Comparative transcriptome analysis of Arabidopsis thaliana infested by diamond back moth (Plutella xylostella) larvae reveals signatures of stress response, secondary metabolism, and signalling.

BACKGROUND: Plants are exposed to attack from a large variety of herbivores. Feeding insects can induce substantial changes of the host plant transcriptome. Arabidopsis thaliana has been established as a relevant system for the discovery of genes associated with response to herbivory, including genes for specialized (i.e. secondary) metabolism as well as genes involved in plant-insect defence signalling. RESULTS: Using a 70-mer oligonucleotide microarray covering 26,090 gene-specific elements, we monitored changes of the Arabidopsis leaf transcriptome in response to feeding by diamond back moth (DBM; Plutella xylostella) larvae. Analysis of samples from a time course of one hour to 24 hours following onset of DBM feeding revealed almost three thousand (2,881) array elements (including 2,671 genes with AGI annotations) that were differentially expressed (>2-fold; p[t-test] < 0.05) of which 1,686 also changed more than twofold in expression between at least two time points of the time course with p(ANOVA) < 0.05. While the majority of these transcripts were up-regulated within 8 h upon onset of insect feeding relative to untreated controls, cluster analysis identified several distinct temporal patterns of transcriptome changes. Many of the DBM-induced genes fall into ontology groups annotated as stress response, secondary metabolism and signalling. Among DBM-induced genes associated with plant signal molecules or phytohormones, genes associated with octadecanoid signalling were clearly overrepresented. We identified a substantial number of differentially expressed genes associated with signal transduction in response to DBM feeding, and we compared there expression profiles with those of previously reported transcriptome responses induced by other insect herbivores, specifically Pieris rapae, Frankliniella occidentalis, Bemisia tabaci,Myzus persicae, and Brevicoryne brassicae. CONCLUSION: Arabidopsis responds to feeding DBM larvae with a drastic reprogramming of the transcriptome, which has considerable overlap with the response induced by other insect herbivores. Based on a meta-analysis of microarray data we identified groups of transcription factors that are either affected by multiple forms of biotic or abiotic stress including DBM feeding or, alternatively, were responsive to DBM herbivory but not to most other forms of stress.

Microarray expression profiling and functional characterization of AtTPS genes: duplicated Arabidopsis thaliana sesquiterpene synthase genes At4g13280 and At4g13300 encode root-specific and wound-inducible (Z)-gamma-bisabolene synthases.

The Arabidopsis thaliana genome contains at least 32 terpenoid synthase (AtTPS) genes [Aubourg et al., Mol. Genet. Genom. 267 (2002) 730] a few of which have recently been characterized. Based on hierarchical cluster analysis of AtTPS gene expression, measured by microarray profiling and validated with published expression data, we identified two groups of predominantly root expressed AtTPS genes containing five members with previously unknown biochemical functions (At4g13280, At4g13300, At5g48110, At1g33750, and At3g29410). Among the root expressed AtTPS genes, a pair of tandem-organized genes, At4g13280 (AtTPS12) and At4g13300 (AtTPS13), shares 91% predicted amino acid identity indicating recent gene duplication. Bacterial expression of cDNAs and enzyme assays showed that both At4g13280 and At4g13300 encode sesquiterpene synthases catalyzing the conversion of farnesyl diphosphate to (Z)-gamma-bisabolene and the additional minor products E-nerolidol and alpha-bisabolol. Expression of beta-glucuronidase (GUS) reporter gene fused to upstream genomic regions of At4g13280 or At4g13300 showed constitutive promoter activities in the cortex and sub-epidermal layers of Arabidopsis roots. In addition, highly localized promoter activities were found in leaf hydathodes and flower stigmata. Mechanical wounding of Arabidopsis leaves induced local expression of At4g13280 and At4g13300. The functional characterization of At4g13280 gene product AtTPS12 and At4g13230 gene product AtTPS13 as (Z)-gamma-bisabolene synthases, together with the recent characterization of two flower-specific AtTPS [At5g23960 and At5g44630; Tholl et al., Plant J. 42 (2005) 757], concludes the biochemical functional annotation of all four predicted Arabidopsis sesquiterpene synthase genes. Our data suggest biological functions for At4g13280 and At4g13300 in the rhizosphere with additional roles in aerial plant tissues.

Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation.

Different stages of vascular and interfascicular fiber differentiation can be identified along the axis of bolting stems in Arabidopsis. To gain insights into the metabolic, developmental, and regulatory events that control this pattern, we applied global transcript profiling employing an Arabidopsis full-genome longmer microarray. More than 5000 genes were differentially expressed, among which more than 3000 changed more than twofold, and were placed into eight expression clusters based on polynomial regression models. Within these, 182 upregulated transcription factors represent candidate regulators of fiber development. A subset of these candidates has been associated with fiber development and/or secondary wall formation and lignification in the literature, making them targets for functional studies and comparative genomic analyses with woody plants. Analysis of differentially expressed phenylpropanoid genes identified a set known to be involved in lignin biosynthesis. These were used to anchor co-expression analyses that allowed us to identify candidate genes encoding proteins involved in monolignol transport and monolignol dehydrogenation and polymerization. Similar analyses revealed candidate genes encoding enzymes that catalyze missing links in the shikimate pathway, namely arogenate dehydrogenase and prephenate aminotransferase.

Isolation of high-quality RNA from white spruce tissue using a three-stage purification method and subsequent cloning of a transcript from the PR-10 gene family.

Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280 nm and 260/230 nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5 microg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picgl, that was very similar to members of the PR10 gene family.