Pineapple proteases, commonly known as bromelains, have been used for many years. In this work we identified and analyzed the protease-coding genes and their products in pineapple. A total of 512 genes encoding putative proteases in pineapple were identified. The encoded proteases are predicted to be distributed in different subcellular compartments, with 152 secreted into extracellular space, 74 localized in cytosol, 67 in nucleus, 60 in chloroplast, 18 in mitochondria, and the remaining in other subcellular locations. The proteases are classified using the conserved domain and protein family database as well as the MEROPS protease classification method. The top large protease families in pineapple were papain family cysteine protease (62 genes), peptidase S8 family (56 genes), aspartyl protease family (38 genes), and serine carboxypeptidase (33 genes). Gene expression analysis revealed that among 512 protease genes, 432 were expressed in various tissues and 72 genes were differentially expressed. Papain family protease (cysteine proteases, bromelain) genes were among the highly expressed genes. Phylogenetic analysis of cysteine proteases showed four large clusters formed among them. The information of identified protease genes with their gene expression profiles along with the predicted subcellular locations of the protease proteins will facilitate the community to further examine their biological roles of proteases in pineapple growth and development and to explore the potential for expressing the recombinant proteases for medical use.

Alternative splicing (AS) has played a major role in defining the protein diversity, which could be linked to phenotypic alternations. It is imperative to have a comparative resolution of AS to understand the pre-mRNAs splicing diversity. In the present research, we present a comparative assessment of the AS events in four different fruit plants including apple (Malus domestica), grape (Vitis vinifera), sweet orange (Citrus sinensis), and woodland strawberry (Fragaria vesca), using spliced mapping of the expressed sequence tags and mRNA sequences. We identified a total of 2039 AS events in apple, 2454 in grape, 1425 in orange, and 631 in strawberry, respectively. In this study grape displayed the maximum number of genes (1588) associated with the splicing, followed by apple (1580), orange (1133) and strawberry (444). Transcripts mapping analysis shows that grape plant has relatively larger intron sizes than introns in other fruit species. The data provide a basis for further functional characterization of the genes undergoing AS and can be accessed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/plant/).

Pineapple, Ananas comosus, is an economically important fruit crop. Recently its genome was completely sequenced and a total of 27,024 protein coding genes were predicted. Using a set of well evaluated bioinformatics tools we have predicted the protein subcellular locations and comparatively analyzed the protein conserved domains of the predicted proteomes in pineapple, Oryza sativa (rice), Sorghum bicolor (sorghum), and Brachypodium distachyson. Our analysis revealed that ~24–26 % of proteins were located in nucleus, 17–21 % in cytosol, 9–11 % in chloroplast, and 8–11 % proteins were secreted in these monocot plants. The secretomes in the four species were analyzed comparatively and a large number of secreted glycosyl hydrolases were identified. As pineapple proteolytic enzymes, knowns as bromelains, have been used for medical treatments, we focused on genome-wide identification and analysis of pineapple genes encoding proteases. A total of 512 pineapple genes encoding putative proteolytic enzymes were identified, with 152 secreted, 74 localized in cytosol, 67 in nucleus, 60 in chloroplast, 18 in mitochondria, and the remaining in other subcellular locations. The top large protease families in pineapple were papain family cysteine protease (62 genes), peptidase S8 family (56 genes), aspartyl protease family (38 genes), and serine carboxypeptidase (33 genes). Gene expression analysis revealed that among 512 protease genes 432 were expressed in various tissues and 72 genes were differentially expressed. The highly expressed protease genes were identified including 7 papain family cysteine proteases. The protease genes with the predicted protein subcellular locations will facilitate the efforts for examining their biological roles in pineapple growth and development and for expressing the recombinant proteases for medical use. The information of protein subcellular location of all plant species can be accessed at the PlantSecKB website (http://proteomics.ysu.edu/secretomes/plant.php).

Kingdom Protista contains a large group of eukaryotic organisms with diverse lifestyles. We developed the Protist Secretome and Subcellular Proteome Knowledgebase (ProtSecKB) to host information of curated and predicted subcellular locations of all protist proteins. The protist protein sequences were retrieved from UniProtKB, consisting of 1.97 million entries generated from 7,024 species with 101 species including 127 organisms having complete proteomes. The protein subcellular locations were based on curated information and predictions using a set of well evaluated computational tools. The database can be searched using several different types of identifiers, gene names or keyword(s). Secretomes and other subcellular proteomes can be searched or downloaded. BLAST searching against the complete set of protist proteins or secretomes is available. Protein family analysis of secretomes from representing protist species, including Dictyostelium discoideum , Phytophthora infestans , and Trypanosoma cruzi , showed that species with different lifestyles had drastic differences of protein families in their secretomes, which may determine their lifestyles. The database provides an important resource for the protist and biomedical research community. The database is available at http://bioinformatics.ysu.edu/secretomes/protist/index.php.

Pineapple (Ananas comosus L. Merrill) is an important tropical and subtropical fruit crop and possesses crassulacean acid metabolism (CAM) photosynthesis. Recent release of its genome sequences makes it possible to identify genes transcribed with alternatively spliced isoforms in this plant. Mapping the assembled transcripts generated by next-generation sequencing technology and existing expressed sequence tags as well as mRNA sequences to the published pineapple genome, we identified and analyzed alternative splicing (AS) events. We identified a total of 10,348 AS events involving 13,449 assembled putative unique transcripts, which were mapped to 5146 pineapple gene models that equivalent to 29.7 % of total expressed gene models. Consistent with previous findings in other plant species, intron retention (61.9 %) remains to be the dominant type among the identified AS events. Comparative genomic analysis of genes which generated pre-mRNAs having AS revealed a total of 481 genes conserved among Oryza sativa (ssp japonica), Sorghum bicolor, Zea mays, and pineapple, with 51 of them were also conserved with Brachypodium distachyon. Gene Ontology classification revealed that the products of these genes which generate AS isoforms are involved in many biological processes with diverse molecular functions. We annotated all assembled transcripts and also associated them with predicted gene models. The annotated information of these data provides a resource for further characterizing these genes and their biological roles. The data can be accessed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/).

Number of conserved alternative splicing genes in cereal crops. (XLS 31 kb)

BACKGROUND:Protein functional diversity at the post-transcriptional level is regulated through spliceosome mediated pre-mRNA alternative splicing (AS) events and that has been widely demonstrated to be a key player in regulating the functional diversity in plants. Identification and analysis of AS genes in cereal crop plants are critical for crop improvement and understanding regulatory mechanisms.RESULTS:We carried out the comparative analyses of the functional landscapes of the AS using the consensus assembly of expressed sequence tags and available mRNA sequences in four cereal plants. We identified a total of 8,734 in Oryza sativa subspecies (ssp) japonica, 2,657 in O. sativa ssp indica, 3,971 in Sorghum bicolor, and 10,687 in Zea mays AS genes. Among the identified AS events, intron retention remains to be the dominant type accounting for 23.5 % in S. bicolor, and up to 55.8 % in O. sativa ssp indica. We identified a total of 887 AS genes that were conserved among Z. mays, S. bicolor, and O. sativa ssp japonica; and 248 AS genes were found to be conserved among all four studied species or ssp. Furthermore, we identified 53 AS genes conserved with Brachypodium distachyon. Gene Ontology classification of AS genes revealed functional assignment of these genes in many biological processes with diverse molecular functions.CONCLUSIONS:AS is common in cereal plants. The AS genes identified in four cereal crops in this work provide the foundation for further studying the roles of AS in regulation of cereal plant growth and development. The data can be accessed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/).