Steven Ralph

The Genome of Black Cottonwood, Populus trichocarpa (Torr. & Gray)

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and accumulation of terpenoid synthase and putative octadecanoid pathway transcripts in Sitka spruce.

Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (−)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (−)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system.

Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (comet) assay.

Pesticides are broadly used for pest control in agriculture despite possible negative impacts they may pose to the environment. Thus, we examined the DNA damage caused by five herbicides commonly used in southern Ontario (Canada). Erythrocytes from Rana catesbeiana (bullfrog) tadpoles were evaluated for DNA damage following exposure to selected herbicides, using the alkaline single‐cell gel DNA electrophoresis (SCG) or “comet” assay [Singh et al. (1988): Exp Cell Res 175:184–191; Ralph et al. (1996): Eviron Mol Mutagen 28:112–120]. This approach involves detection, under alkaline conditions, of DNA fragments that upon electrophoresis migrate from the nuclear core, resulting in a comet formation. The herbicides tested, along with their active ingredients, were AAtrex Nine‐O (atrazine), Dual‐960E (metalochlor), Roundup(glyphosate), Sencor‐500F (metribuzin), and Amsol (2,4‐D amine). Tadpoles were exposed in the laboratory for a 24‐hr period to several concentrations of the herbicides dissolved in dechlorinated water. Methyl methanesulphonate was used as a positive control. The herbicides AAtrex Nine‐O‐, Dual‐960E‐,Roundup‐, and Sencor‐500F‐treated tadpolesshowed significant DNA damage when compared with unexposed control animals, whereas, Amsol‐treated tadpoles did not. Unlike the other responding herbicides, Sencor‐500F did not show a relationship between dosage and DNA damage. In summary, the results indicate that at least some of the herbicides currently used in southern Ontario are capable of inducing DNA damage in tadpoles. Environ. Mol. Mutagen. 29:277‐288, 1997

Genomics of hybrid poplar (Populus trichocarpa× deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar

As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore‐ and elicitor‐treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full‐length cDNA libraries, we generated 139 007 3′‐ or 5′‐end sequenced ESTs, representing more than one‐third of the c. 385 000 publicly available Populus ESTs. Clustering and assembly of 107 519 3′‐end ESTs resulted in 14 451 contigs and 20 560 singletons, altogether representing 35 011 putative unique transcripts, or potentially more than three‐quarters of the predicted c. 45 000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15 496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up‐regulated, compared to only 537 down‐regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1‐aminocyclopropane‐1‐carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (–)‐germacrene D synthase] and transcriptional regulation [e.g. leucine‐rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome‐scale approach to characterize insect‐induced defences in a woody perennial providing a solid platform for functional investigation of plant–insect interactions in poplar.

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.

Robust simple sequence repeat markers for spruce (Picea spp.) from expressed sequence tags

Traditionally, simple sequence repeat (SSR) markers have been developed from libraries of genomic DNA. However, the large, repetitive nature of conifer genomes makes development of robust, single-copy SSR markers from genomic DNA difficult. Expressed sequence tags (ESTs), or sequences of messenger RNA, offer the opportunity to exploit single, low-copy, conserved sequence motifs for SSR development. From a 20,275-unigene spruce EST set, we identified 44 candidate EST-SSR markers. Of these, 25 amplified and were polymorphic in white, Sitka, and black spruce; 20 amplified in all 23 spruce species tested; the remaining five amplified in all except one species. In addition, 101 previously described spruce SSRs (mostly developed from genomic DNA), were tested. Of these, 17 amplified across white, Sitka, and black spruce. The 25 EST-SSRs had approximately 9% less heterozygosity than the 17 genomic-derived SSRs (mean H=0.65 vs 0.72), but appeared to have less null alleles, as evidenced by much lower apparent inbreeding (mean F=0.046 vs 0.126). These robust SSRs are of particular use in comparative studies, and as the EST-SSRs are within the expressed portion of the genome, they are more likely to be associated with a particular gene of interest, improving their utility for quantitative trait loci mapping and allowing detection of selective sweeps at specific genes.

Isolation of high-quality RNA from gymnosperm and angiosperm trees.

An improved protocol was developed for efficient and reliable extraction of high-quality total RNA and mRNA from various tissues of spruce (Picea spp.) and poplar (Populus spp.) trees, as well as other plant species. This method was specifically optimized for tissues with high content of polysaccharides, oleoresin terpenoids, and phenolic secondary metabolites, which often co-precipitate with RNA and inhibit subsequent reverse transcription. The improved protocol yielded up to 600 micrograms of total RNA per gram of tissue suitable for standard expressed sequence tags (ESTs), full-length cDNA library construction, and for microarray applications.

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.

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

BackgroundIn 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.ResultsThe 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.ConclusionsThe 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.

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.