Current 22K Microarray Construction and Validation
This project uses a 21.8k non-redundant cDNA microarray platform developed under the Treenomix I and Treenomix II projects. This cDNA array platform has been built by extension of our earlier 9.6k and 15.7 array platforms (see Ralph et al., 2006 and Friedmann et al., 2007). Based on the generation of ESTs, we developed a spruce cDNA microarray composed of 21,843 cDNA elements selected from 12 cDNA libraries representing developmental stages of xylem, phloem, bark and roots, as well as elicitor-treated bark. Clones on the array were selected from a CAP3 assembly of 50,770 hq 3’ ESTs, and were carefully chosen to represent a minimally redundant gene set. Functional annotation of array elements has been assigned according to the TAIR Arabidopsis protein set using BLASTX.
For validation of the new 21.8k microarray, total RNA was isolated from bark of four 2-year-old trees, pooled, and used to evaluate the false change rate. Four technical replicate hybridizations were performed using this same RNA source, revealing that the median foreground signal intensity for blank elements (Cy3-89.6; Cy5-90.7), DMSO buffer-only elements (Cy3-79.1; Cy5-68.6), and DNA-based negative control elements (Cy3-33.9; Cy5-36.0) was low compared to that of EST elements (Cy3-2,942.5; Cy5-4,517.0), indicating a low level of nonspecific hybridization. Among the three possible combinations for randomly assigning RNA in each channel to treatment groups when comparing the four slides in self-self hybridizations, no differentially expressed (DE; fold-change > 1.5x; P value < 0.05; Q value < 0.05) EST array elements were identified, indicating no false changes due to technical variation on the array.
In the current project, gene expression profiling is integrated with research activities on Conifer Defence Mechanisms, Provenance Expression Profiling, eQTL Mapping, and for activities related to Conifer Adaptation. For further information, please contact Joerg Bohlmann.
Conifer Defence Mechanisms
As part of the characterization of Conifer Defence Mechanisms, we have completed two microarray analyses of bark tissue from shoots of Sitka spruce exposed to controlled weevil attack (Ralph et al., 2006, Ralph et al., manuscript in preparation). In a third study we compared the effect of wounding, weevil-attack, and fungal inoculation in interior spruce.
- - We find that weevil attack results in a major redirection of the spruce transcriptome. For example, after 48 h of weevil feeding 1,857 (8.5%) microarray elements identified transcripts as up-regulated, compared to 1,374 (6.3%) down-regulated.
- - Elevated transcript levels of several key steps of terpenoid and phenolic secondary metabolism were detected in Sitka spruce bark tissue.
- - Expression data has been mapped onto comprehensive pathway maps. For example, regulated steps in terpenoid defence were identified as deoxy-xylulose phosphate synthase (DXS) and terpenoid synthases (TPS). In the phenolic metabolisms, many genes had increased transcript levels including the poorly characterized family of dirigent proteins (DIR).
Since we identified several gene families that responded strongly to insect attack, we have used gene-specific, quantitative RT-PCR to obtain expression data for individual members of gene families. These include analyses of 35 genes encoding DIR proteins putatively involved in phenolic defence; ACC-synthase and ACC-oxidase in wound- and insect-induced defence signalling (details in Ralph et al., 2007 and Hudgins et al., 2006); as well as more than 50 defence-related genes in terpenoid and phenolic metabolism . Current targets for gene specific expression analysis using gene-specific primers are the large TPS family and cytochrome P450s (P450) of the CYP85 clan identified in our EST/FLcDNA program (see Hamberger and Bohlmann, 2006; Keeling and Bohlmann, 2006). The TPS and P450s as well as DIR proteins are also targets in our Functional Gene Characterization program.
In collaboration with the co-funding Max Planck Institute, we have conducted transcriptome profiling on elicitor-treated Norway spruce (P. abies) cell cultures. Chitosan and heat-treated Ceratocystis polonica (insect-associated blue stain fungus) were used as elicitors in a 48-slide experiment. We focused on the early response to chitosan elicitation, and are currently working on the biological interpretation of expression changes, followed-up by gene-specific real-time PCR, and comparison to patterns of protein expression. In the same project, these collaborators conducted a 36-slide experiment in our lab using samples from Norway spruce trees inoculated by the Ceratocystis polonica fungus. For further information, please contact Joerg Bohlmann.