenic leaves suggested in vivo utilization of cinnamic acid by Pinta4CL3. Lignin was unaffected in transgenic Populus, consistent with Pinta4CL3 involvement in biosynthesis of non-structural phenylpropanoids. We discuss the in vivo cinnamic acid utilization activity of Pinta4CL3 and its adaptive significance in conifer defense. Together with phylogenetic inference, our data support an ancient origin of Class II 4CLs that pre-dates the angiosperm-gymnosperm split.

Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 µM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.

Properties and Functional Analysis of Two Chorismate Mutases from Maritime Pine.

Through the shikimate pathway, a massive metabolic flux connects the central carbon metabolism with the synthesis of chorismate, the common precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as other compounds, including salicylate or folate. The alternative metabolic channeling of chorismate involves a key branch-point, finely regulated by aromatic amino acid levels. Chorismate mutase catalyzes the conversion of chorismate to prephenate, a precursor of phenylalanine and tyrosine and thus a vast repertoire of fundamental derived compounds, such as flavonoids or lignin. The regulation of this enzyme has been addressed in several plant species, but no study has included conifers or other gymnosperms, despite the importance of the phenolic metabolism for these plants in processes such as lignification and wood formation. Here, we show that maritime pine (Pinus pinaster Aiton) has two genes that encode for chorismate mutase, PpCM1 and PpCM2. Our investigations reveal that these genes encode plastidial isoenzymes displaying activities enhanced by tryptophan and repressed by phenylalanine and tyrosine. Using phylogenetic studies, we have provided new insights into the possible evolutionary origin of the cytosolic chorismate mutases in angiosperms involved in the synthesis of phenylalanine outside the plastid. Studies based on different platforms of gene expression and co-expression analysis have allowed us to propose that PpCM2 plays a central role in the phenylalanine synthesis pathway associated with lignification.

Transcriptome resources and functional characterization of monoterpene synthases for two host species of the mountain pine beetle, lodgepole pine (Pinus contorta) and jack pine (Pinus banksiana).

BACKGROUND: The mountain pine beetle (MPB, Dendroctonus ponderosae) epidemic has affected lodgepole pine (Pinus contorta) across an area of more than 18 million hectares of pine forests in western Canada, and is a threat to the boreal jack pine (Pinus banksiana) forest. Defence of pines against MPB and associated fungal pathogens, as well as other pests, involves oleoresin monoterpenes, which are biosynthesized by families of terpene synthases (TPSs). Volatile monoterpenes also serve as host recognition cues for MPB and as precursors for MPB pheromones. The genes responsible for terpene biosynthesis in jack pine and lodgepole pine were previously unknown. RESULTS: We report the generation and quality assessment of assembled transcriptome resources for lodgepole pine and jack pine using Sanger, Roche 454, and Illumina sequencing technologies. Assemblies revealed transcripts for approximately 20,000 - 30,000 genes from each species and assembly analyses led to the identification of candidate full-length prenyl transferase, TPS, and P450 genes of oleoresin biosynthesis. We cloned and functionally characterized, via expression of recombinant proteins in E. coli, nine different jack pine and eight different lodgepole pine mono-TPSs. The newly identified lodgepole pine and jack pine mono-TPSs include (+)-α-pinene synthases, (-)-α-pinene synthases, (-)-ß-pinene synthases, (+)-3-carene synthases, and (-)-ß-phellandrene synthases from each of the two species. CONCLUSION: In the absence of genome sequences, transcriptome assemblies are important for defence gene discovery in lodgepole pine and jack pine, as demonstrated here for the terpenoid pathway genes. The product profiles of the functionally annotated mono-TPSs described here can account for the major monoterpene metabolites identified in lodgepole pine and jack pine.

Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants.

2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ~90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K(+), whereas isoprene production is inhibited by K(+) such that, at physiologically relevant [K(+)], little or no isoprene emission should be detected from MBO-emitting trees. The K(m) of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site.

CCoAOMT suppression modifies lignin composition in Pinus radiata.

A cDNA clone encoding the lignin-related enzyme caffeoyl CoA 3-O-methyltransferase (CCoAOMT) was isolated from a Pinus radiata cDNA library derived from differentiating xylem. Suppression of PrCCoAOMT expression in P. radiata tracheary element cultures affected lignin content and composition, resulting in a lignin polymer containing p-hydroxyphenyl (H), catechyl (C) and guaiacyl (G) units. Acetyl bromide-soluble lignin assays revealed reductions in lignin content of up to 20% in PrCCoAOMT-deficient transgenic lines. Pyrolysis-GC/MS and 2D-NMR studies demonstrated that these reductions were due to depletion of G-type lignin. Correspondingly, the proportion of H-type lignin in PrCCoAOMT-deficient transgenic lines increased, resulting in up to a 10-fold increase in the H/G ratio relative to untransformed controls. 2D-NMR spectra revealed that PrCCoAOMT suppression resulted in formation of benzodioxanes in the lignin polymer. This suggested that phenylpropanoids with an ortho-diphenyl structure such as caffeyl alcohol are involved in lignin polymerization. To test this hypothesis, synthetic lignins containing methyl caffeate or caffeyl alcohol were generated and analyzed by 2D-NMR. Comparison of the 2D-NMR spectra from PrCCoAOMT-RNAi lines and synthetic lignins identified caffeyl alcohol as the new lignin constituent in PrCCoAOMT-deficient lines. The incorporation of caffeyl alcohol into lignin created a polymer containing catechyl units, a lignin type that has not been previously identified in recombinant lignin studies. This finding is consistent with the theory that lignin polymerization is based on a radical coupling process that is determined solely by chemical processes.

Can insect egg deposition 'warn' a plant of future feeding damage by herbivorous larvae?

Plant anti-herbivore defence is inducible by both insect feeding and egg deposition. However, little is known about the ability of insect eggs to induce defences directed not against the eggs themselves, but against larvae that subsequently hatch from the eggs. We studied how oviposition (OP) by the sawfly Diprion pini on Pinus sylvestris foliage affects the plant's defensive potential against sawfly larvae. Larvae that initiated their development on P. sylvestris twigs on which they hatched from eggs gained less weight and suffered higher mortality than those fed on egg-free twigs. The poor performance of these larvae also affected the next herbivore generation since fecundity of resulting females was lower than that of females which spent their larval development on egg-free pine. Transcript levels of P. sylvestris sesquiterpene synthases (PsTPS1, PsTPS2) were increased by D. pini OP, reached their highest levels just before larval hatching, and decreased when larvae started to feed. However, concentrations of terpenoid and phenolic metabolites presumed to act as feeding deterrents or toxins for herbivores did not change significantly after OP and feeding. Nevertheless, our performance data suggest that insect egg deposition may act to 'warn' a plant of upcoming feeding damage by larvae.

Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects.

Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of ent-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ent-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism.

Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum.

Chlorophyll biosynthesis is catalyzed by two multi subunit enzymes; a light-dependent and a light-independent protochlorophyllide oxidoreductase. The light-independent enzyme consists of three subunits (ChlL, ChlN and ChlB) in photosynthetic bacteria and plastids in which the chlB gene encodes the major subunit that catalyzes the reduction of protochlorophyllide to chlorophyllide. We report here stable integration of the chlB gene from Pinus thunbergii into the chloroplast genome of tobacco. Using helium-driven biolistic gun, transplastomic clones were developed in vitro. The stable integration and homoplasmy for transgenes was confirmed by using PCR and Southern blotting techniques. Nodal cuttings of the homoplasmic transgenic and untransformed wild type shoots were cultured on MS medium in the dark. As expected, shoots developed from the cuttings of the wild type plants in the dark showed etiolated growth with no roots whereas shoots from the cuttings of the transgenic plants developed early and more roots. Upon shifting from dark to light in growth room, leaves of the transgenic shoots showed early development of chlorophyll pigments compared to the wild type shoots. Further, photosynthetically indistinguishable transgenic shoots also showed significant difference in root development from untransformed wild type shoots when cuttings were grown in the light. Therefore, it may be concluded that the chlB gene is involved, directly or indirectly, in the root development of tobacco. Further, the gene promotes early development of chlorophyll pigments, upon illumination from dark, in addition to its role in the light-independent chlorophyll formation when expressed together with subunits L&N in other organisms.