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.

ABSTRACTPlant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogenRalstonia solanacearumis well adapted to it, growing to 108to 109 CFU/g tomato stem. To better understand howR. solanacearumsucceeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinctR. solanacearumstrains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 108 CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly alteredin plantaversus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in thescrABYcluster. A UW551scrAmutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed hostSolanum dulcamara. FunctionalscrAcontributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose.scrAexpression was induced by sucrose, but to a much greater degree by growthin planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulatedin plantaat high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle.IMPORTANCERalstonia solanacearumis a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions thatR. solanacearumexpresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen’s dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further,R. solanacearumwas highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783-800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in b-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid a-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps− mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps− mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps− mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL.) and a functional PTS2 (RLx 5 HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one-and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions.