Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

HybridPoplar_H11-11_Leaves_MalacosomaDisstria_Herbivory

ABSTRACT: 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. Ten hybridizations were performed comparing FTC-treated poplar leaves after 24 h of continuous feeding and untreated control leaves harvested at the same time. RNA isolated from each of the five individual FTC-treated trees was compared directly against the five individual untreated control trees using two hybridizations with a dye flip for each tree pair.

ORGANISM(S): Populus trichocarpa x Populus deltoides

SUBMITTER: Joerg Bohlmann

PROVIDER: E-GEOD-9522 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genomics of hybrid poplar (Populus trichocarpax 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.

Ralph Steven S Oddy Claire C Cooper Dawn D Yueh Hesther H Jancsik Sharon S Kolosova Natalia N Philippe Ryan N RN Aeschliman Dana D White Rick R Huber Dezene D Ritland Carol E CE Benoit François F Rigby Tracey T Nantel André A Butterfield Yaron S N YS Kirkpatrick Robert R Chun Elizabeth E Liu Jerry J Palmquist Diana D Wynhoven Brian B Stott Jeffrey J Yang George G Barber Sarah S Holt Robert A RA Siddiqui Asim A Jones Steven J M SJ Marra Marco A MA Ellis Brian E BE Douglas Carl J CJ Ritland Kermit K Bohlmann Jörg J

Molecular ecology 20060401 5

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 ...[more]

PMID: 16626454

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Project description: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. In spite of 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, we monitored within and among-population gene expression during the fall. Microarray data were validated for selected genes using real-time PCR. Similar numbers of genes were significantly two-fold upregulated (1,257) 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. Our 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 in conifers. Keywords: Time course Foliage for RNA extraction was obtained from four-year-old Sitka spruce seedlings, which were grown from seed collected across the species range in a raised-bed outdoor common garden in Vancouver, BC, Canada (49Âº N) (Mimura and Aitken, 2007, Heredity). Needle tissues from current year upper lateral shoots, separated from stem and bark tissue, were collected at five time points between late summer and early winter, 2004. Three populations were chosen for sampling: Valdez, Alaska, USA (61Âº N) (AK), Prince Rupert, British Columbia, Canada (54Âº N) (BC) and Redwood, California, USA (41Âº N) (CA) (Figure 1a). Needle samples (~1g) were taken from eight individuals in the BC population on each of the five dates (Aug. 30, Oct. 18, Nov. 24, Dec. 1, and Dec. 13). Eight individuals from the AK and CA populations were also sampled on the second and fourth dates, as well as eight additional individuals from the BC population. Tissues were flash frozen in an N2 vapor tank immediately upon collection, and subsequently stored at -80Â°C until processing. To decrease the effects of biological variance among individual seedlings within populations, equal amounts of foliar tissue were pooled from four individuals prior to RNA extraction. Two pools were collected at each time point for each population, and total RNA was extracted following a previously published protocol (Kolosova et al., 2004). Slides were scanned and spot intensity quantified using ImaGene software (BioDiscovery, Inc., El Segundo, CA). To correct for background intensity, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities. Data were then normalized by variance stabilizing normalization (VSN) to compensate for nonlinearity of intensity distributions (Huber et al., 2002). To identify significant changes in gene expression, a linear mixed-effects model was fit to the normalized intensities in the Cy3 and Cy5 channels of the 32 microarray slides. The model contained an adjustment for dye bias, an array effect indicating which Cy5/Cy3 pair was on each array, a treatment effect indicating sample population and time point, and a random effect to adjust for repeated measures on the same biological sample (Kerr et al., 2000). P-values were computed for each gene-by-treatment effect and Q-values were calculated to adjust for the false discovery rate (FDR) (Storey & Tibshirani, 2003).

Project description:Over recent decades the progression of natural autumnal senescence has been increasingly delayed across wide areas of the Northern Hemisphere and is thought to be caused by rising temperature. Recently this delay has been shown to be partly attributable to rising atmospheric carbon dioxide concentrations [CO2]. Here, for the first time, we present a possible mechanistic explanation for this phenomenon. Using a plantation of Populus x euramericana grown at elevated [CO2] (e[CO2]) using Free-Air CO2 Enrichment (FACE) technology, we investigated the molecular and biochemical basis underlying this response. Using a poplar cDNA microarray where late growing season and senescing leaves from ambient [CO2] (a[CO2]) and e[CO2] were compared, revealed unique increases and decreases in transcript abundance in the e[CO2] treatment. Growth at e[CO2]caused the greatest increase in the abundance of transcripts catalysing steps in the anthocyanin biosynthetic pathway and an increase in leaf anthocyanin content. Leaf sucrose and starch content were also increased during senescence in e[CO2] and associated with a higher abundance of transcripts in the sucrose and starch biosynthetic pathways. We propose that in e[CO2], autumnal photosynthesis and sugar accumulation results in changes in genes expression that include further investment in secondary carbon metabolism leading to anthocyanin production. This enables prolonged leaf longevity during natural autumnal senescence through increased availability of carbon and improved stress tolerance while in contrast this may also have a negative effect on the control of dormancy. Samples GSM398292..GSM398314 (late senescence samples only): Six replicate trees were labelled in the unfertilised sub-plots of plots 1-4 in the POP/EUROFACE site (http://www.unitus.it/euroface/) and the 10th leaf down from the closed apical bud of the dominant branch were labelled. Leaves were sampled to liquid nitrogen (18th October 2004) and stored at -80 oC. Extracted RNA was tested for quality and quantity and of that passing the test equal quantities of RNA from each replicate were taken and each sample from the control plots (plots 2 and 3) were randomly paired with a sample from and elevated CO2 plot (plots 1 and 4). From the resulting 9 dual channel hybridisations 6 had the control sample in the Cy3 channel and 3 had the elevated CO2 sample in the Cy3 channel. The 6 samples with Cydye orientation as Cy3=control (ambient CO2) and Cy5 = treated (elevated CO2) are as follows: GSM398296, GSM398301, GSM398303, GSM398304, GSM398305 and GSM398306. The 3 samples with dye swap i.e Cy3 = treated (elevated CO2) are as follows and Cy5 = control (ambient CO2) are as follows: GSM398292, GSM398313 and GSM398314. Samples GSM398426..GSM398474: Four replicate leaves per sub-plot were sampled and flash frozen into liquid nitrogen, on 31 August 2004, and prior to visible senescence but around the time of bud-set as given by Calfapietra et al., (2003) [1]. The tenth leaf down the dominant stem (which represented the first fully expanded mature leaf of maximum size) from a reference leaf of 30 mm in length (leaf one) were selected. On the 14th September twenty trees from within each experimental plot were taken. The tenth leaf down the dominant stem (which represented the first fully expanded mature leaf of maximum size) from a reference leaf of 30 mm in length (leaf one) were labelled on the main-stem above the petiole. All labelled leaves were a uniform height (approx 0.3 m) from the canopy top across all treatments and not shaded by the canopy. Four replicate leaves from within each sub-plot were sampled during each harvest (except on 18th October when six replicate leaves were sampled). Each sampling time was restricted to two hours within the same day and across sampling day’s uniform cloud free skies were chosen. September samplings took place between 15.00 and 17.00 when climatic variables were generally consistent; later in the season time of sampling was adjusted to an earlier two hour period after mid-day when climatic variables were generally consistent. A chlorotic canopy was evident in plots five and six, as described by Liberloo et al. (2007) therefore any subsequent analysis was for plots one to four only. Six replicate labelled leaves were sampled (as above) from each sub-plot on 18th October 2004

Dataset Information

HybridPoplar_H11-11_Leaves_MalacosomaDisstria_Herbivory

Publications

Genomics of hybrid poplar (Populus trichocarpax 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.

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