Production of a viral surface protein in Nannochloropsis oceanica for fish vaccination against infectious pancreatic necrosis virus.

Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet. KEY POINTS: • We engineered N. oceanica for recombinant protein production. • The antigenic surface protein 2 of IPN virus could indeed be expressed in the host. • A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.

Microalgae and Bacteria Interaction-Evidence for Division of Diligence in the Alga Microbiota.

Microalgae are one of the most dominant forms of life on earth that is tightly associated with a distinct and specialized microbiota. We have previously shown that the microbiota of Scenedesmus quadricauda harbors less than 10 distinct microbial species. Here, we provide evidence that dominant species are affiliated with the genera of Variovorax, Porphyrobacter, and Dyadobacter. Experimental and transcriptome-based evidence implies that within this multispecies interaction, Dyadobacter is a key to alga growth and fitness and is highly adapted to live in the phycosphere. While presumably under light conditions the alga provides the energy source to the bacteria, Dyadobacter produces and releases mainly a large variety of polysaccharides modifying enzymes. This is coherent with high-level expression of the T9SS in alga cocultures. The transcriptome data further imply that quorum-quenching proteins (QQ) and biosynthesis of vitamins B1, B2, B5, B6, and B9 are expressed by Dyadobacter at high levels in comparison to Variovorax and Porphyrobacter. Notably, Dyadobacter produces a significant number of leucine-rich repeat (LRR) proteins and enzymes involved in bacterial reactive oxygen species (ROS) tolerance. Complementary to this, Variovorax expresses the genes of the biosynthesis of vitamins B2, B5, B6, B7, B9, and B12, and Porphyrobacter is specialized in the production of vitamins B2 and B6. Thus, the shared currency between partners are vitamins, microalgae growth-promoting substances, and dissolved carbon. This work significantly enlarges our knowledge on alga-bacteria interaction and demonstrates physiological investigations of microalgae and associated bacteria, using microscopy observations, photosynthetic activity measurements, and flow cytometry. IMPORTANCE The current study gives a detailed insight into mutualistic collaboration of microalgae and bacteria, including the involvement of competitive interplay between bacteria. We provide experimental evidence that Gram-negative bacteria belonging to the Dyadobacter, Porphyrobacter, and Variovorax are the key players in a Scenedesmus quadricauda alga-bacteria interaction. We impart strong evidence that Dyadobacter produces and releases polysaccharides degradation enzymes and leucine-rich repeat proteins; Variovorax supplies the consortium with auxins and vitamin B12, while Porphyrobacter produces a broad spectrum of B vitamins. We show not only that the microalgae collaborate with the bacteria and vice versa but also that the bacteria interact with each other via quorum-sensing and secretion system mechanisms. The shared currency between partners appears to be vitamins, microalgae growth-promoting substances, and dissolved carbon.

The essential role of fungal peroxisomes in plant infection.

Several filamentous fungi are ecologically and economically important plant pathogens that infect a broad variety of crops. They cause high annual yield losses and contaminate seeds and fruits with mycotoxins. Not only powerful infection structures and detrimental toxins, but also cell organelles, such as peroxisomes, play important roles in plant infection. In this review, we summarize recent research results that revealed novel peroxisomal functions of filamentous fungi and highlight the importance of peroxisomes for infection of host plants. Central for fungal virulence are two primary metabolic pathways, fatty acid ß-oxidation and the glyoxylate cycle, both of which are required to produce energy, acetyl-CoA, and carbohydrates. These are ultimately needed for the synthesis of cell wall polymers and for turgor generation in infection structures. Most novel results stem from different routes of secondary metabolism and demonstrate that peroxisomes produce important precursors and house various enzymes needed for toxin production and melanization of appressoria. All these peroxisomal functions in fungal virulence might represent elegant targets for improved crop protection.

Stramenopile microalgae as "green biofactories" for recombinant protein production.

Photoautotrophic microalgae have become intriguing hosts for recombinant protein production because they offer important advantages of both prokaryotic and eukaryotic expression systems. Advanced molecular tools have recently been established for the biotechnologically relevant group of stramenopile microalgae, particularly for several Nannochloropsis species and diatoms. Strategies for the selection of powerful genetic elements and for optimization of protein production have been reported. Much needed high-throughput techniques required for straight-forward identification and selection of the best expression constructs and transformants have become available and are discussed. The first recombinant proteins have already been produced successfully in stramenopile microalgae and include not only several subunit vaccines but also one antimicrobial peptide, a fish growth hormone, and an antibody. These research results offer interesting future applications in aquaculture and as biopharmaceuticals. In this review we highlight recent progress in genetic technology development for recombinant protein production in the most relevant Nannochloropsis species and diatoms. Diverse realistic biotechnological applications of these proteins are emphasized that have the potential to establish stramenopile algae as sustainable green factories for an economically competitive production of high-value biomolecules.

Evolutionary Maintenance of the PTS2 Protein Import Pathway in the Stramenopile Alga Nannochloropsis.

The stramenopile alga Nannochloropsis evolved by secondary endosymbiosis of a red alga by a heterotrophic host cell and emerged as a promising organism for biotechnological applications, such as the production of polyunsaturated fatty acids and biodiesel. Peroxisomes play major roles in fatty acid metabolism but experimental analyses of peroxisome biogenesis and metabolism in Nannochloropsis are not reported yet. In fungi, animals, and land plants, soluble proteins of peroxisomes are targeted to the matrix by one of two peroxisome targeting signals (type 1, PTS1, or type 2, PTS2), which are generally conserved across kingdoms and allow the prediction of peroxisomal matrix proteins from nuclear genome sequences. Because diatoms lost the PTS2 pathway secondarily, we investigated its presence in the stramenopile sister group of diatoms, the Eustigmatophyceae, represented by Nannochloropsis. We detected a full-length gene of a putative PEX7 ortholog coding for the cytosolic receptor of PTS2 proteins and demonstrated its expression in Nannochloropsis gaditana. The search for predicted PTS2 cargo proteins in N. gaditana yielded several candidates. In vivo subcellular targeting analyses of representative fusion proteins in different plant expression systems demonstrated that two predicted PTS2 domains were indeed functional and sufficient to direct a reporter protein to peroxisomes. Peroxisome targeting of the predicted PTS2 cargo proteins was further confirmed in Nannochloropsis oceanica by confocal and transmission electron microscopy. Taken together, the results demonstrate for the first time that one group of stramenopile algae maintained the import pathway for PTS2 cargo proteins. To comprehensively map and model the metabolic capabilities of Nannochloropsis peroxisomes, in silico predictions needs to encompass both the PTS1 and the PTS2 matrix proteome.

Development of a constitutive and an auto-inducible high-yield expression system for recombinant protein production in the microalga Nannochloropsis oceanica.

Photoautotrophic microalgae offer a great potential as novel hosts for efficient recombinant protein production. Nannochloropsis oceanica produces an extraordinarily high content of polyunsaturated fatty acids, and its robust growth characteristics, published genome sequence and efficient nuclear transformation make N. oceanica a promising candidate for biotechnological applications. To establish a robust and flexible system for recombinant protein production, we cloned six endogenous, potentially constitutive or inducible promoters from N. oceanica strain CCMP1779 and investigated their strength using monomeric Venus as reporter gene. Microscopic pre-screening of individual transformants revealed that the promoters of elongation factor (EF), tubulin (TUB) and nitrate reductase (NR) enabled high reporter gene expression. Comparative quantitative analyses of transformant populations by flow cytometry and qRT-PCR demonstrated the highest Venus expression from the EF promoter and the NR promoter if extended by an N-terminal 14-amino acid leader sequence. The kinetics of reporter gene expression were analysed during photobioreactor cultivation, achieving Venus yields of 0.3% (for EF) and 4.9% (for NR::LS) of total soluble protein. Since inducible expression systems enable the production of toxic proteins, we developed an auto-induction medium for the NR promoter transformants. By switching the N source from ammonium to nitrate in the presence of low ammonium concentrations, the starting point of Venus induction could be fine-tuned and shifted towards exponential growth phase while maintaining high recombinant protein yields. Taken together, we demonstrate that a model recombinant protein can be produced robustly and at very high levels in N. oceanica not only under constitutive but also under auto-inducible cultivation conditions. KEY POINTS: • Nannochloropsis oceanica might serve as host for recombinant protein production. • Comparative promoter strength analyses were conducted for twelve different constructs. • Robust high-yield recombinant protein production was achieved under constitutive conditions. • The nitrate reductase promoter enabled protein production under auto-induction conditions.

New guidelines for fluorophore application in peroxisome targeting analyses in transient plant expression systems.

Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein (YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co-cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co-transformation with the peroxisome marker, CFP-PTS1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP-PTS1, allowing piggy-back import into peroxisomes. Indeed, if co-expressed with monomeric Cerulean-PTS1 (mCer-PTS1), the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization-based piggy-back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co-expressed with CFP-PTS1 but not with mCer-PTS1. By contrast, piggy-back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.

Prediction of Peroxisomal Matrix Proteins in Plants.

Our knowledge of the proteome of plant peroxisomes is far from being complete, and the functional complexity and plasticity of this cell organelle are amazingly high particularly in plants, as exemplified by the model species Arabidopsis thaliana. Plant-specific peroxisome functions that have been uncovered only recently include, for instance, the participation of peroxisomes in phylloquinone and biotin biosynthesis. Experimental proteome studies have been proved very successful in defining the proteome of Arabidopsis peroxisomes but this approach also faces significant challenges and limitations. Complementary to experimental approaches, computational methods have emerged as important powerful tools to define the proteome of soluble matrix proteins of plant peroxisomes. Compared to other cell organelles such as mitochondria, plastids and the ER, the simultaneous operation of two major import pathways for soluble proteins in peroxisomes is rather atypical. Novel machine learning prediction approaches have been developed for peroxisome targeting signals type 1 (PTS1) and revealed high sensitivity and specificity, as validated by in vivo subcellular targeting analyses in diverse transient plant expression systems. Accordingly, the algorithms allow the correct prediction of many novel peroxisome-targeted proteins from plant genome sequences and the discovery of additional organelle functions. In contrast, the prediction of PTS2 proteins largely remains restricted to genome searches by conserved patterns contrary to more advanced machine learning methods. Here, we summarize and discuss the capabilities and accuracies of available prediction algorithms for PTS1 and PTS2 carrying proteins.

Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis leaves.

Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the biosynthesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry (MS)-based proteomics has been performed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of peroxisomes from Arabidopsis leaves given a 48-h dark treatment. Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identified a total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the full function of plant peroxisomes.

The levels of peroxisomal catalase protein and activity modulate the onset of cell death in tobacco BY-2 cells via reactive oxygen species levels and autophagy.

In plant cells, peroxisomes participate in the metabolism of reactive oxygen species (ROS). One of the major regulators of cellular ROS levels - catalase (CAT) - occurs exclusively in peroxisomes. CAT activity is required for immunity-triggered autophagic programmed cell death (PCD). Autophagy has been recently demonstrated to represent a route for degradation of peroxisomes in plant cells. In the present study, the dynamics of the cellular peroxisome pool in tobacco BY-2 cell suspension cultures were used to analyse the effects of inhibition of basal autophagy with special attention to CAT activity. Numbers of peroxisomes per cell, levels of CAT protein and activity, cell viability, ROS levels and expression levels of genes encoding components of antioxidant system were analysed upon application of 3-methyladenine (3-MA), an inhibitor of autophagy, and/or aminotriazole (AT), an inhibitor of CAT. When applied separately, 3-MA and AT led to an increase in cell death, but this effect was attenuated by their simultaneous application. The obtained data suggest that both the levels of CAT protein in peroxisomes as well as CAT activity modulate the onset of cell death in tobacco BY-2 cells via ROS levels and autophagy.

Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane.

To date, less than 150 proteins have been located to plant peroxisomes, indicating that unbiased large-scale approaches such as experimental proteome research are required to uncover the remaining yet unknown metabolic functions of this organelle as well as its regulatory mechanisms and membrane proteins. For experimental proteome research, Arabidopsis thaliana is the model plant of choice and an isolation methodology that obtains peroxisomes of sufficient yield and high purity is vital for research on this organelle. However, organelle enrichment is more difficult from Arabidopsis when compared to other plant species and especially challenging for peroxisomes. Leaf peroxisomes from Arabidopsis are very fragile in aqueous solution and show pronounced physical interactions with chloroplasts and mitochondria in vivo that persist in vitro and decrease peroxisome purity. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes using two different types of density gradients (Percoll and sucrose) sequentially that yields approximately 120 µg of peroxisome proteins from 60 g of fresh leaf material. A method is also provided to assess the relative purity of the isolated peroxisomes by immunoblotting to allow selection of the purest peroxisome isolates. To enable the analysis of peroxisomal membrane proteins, an enrichment strategy using sodium carbonate treatment of isolated peroxisome membranes has been adapted to suit isolated leaf peroxisomes and is described here.

Plant peroxisomes: recent discoveries in functional complexity, organelle homeostasis, and morphological dynamics.

Peroxisomes are essential for life in plants. These organelles house a variety of metabolic processes that generate and inactivate reactive oxygen species. Our knowledge of pathways and mechanisms that depend on peroxisomes and their constituent enzymes continues to grow, and in this review we highlight recent advances in understanding the identity and biological functions of peroxisomal enzymes and metabolic processes. We also review how peroxisomal matrix and membrane proteins enter the organelle from their sites of synthesis. Peroxisome homeostasis is regulated by specific degradation mechanisms, and we discuss the contributions of specialized autophagy and a peroxisomal protease to the degradation of entire peroxisomes and peroxisomal enzymes that are damaged or superfluous. Finally, we review how peroxisomes can flexibly change their morphology to facilitate inter-organellar contacts.

Experimental validation of plant peroxisomal targeting prediction algorithms by systematic comparison of in vivo import efficiency and in vitro PTS1 binding affinity.

Most peroxisomal matrix proteins possess a C-terminal targeting signal type 1 (PTS1). Accurate prediction of functional PTS1 sequences and their relative strength by computational methods is essential for determination of peroxisomal proteomes in silico but has proved challenging due to high levels of sequence variability of non-canonical targeting signals, particularly in higher plants, and low levels of availability of experimentally validated non-canonical examples. In this study, in silico predictions were compared with in vivo targeting analyses and in vitro thermodynamic binding of mutated variants within the context of one model targeting sequence. There was broad agreement between the methods for entire PTS1 domains and position-specific single amino acid residues, including residues upstream of the PTS1 tripeptide. The hierarchy Leu>Met>Ile>Val at the C-terminal position was determined for all methods but both experimental approaches suggest that Tyr is underweighted in the prediction algorithm due to the absence of this residue in the positive training dataset. A combination of methods better defines the score range that discriminates a functional PTS1. In vitro binding to the PEX5 receptor could discriminate among strong targeting signals while in vivo targeting assays were more sensitive, allowing detection of weak functional import signals that were below the limit of detection in the binding assay. Together, the data provide a comprehensive assessment of the factors driving PTS1 efficacy and provide a framework for the more quantitative assessment of the protein import pathway in higher plants.

Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells.

Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism.

Isolation of leaf peroxisomes from Arabidopsis for organelle proteome analyses.

The isolation of cell organelles from model organisms in high purity is important for biochemical analyses of single proteins, entire metabolic pathways, and protein complexes and is absolutely essential for organelle proteome analyses. The efficient enrichment of nearly all cell organelles is more difficult from Arabidopsis as compared to traditional model plants and especially challenging for peroxisomes. Leaf peroxisomes are generally very instable in aqueous solution due to the presence of a single membrane and (para-)crystalline inclusions in the matrix. Leaf peroxisomes from Arabidopsis are particularly fragile and, moreover, strongly physically adhere to chloroplasts and mitochondria for largely unknown reasons. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes by Percoll followed by sucrose density gradient centrifugation that yields high purity suitable for proteome analyses. Diverse enzymatic and immuno-biochemical methods are summarized to assess purity and intactness.

Biosynthesis of vitamin K1 (phylloquinone) by plant peroxisomes and its integration into signaling molecule synthesis pathways.

Vitamin K1 (phylloquinone) is a substituted membrane-anchored naphthoquinone that functions as an essential electron carrier in photosystem I in photosynthetic organisms. While plants can synthesize phylloquinone de novo, humans rely on vitamin K1 uptake from green leafy vegetables as a precursor for the synthesis of its structural derivative, menaquinone-4 (vitamin K2). In vertebrates, menaquinone-4 serves as an enzymatic co-factor that is required for posttranslational protein modification, i.e. the γ-carboxylation of glutamate residues in specific proteins involved in blood coagulation, bone metabolism and vascular biology. Comprehensive knowledge of the subcellular compartmentalization of vitamin K biosynthesis in plants, pathway regulation and its integration in cellular metabolic networks is important to design functional food with elevated vitamin levels and health benefits to human consumers. It had long been assumed that plants obtained all enzymes for phylloquinone biosynthesis from the ancient cyanobacterial endosymbiont and that, upon gene transfer to the nucleus, all biosynthetic enzymes were re-directed to the plastid. This view, however, has been recently challenged by the exclusive localization of the 6th pathway enzyme (MenB/NS) to peroxisomes in Arabidopsis. Soon afterwards, not only the preceding enzyme, acyl-activating enzyme 14 (MenE/AAE14), but also the succeeding thioesterase (DHNAT) were also shown to be peroxisomal. Phylogenetic analysis revealed a heterogeneous evolutionary origin of the peroxisomal enzymes. Phylloquinone biosynthesis reveals several branching points leading to the synthesis of important defence signalling molecules, such as salicylic acid and benzoic acid derivatives. Recent research data demonstrate that, of the two phenylalanine-dependent pathways for benzoic and salicylic acid biosynthesis, the CoA-dependent ß-oxidative pathway, which is peroxisomal, is the major route. Hence, peroxisomes emerge as an important cell compartment for the interconnected networks of phylloquinone, benzoic and salicylic acid biosynthesis. Numerous mechanisms to regulate intermediate flux and the fine-tuned inducible production of secondary metabolites, including signalling molecules, await their characterization at the molecular level.

PredPlantPTS1: A Web Server for the Prediction of Plant Peroxisomal Proteins.

Prediction of subcellular protein localization is essential to correctly assign unknown proteins to cell organelle-specific protein networks and to ultimately determine protein function. For metazoa, several computational approaches have been developed in the past decade to predict peroxisomal proteins carrying the peroxisome targeting signal type 1 (PTS1). However, plant-specific PTS1 protein prediction methods have been lacking up to now, and pre-existing methods generally were incapable of correctly predicting low-abundance plant proteins possessing non-canonical PTS1 patterns. Recently, we presented a machine learning approach that is able to predict PTS1 proteins for higher plants (spermatophytes) with high accuracy and which can correctly identify unknown targeting patterns, i.e., novel PTS1 tripeptides and tripeptide residues. Here we describe the first plant-specific web server PredPlantPTS1 for the prediction of plant PTS1 proteins using the above-mentioned underlying models. The server allows the submission of protein sequences from diverse spermatophytes and also performs well for mosses and algae. The easy-to-use web interface provides detailed output in terms of (i) the peroxisomal targeting probability of the given sequence, (ii) information whether a particular non-canonical PTS1 tripeptide has already been experimentally verified, and (iii) the prediction scores for the single C-terminal 14 amino acid residues. The latter allows identification of predicted residues that inhibit peroxisome targeting and which can be optimized using site-directed mutagenesis to raise the peroxisome targeting efficiency. The prediction server will be instrumental in identifying low-abundance and stress-inducible peroxisomal proteins and defining the entire peroxisomal proteome of Arabidopsis and agronomically important crop plants. PredPlantPTS1 is freely accessible at ppp.gobics.de.

Non-canonical peroxisome targeting signals: identification of novel PTS1 tripeptides and characterization of enhancer elements by computational permutation analysis.

BACKGROUND: High-accuracy prediction tools are essential in the post-genomic era to define organellar proteomes in their full complexity. We recently applied a discriminative machine learning approach to predict plant proteins carrying peroxisome targeting signals (PTS) type 1 from genome sequences. For Arabidopsis thaliana 392 gene models were predicted to be peroxisome-targeted. The predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal. RESULTS: In this study, we experimentally validated the predictions in greater depth by focusing on the most challenging Arabidopsis proteins with unknown non-canonical PTS1 tripeptides and prediction scores close to the threshold. By in vivo subcellular targeting analysis, three novel PTS1 tripeptides (QRL>, SQM>, and SDL>) and two novel tripeptide residues (Q at position -3 and D at pos. -2) were identified. To understand why, among many Arabidopsis proteins carrying the same C-terminal tripeptides, these proteins were specifically predicted as peroxisomal, the residues upstream of the PTS1 tripeptide were computationally permuted and the changes in prediction scores were analyzed. The newly identified Arabidopsis proteins were found to contain four to five amino acid residues of high predicted targeting enhancing properties at position -4 to -12 in front of the non-canonical PTS1 tripeptide. The identity of the predicted targeting enhancing residues was unexpectedly diverse, comprising besides basic residues also proline, hydroxylated (Ser, Thr), hydrophobic (Ala, Val), and even acidic residues. CONCLUSIONS: Our computational and experimental analyses demonstrate that the plant PTS1 tripeptide motif is more diverse than previously thought, including an increasing number of non-canonical sequences and allowed residues. Specific targeting enhancing elements can be predicted for particular sequences of interest and are far more diverse in amino acid composition and positioning than previously assumed. Machine learning methods become indispensable to predict which specific proteins, among numerous candidate proteins carrying the same non-canonical PTS1 tripeptide, contain sufficient enhancer elements in terms of number, positioning and total strength to cause peroxisome targeting.

Plant peroxisomes: biogenesis and function.

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle's dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.

Experimental and statistical post-validation of positive example EST sequences carrying peroxisome targeting signals type 1 (PTS1).

We recently developed the first algorithms specifically for plants to predict proteins carrying peroxisome targeting signals type 1 (PTS1) from genome sequences. As validated experimentally, the prediction methods are able to correctly predict unknown peroxisomal Arabidopsis proteins and to infer novel PTS1 tripeptides. The high prediction performance is primarily determined by the large number and sequence diversity of the underlying positive example sequences, which mainly derived from EST databases. However, a few constructs remained cytosolic in experimental validation studies, indicating sequencing errors in some ESTs. To identify erroneous sequences, we validated subcellular targeting of additional positive example sequences in the present study. Moreover, we analyzed the distribution of prediction scores separately for each orthologous group of PTS1 proteins, which generally resembled normal distributions with group-specific mean values. The cytosolic sequences commonly represented outliers of low prediction scores and were located at the very tail of a fitted normal distribution. Three statistical methods for identifying outliers were compared in terms of sensitivity and specificity." Their combined application allows elimination of erroneous ESTs from positive example data sets. This new post-validation method will further improve the prediction accuracy of both PTS1 and PTS2 protein prediction models for plants, fungi, and mammals.