Biosynthesis of the major tetrahydroxystilbenes in spruce, astringin and isorhapontin, proceeds via resveratrol and is enhanced by fungal infection.

Stilbenes are dibenzyl polyphenolic compounds produced in several unrelated plant families that appear to protect against various biotic and abiotic stresses. Stilbene biosynthesis has been well described in economically important plants, such as grape (Vitis vinifera), peanut (Arachis hypogaea), and pine (Pinus species). However, very little is known about the biosynthesis and ecological role of stilbenes in spruce (Picea), an important gymnosperm tree genus in temperate and boreal forests. To investigate the biosynthesis of stilbenes in spruce, we identified two similar stilbene synthase (STS) genes in Norway spruce (Picea abies), PaSTS1 and PaSTS2, which had orthologs with high sequence identity in sitka (Picea sitchensis) and white (Picea glauca) spruce. Despite the conservation of STS sequences in these three spruce species, they differed substantially from angiosperm STSs. Several types of in vitro and in vivo assays revealed that the P. abies STSs catalyze the condensation of p-coumaroyl-coenzyme A and three molecules of malonyl-coenzyme A to yield the trihydroxystilbene resveratrol but do not directly form the dominant spruce stilbenes, which are tetrahydroxylated. However, in transgenic Norway spruce overexpressing PaSTS1, significantly higher amounts of the tetrahydroxystilbene glycosides, astringin and isorhapontin, were produced. This result suggests that the first step of stilbene biosynthesis in spruce is the formation of resveratrol, which is further modified by hydroxylation, O-methylation, and O-glucosylation to yield astringin and isorhapontin. Inoculating spruce with fungal mycelium increased STS transcript abundance and tetrahydroxystilbene glycoside production. Extracts from STS-overexpressing lines significantly inhibited fungal growth in vitro compared with extracts from control lines, suggesting that spruce stilbenes have a role in antifungal defense.

Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.).

BACKGROUND: In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress. RESULTS: The availability of extensive transcriptome resources in the form of expressed sequence tags (ESTs) and full-length cDNAs in several spruce (Picea) species allowed us to estimate that a conifer genome contains at least 69 unique and transcriptionally active TPS genes. This number is comparable to the number of TPSs found in any of the sequenced and well-annotated angiosperm genomes. We functionally characterized a total of 21 spruce TPSs: 12 from Sitka spruce (P. sitchensis), 5 from white spruce (P. glauca), and 4 from hybrid white spruce (P. glauca × P. engelmannii), which included 15 monoterpene synthases, 4 sesquiterpene synthases, and 2 diterpene synthases. CONCLUSIONS: The functional diversity of these characterized TPSs parallels the diversity of terpenoids found in the oleoresin and volatile emissions of Sitka spruce and provides a context for understanding this chemical diversity at the molecular and mechanistic levels. The comparative characterization of Sitka spruce and Norway spruce diterpene synthases revealed the natural occurrence of TPS sequence variants between closely related spruce species, confirming a previous prediction from site-directed mutagenesis and modelling.

Transcriptome profiles of hybrid poplar (Populus trichocarpa × deltoides) reveal rapid changes in undamaged, systemic sink leaves after simulated feeding by forest tent caterpillar (Malacosoma disstria).

• Poplar has been established as a model tree system for genomic research of the response to biotic stresses. This study describes a series of induced transcriptome changes and the associated physiological characterization of local and systemic responses in hybrid poplar (Populus trichocarpa × deltoides) after simulated herbivory. • Responses were measured in local source (LSo), systemic source (SSo), and systemic sink (SSi) leaves following application of forest tent caterpillar (Malacosoma disstria) oral secretions to mechanically wounded leaves. • Transcriptome analyses identified spatially and temporally dynamic, distinct patterns of local and systemic gene expression in LSo, SSo and SSi leaves. Galactinol synthase was strongly and rapidly upregulated in SSi leaves. Genome analyses and full-length cDNA cloning established an inventory of poplar galactinol synthases. Induced changes of galactinol and raffinose oligosaccharides were detected by anion-exchange high-pressure liquid chromatography. • The LSo leaves showed a rapid and strong transcriptome response compared with a weaker and slower response in adjacent SSo leaves. Surprisingly, the transcriptome response in distant, juvenile SSi leaves was faster and stronger than that observed in SSo leaves. Systemic transcriptome changes of SSi leaves have signatures of rapid change of metabolism and signaling, followed by later induction of defense genes.

Endogenous overexpression of Populus MYB186 increases trichome density, improves insect pest resistance, and impacts plant growth.

Trichomes are specialized epidermal cells that generally play a role in reducing transpiration and act as a deterrent to herbivory. In a screen of activation-tagged Populus tremula × Populus alba 717-1B4 trees, we identified a mutant line, fuzzy, with increased foliar trichome density. This mutant also had a 35% increase in growth rate and a 200% increase in the rate of photosynthesis as compared with wild-type poplar. The fuzzy mutant had significant resistance to feeding by larvae of the white-spotted tussock moth (Orgyia leucostigma), a generalist insect pest of poplar trees. The fuzzy trichome phenotype is attributable to activation tagging and increased expression of the gene encoding PtaMYB186, which is related to Arabidopsis thaliana MYB106, a known regulator of trichome initiation. The fuzzy phenotype can be recapitulated by overexpressing PtaMYB186 in poplar. PtaMYB186 overexpression results in reconfiguration of the poplar transcriptome, with changes in the transcript abundance of suites of genes that are related to trichome differentiation. It is notable that a plant with misexpression of a gene responsible for trichome development also had altered traits related to growth rate and pest resistance, suggesting that non-intuitive facets of plant development might be useful targets for plant improvement.

Identification and functional characterization of monofunctional ent-copalyl diphosphate and ent-kaurene synthases in white spruce reveal different patterns for diterpene synthase evolution for primary and secondary metabolism in gymnosperms.

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

Poplar defense against insects: genome analysis, full-length cDNA cloning, and transcriptome and protein analysis of the poplar Kunitz-type protease inhibitor family.

*Kunitz protease inhibitors (KPIs) feature prominently in poplar defense responses against insects. The increasing availability of genomics resources enabled a comprehensive analysis of the poplar (p)KPI family. *Using genome analysis, expressed sequence tag (EST) mining and full-length (FL)cDNA cloning we established an inventory and phylogeny of pKPIs. Microarray and real-time PCR analyses were used to profile pKPI gene expression following real or simulated insect attack. Proteomics of insect midgut content was used to monitor stability of pKPI protein. *We identified 31 pKPIs in the genome and validated gene models by EST mining and cloning of 41 unique FLcDNAs. Genome organization of the pKPI family, with six poplar-specific subfamilies, suggests that tandem duplications have played a major role in its expansion. pKPIs are expressed throughout the plant and many are strongly induced by insect attack, although insect-specific signals seem initially to suppress the tree pKPI response. We found substantial peptide coverage for a potentially intact pKPI protein in insect midgut after eating poplar leaves. *These results highlight the complexity of an important defense gene family in poplar with regard to gene family size, differential constitutive and insect-induced gene expression, and resilience of at least one pKPI protein to digestion by herbivores.

Quantitative iTRAQ proteome and comparative transcriptome analysis of elicitor-induced Norway spruce (Picea abies) cells reveals elements of calcium signaling in the early conifer defense response.

Long-lived conifer trees depend on both constitutive and induced defenses for resistance against a myriad of potential pathogens and herbivores. In species of spruce (Picea spp.), several of the late events of pathogen-, insect-, or elicitor-induced defense responses have previously been characterized at the anatomical, biochemical, transcriptome, and proteome levels in stems and needles. However, accurately measuring the early events of induced cellular responses in a conifer is technically challenging due to limitations in the precise timing of induction and tissue sampling from intact trees following insect or fungal treatment. In the present study, we used the advantages of Norway spruce (Picea abies) cell suspensions combined with chitosan elicitation to investigate the early proteome response in a conifer. A combination of iTRAQ labeling and a new design of iterative sample analysis employing data-dependent exclusion lists were used for proteome analysis. This approach improved the coverage of the spruce proteome beyond that achieved in any prior study in a conifer system. Comparison of elicitor-induced proteome and transcriptome responses in Norway spruce cells consistently identified features associated with calcium-mediated signaling and response to oxidative stress that have not previously been observed in the response of intact trees to fungal attack.

A conifer genomics resource of 200,000 spruce (Picea spp.) ESTs and 6,464 high-quality, sequence-finished full-length cDNAs for Sitka spruce (Picea sitchensis).

BACKGROUND: Members of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests. These conifer forests are of critical importance for global ecosystem stability and biodiversity. They also provide the majority of the world's wood and fiber supply and serve as a renewable resource for other industrial biomaterials. In contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. Sequence-finished full-length (FL)cDNAs and large collections of expressed sequence tags (ESTs) are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation. RESULTS: As part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed 20 standard, normalized or full-length enriched cDNA libraries from Sitka spruce (P. sitchensis), white spruce (P. glauca), and interior spruce (P. glauca-engelmannii complex). We sequenced and analyzed 206,875 3'- or 5'-end ESTs from these libraries, and developed a resource of 6,464 high-quality sequence-finished FLcDNAs from Sitka spruce. Clustering and assembly of 147,146 3'-end ESTs resulted in 19,941 contigs and 26,804 singletons, representing 46,745 putative unique transcripts (PUTs). The 6,464 FLcDNAs were all obtained from a single Sitka spruce genotype and represent 5,718 PUTs. CONCLUSION: This paper provides detailed annotation and quality assessment of a large EST and FLcDNA resource for spruce. The 6,464 Sitka spruce FLcDNAs represent the third largest sequence-verified FLcDNA resource for any plant species, behind only rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the only substantial FLcDNA resource for a gymnosperm. Our emphasis on capturing FLcDNAs and ESTs from cDNA libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. Sequence comparisons against five plant genomes and the non-redundant GenBank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. Opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed.

Genome-wide analysis of a land plant-specific acyl:coenzyme A synthetase (ACS) gene family in Arabidopsis, poplar, rice and Physcomitrella.

The plant enzyme 4-coumarate:coenzyme A ligase (4CL) is part of a family of adenylate-forming enzymes present in all organisms. Analysis of genome sequences shows the presence of '4CL-like' enzymes in plants and other organisms, but their evolutionary relationships and functions remain largely unknown. 4CL and 4CL-like genes were identified by BLAST searches in Arabidopsis, Populus, rice, Physcomitrella, Chlamydomonas and microbial genomes. Evolutionary relationships were inferred by phylogenetic analysis of aligned amino acid sequences. Expression patterns of a conserved set of Arabidopsis and poplar 4CL-like acyl-CoA synthetase (ACS) genes were assayed. The conserved ACS genes form a land plant-specific class. Angiosperm ACS genes grouped into five clades, each of which contained representatives in three fully sequenced genomes. Expression analysis revealed conserved developmental and stress-induced expression patterns of Arabidopsis and poplar genes in some clades. Evolution of plant ACS enzymes occurred early in land plants. Differential gene expansion of angiosperm ACS clades has occurred in some lineages. Evolutionary and gene expression data, combined with in vitro and limited in vivo protein function data, suggest that angiosperm ACS enzymes play conserved roles in octadecanoid and fatty acid metabolism, and play roles in organ development, for example in anthers.

Global monitoring of autumn gene expression within and among phenotypically divergent populations of Sitka spruce (Picea sitchensis).

Cold acclimation in conifers is a complex process, the timing and extent of which reflects local adaptation and varies widely along latitudinal gradients for many temperate and boreal tree species. Despite their ecological and economic importance, little is known about the global changes in gene expression that accompany autumn cold acclimation in conifers. Using three populations of Sitka spruce (Picea sitchensis) spanning the species range, and a Picea cDNA microarray with 21,840 unique elements, within- and among-population gene expression was monitored during the autumn. Microarray data were validated for selected genes using real-time PCR. Similar numbers of genes were significantly twofold upregulated (1257) and downregulated (967) between late summer and early winter. Among those upregulated were dehydrins, pathogenesis-related/antifreeze genes, carbohydrate and lipid metabolism genes, and genes involved in signal transduction and transcriptional regulation. Among-population microarray hybridizations at early and late autumn time points revealed substantial variation in the autumn transcriptome, some of which may reflect local adaptation. These results demonstrate the complexity of cold acclimation in conifers, highlight similarities and differences to cold tolerance in annual plants, and provide a solid foundation for functional and genetic studies of this important adaptive process.

Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding.

BACKGROUND: The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. RESULTS: As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa x P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in poplar leaves attacked by forest tent caterpillars. CONCLUSION: This study has generated a high-quality FLcDNA resource for poplar and the third largest FLcDNA collection published to date for any plant species. We successfully used the FLcDNA sequences to reassess gene prediction in the poplar genome sequence, perform comparative sequence annotation, and identify differentially expressed transcripts associated with defense against insects. The FLcDNA sequences will be essential to the ongoing curation and annotation of the poplar genome, in particular for targeting gaps in the current genome assembly and further improvement of gene predictions. The physical FLcDNA clones will serve as useful reagents for functional genomics research in areas such as analysis of gene functions in defense against insects and perennial growth. Sequences from this study have been deposited in NCBI GenBank under the accession numbers EF144175 to EF148838.

Functional identification and differential expression of 1-deoxy-D-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies).

Conifers produce terpenoid-based oleoresins as constitutive and inducible defenses against herbivores and pathogens. Much information is available about the genes and enzymes of the late steps of oleoresin terpenoid biosynthesis in conifers, but almost nothing is known about the early steps which proceed via the methylerythritol phosphate (MEP) pathway. Here we report the cDNA cloning and functional identification of three Norway spruce (Picea abies) genes encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, and their differential expression in the stems of young saplings. Among them are representatives of both types of plant DXS genes. A single type I DXS gene is constitutively expressed in bark tissue and not affected by wounding or fungal application. In contrast, two distinct type II DXS genes, PaDXS2A and PaDXS2B, showed increased transcript abundance after these treatments as did two other genes of the MEP pathway tested, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxyl 3-methylbutenyl diphosphate reductase (HDR). We also measured gene expression in a Norway spruce cell suspension culture system that, like intact trees, accumulates monoterpenes after treatment with methyl jasmonate. These cell cultures were characterized by an up-regulation of monoterpene synthase gene transcripts and enzyme activity after elicitor treatment, as well as induced formation of octadecanoids, including jasmonic acid and 12-oxophytodienoic acid. Among the Type II DXS genes in cell cultures, PaDXS2A was induced by treatment with chitosan, methyl salicylate, and Ceratocystis polonica (a bark beetle-associated, blue-staining fungal pathogen of Norway spruce). However, PaDXS2B was induced by treatment with methyl jasmonate and chitosan, but was not affected by methyl salicylate or C. polonica. Our results suggest distinct functions of the three DXS genes in primary and defensive terpenoid metabolism in Norway spruce.

The transcriptional response of hybrid poplar (Populus trichocarpa x P. deltoides) to infection by Melampsora medusae leaf rust involves induction of flavonoid pathway genes leading to the accumulation of proanthocyanidins.

The transcriptional response of hybrid poplar (Populus trichocarpa x P. deltoides) to poplar leaf rust (Melampsora medusae) infection was studied using the Populus 15.5K cDNA microarray. Pronounced changes in the transcriptome were observed, with approximately 20% of genes on the array showing either induction or repression of transcription within the 9-day infection timecourse. A small number of pathogen-defense genes encoding PR-1, chitinases, and other pathogenesis-related proteins were consistently upregulated throughout the experimental period, but most genes were affected only at individual timepoints. The largest number of changes in gene expression was observed late in the infection at 6 to 9 days postinoculation (dpi). At these timepoints, genes encoding enzymes required for proanthocyanidin (condensed tannin) synthesis were upregulated dramatically. Phytochemical analysis confirmed that, late in the infection, proanthocyanidin levels increased in infected leaves. Strongly M. medusae-repressed genes at 9 dpi included previously characterized wound- and herbivore-induced defense genes, which suggests antagonism between the tree responses to insect feeding and M. medusae infection. In this highly compatible plant-pathogen interaction, we postulate that the biotrophic pathogen evades detection and suppresses early host responses.

Dirigent proteins in conifer defense II: Extended gene discovery, phylogeny, and constitutive and stress-induced gene expression in spruce (Picea spp.).

Analysis of expressed sequence tags (ESTs) and full-length (FL)cDNAs from species of spruce (Picea spp.) revealed a family of 35 unique dirigent proteins (DIR) and DIR-like proteins. Phylogenetic analysis indicates the spruce DIR and DIR-like genes cluster into three distinct subfamilies, DIR-a, DIR-b/d, and DIR-f, of a larger plant DIR and DIR-like gene family. Gene-specific primers were designed for 31 unique spruce DIR family genes, and closely related isoforms, and used to evaluate patterns of constitutive expression, as well as responses to herbivory by stem-boring insects (i.e., white pine weevil, Pissodes strobi) in bark tissue and defoliating insects (i.e., western spruce budworm, Choristoneura occidentalis) in green apical shoots. Furthermore, meta-analysis of microarray gene expression data obtained from a series of independent experiments using the same 16.7K cDNA array platform identified several distinct expression clusters of the spruce DIR transcriptome closely matching phylogenetic clusters of sequence similarity. Members of the DIR-a family, which also contains functionally characterized DIR from other plant species, are most prominent for their induced response to feeding by weevils on Sitka spruce bark.

Generation and annotation of lodgepole pine and oleoresin-induced expressed sequences from the blue-stain fungus Ophiostoma clavigerum, a Mountain Pine Beetle-associated pathogen.

Ophiostoma clavigerum is a destructive pathogen of lodgepole pine (Pinus contorta) forests in western North America. It is therefore a relevant system for a genomics analysis of fungi vectored by bark beetles. To begin characterizing molecular interactions between the pathogen and its conifer host, we created an expressed sequence tag (EST) collection for O. clavigerum. Lodgepole pine sawdust and oleoresin media were selected to stimulate gene expression that would be specific to this host interaction. Over 6500 cDNA clones, derived from four normalized cDNA libraries, were single-pass sequenced from the 3' end. After quality screening, we identified 5975 high-quality reads with an average PHRED 20 of greater than 750 bp. Clustering and assembly of this high-quality EST set resulted in the identification of 2620 unique putative transcripts. BLASTX analysis revealed that only 67% of these unique transcripts could be matched to known or predicted protein sequences in public databases. Functional classification of these sequences provided initial insights into the transcriptome of O. clavigerum. Of particular interest, our ESTs represent an extensive collection of cytochrome P450 s, ATP-binding-cassette-type transporters and genes involved in 1,8-dihydroxynaphthalene-melanin biosynthesis. These results are discussed in the context of detoxification of conifer oleoresins and fungal pathogenesis.

Microarray gene expression profiling of developmental transitions in Sitka spruce (Picea sitchensis) apical shoots.

The apical shoot drives the yearly new stem growth of conifer trees, is the primary site for the establishment of chemical and physical defences, and is important in establishing subsequent perennial growth. This organ presents an interesting developmental system, with growth and development progressing from a meristematic tip through development of a primary vascular system, to a base with fully differentiated and lignified secondary xylem on the inside and bark tissue with constitutive defence structures such as resin, polyphenolic phloem parenchyma cells, and sclereids on the outside. A spruce (Picea spp.) microarray containing approximately 16.7K unique cDNAs was used to study transcript profiles that characterize the developmental transition in apical shoots of Sitka spruce (Picea sitchensis) from their vegetative tips to their woody bases. Along with genes involved in cell-wall modification and lignin biosynthesis, a number of differentially regulated genes encoding protein kinases and transcription factors with base-preferred expression patterns were identified, which could play roles in the formation of woody tissues inside the apical shoot, as well as in regulating other developmental transitions associated with organ maturation. Preferential expression of known conifer defence genes, genes encoding defence-related proteins, and genes encoding regulatory proteins was observed at the apical shoot tip and in the green bark tissues at the apical shoot base, suggesting a commitment to constitutive defence in the apical shoot that is co-ordinated with rapid development of secondary xylem.

Aminocyclopropane carboxylic acid synthase is a regulated step in ethylene-dependent induced conifer defense. Full-length cDNA cloning of a multigene family, differential constitutive, and wound- and insect-induced expression, and cellular and subcellular localization in spruce and Douglas fir.

In conifer stems, formation of chemical defenses against insects or pathogens involves specialized anatomical structures of the phloem and xylem. Oleoresin terpenoids are formed in resin duct epithelial cells and phenolics accumulate in polyphenolic parenchyma cells. Ethylene signaling has been implicated in the induction of these chemical defenses. Recently, we reported the cloning of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from spruce (Picea spp.) and Douglas fir (Pseudotsuga menziesii). ACO protein was constitutively expressed in Douglas fir and only weakly induced upon wounding. We now cloned seven full-length and one near full-length cDNA representing four distinct 1-aminocyclopropane-1-carboxylic acid synthases (ACS; ACS1, ACS2, ACS3, and ACS4) from spruce and Douglas fir. Cloning of ACS has not previously been reported for any gymnosperm. Using gene-specific, quantitative real-time polymerase chain reaction, we measured constitutive expression for the four ACS genes and the single-copy ACO gene in various tissues of Sitka spruce (Picea sitchensis) and in white spruce (Picea glauca) somatic embryos. ACO and ACS4 were ubiquitously expressed at high levels; ACS1 was predominantly expressed in developing embryos and ACS2 and ACS3 were expressed only at very low levels. Insect attack or mechanical wounding caused strong induction of ACS2 and ACS3 in Sitka spruce bark, a moderate increase in ACO transcripts, but had no effect on ACS1 and ACS4. ACS protein was also strongly induced following mechanical wounding in Douglas fir and was highly abundant in resin duct epithelial cells and polyphenolic parenchyma cells. These results suggest that ACS, but not ACO, is a regulated step in ethylene-induced conifer defense.

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome.

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells.

Members of the Pinaceae family have complex chemical defense strategies. Conifer defenses associated with specialized cell types of the bark involve constitutive and inducible accumulation of phenolic compounds in polyphenolic phloem parenchyma cells and oleoresin terpenoids in resin ducts. These defenses can protect trees against insect herbivory and fungal colonization. The phytohormone ethylene has been shown to induce the same anatomical and cellular defense responses that occur following insect feeding, mechanical wounding, or fungal inoculation in Douglas fir (Pseudotsuga menziesii) stems (Hudgins and Franceschi in Plant Physiol 135:2134-2149, 2004). However, very little is known about the genes involved in ethylene formation in conifer defense or about the temporal and spatial patterns of their protein expression. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the final step in ethylene biosynthesis. We cloned full-length and near full-length ACO cDNAs from three conifer species, Sitka spruce (Picea sitchensis), white spruce (P. glauca), and Douglas fir, each with high similarity to Arabidopsis thaliana ACO proteins. Using an Arabidopsis anti-ACO antibody we determined that ACO is constitutively expressed in Douglas fir stem tissues and is up-regulated by mechanical wounding, consistent with the wound-induced increase of ethylene levels. Immunolocalization showed cytosolic ACO is predominantly present in specialized cell types of the wound-induced bark, specifically in epithelial cells of terpenoid-producing cortical resin ducts, in polyphenolic phloem parenchyma cells, and in ray parenchyma cells.