We have established a protocol for the isolation of highly purified peroxisomes from mature Arabidopsis thaliana leaves and analyzed the proteome by complementary gel-based and gel-free approaches. Seventy-eight nonredundant proteins were identified, of which 42 novel proteins had previously not been associated with plant peroxisomes. Seventeen novel proteins carried predicted peroxisomal targeting signals (PTS) type 1 or type 2; 11 proteins contained PTS-related peptides. Peroxisome targeting was supported for many novel proteins by in silico analyses and confirmed for 11 representative fulllength fusion proteins by fluorescence microscopy. The targeting function of predicted and unpredicted signals was investigated and SSL>, SSI>, and ASL> were established as novel functional PTS1 peptides. In contrast with the generally accepted confinement of PTS2 peptides to the N-terminal domain, the bifunctional transthyretin-like protein was demonstrated to carry internally a functional PTS2. The novel enzymes include numerous enoyl-CoA hydratases, short-chain dehydrogenases, and several enzymes involved in NADP and glutathione metabolism. Seven proteins, including b-glucosidases and myrosinases, support the currently emerging evidence for an important role of leaf peroxisomes in defense against pathogens and herbivores. The data provide new insights into the biology of plant peroxisomes and improve the prediction accuracy of peroxisome-targeted proteins from genome sequences.

The plant signaling molecule salicylic acid (SA) and/or xenobiotic chemicals like the auxin mimic 2,4-D induce transcriptional activation of defense-and stress-related genes that contain activation sequence-1 (as-1)-like cis-elements in their promoters. as-1-like sequences are recognized by basic/leucine zipper transcription factors of the TGA family. Expression of genes related to the SA-dependent defense program systemic acquired resistance requires the TGA-interacting protein NPR1. However, a number of as-1-containing promoters can be activated independently from NPR1. Here, we report the identification of Arabidopsis thaliana SCARECROW-like 14 (SCL14), a member of the GRAS family of regulatory proteins, as a TGA-interacting protein that is required for the activation of TGA-dependent but NPR1-independent SA-and 2,4-Dinducible promoters. Chromatin immunoprecipitation experiments revealed that class II TGA factors TGA2, TGA5, and/or TGA6 are needed to recruit SCL14 to promoters of selected SCL14 target genes identified by whole-genome transcript profiling experiments. The coding regions and the expression profiles of the SCL14-dependent genes imply that they might be involved in the detoxification of xenobiotics and possibly endogenous harmful metabolites. Consistently, plants ectopically expressing SCL14 showed increased tolerance to toxic doses of the chemicals isonicotinic acid and 2,4,6-triiodobenzoic acid, whereas the scl14 and the tga2 tga5 tga6 mutants were more susceptible. Hence, the TGA/SCL14 complex seems to be involved in the activation of a general broad-spectrum detoxification network upon challenge of plants with xenobiotics.

In the postgenomic era, accurate prediction tools are essential for identification of the proteomes of cell organelles. Prediction methods have been developed for peroxisome-targeted proteins in animals and fungi but are missing specifically for plants. For development of a predictor for plant proteins carrying peroxisome targeting signals type 1 (PTS1), we assembled more than 2500 homologous plant sequences, mainly from EST databases. We applied a discriminative machine learning approach to derive two different prediction methods, both of which showed high prediction accuracy and recognized specific targeting-enhancing patterns in the regions upstream of the PTS1 tripeptides. Upon application of these methods to the Arabidopsis thaliana genome, 392 gene models were predicted to be peroxisome targeted. These predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal. The prediction methods were able to correctly infer novel PTS1 tripeptides, which even included novel residues. Twenty-three newly predicted PTS1 tripeptides were experimentally confirmed, and a high variability of the plant PTS1 motif was discovered. These prediction methods will be instrumental in identifying lowabundance and stress-inducible peroxisomal proteins and defining the entire peroxisomal proteome of Arabidopsis and agronomically important crop plants.

Einleitung 2.1 Die pflanzliche Reaktion auf abiotischen und biotischen Stress 2.2 Reaktive Sauerstoffspezies als Signaltransduktoren 2.3 Das an pflanzlicher Stressantwort beteiligte cis-Element as-1 2.4 Die Proteine der TGA-Familie binden an das as-1 Element 2.5 Protein-Protein-Interaktionspartner der TGA-Familie 2.6 Die GRAS-Genfamilie 4.4 Verwendete Computerprogramme 5 Ergebnisse 5.1 Analyse der Interaktion von SCL14 und TGA2 im Hefe-Ein-und Zweihybridsystem 5.2 Interaktionsstudien zwischen TGA2 und verwandten Scarecrow-like Proteinen 5.3 Pull-down-Analyse von SCL14 und TGA2 5.4 Transiente Analysen der as-1-Aktivität in BY-2-Protoplasten 5.5 Analysen der subzellulären Lokalisation in transient transformierten BY-2-Protoplasten 5.5.1 Subzelluläre Lokalisierung von SCL14 und TGA2 5.5.2 Subzelluläre Lokalisierung von SCL14 nach Inhibierung des Kernexports durch LMB 5.6 Herstellung und Charakterisierung eines SCL14-Antiserums