Analysis of the oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry

To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LC-MS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membrane-spanning β-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signaling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally unclassified despite being predicted to be membrane proteins.Siria H. A. Natera, Kristina L. Ford, Andrew M. Cassin, John H. Patterson, Edward J. Newbigin and Antony Baci

Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate

Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes-notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodiumhighlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite\u27s host. Hematodinium sp. thus represents a further dimension of endosymbiosis-life after the organelle

Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate.

Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes--notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium--highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite's host. Hematodinium sp. thus represents a further dimension of endosymbiosis--life after the organelle.We thank Nick Katris for assistance with Toxoplasma transformation and Ellen Nisbet for critically reading this report. This work was supported by Australian Research Council (ARC) Grants DP130100572 and DP1093395; a King Abdullah University of Science and Technology Faculty Baseline Research Fund; and Victorian Life Sciences Computation Initiative Grant VR0254. S.G.G. was supported by Science Foundation Ireland Grant 13/SIRG/2125; F. was supported by an Australia Award; A.M.C. and A.B. were supported by ARC Centre of Excellence in Plant Cell Walls Grant CE110001007; and M.J.M. was supported by the National Health and Medical Research Council as a Principal Research Fellow.This is the accepted manuscript of a paper published in the Proceedings of the National Academy of Sciences (Gornik et al., PNAS 2015, 112, 18, 5767-5772, doi:10.1073/pnas.1423400112). The final version is available at http://dx.doi.org/10.1073/pnas.142340011

In vitro grown pollen tubes of Nicotiana alata actively synthesise a fucosylated xyloglucan.

Nicotiana alata pollen tubes are a widely used model for studies of polarized tip growth and cell wall synthesis in plants. To better understand these processes, RNA-Seq and de novo assembly methods were used to produce a transcriptome of N. alata pollen grains. Notable in the reconstructed transcriptome were sequences encoding proteins that are involved in the synthesis and remodelling of xyloglucan, a cell wall polysaccharide previously not thought to be deposited in Nicotiana pollen tube walls. Expression of several xyloglucan-related genes in actively growing pollen tubes was confirmed and xyloglucan epitopes were detected in the wall with carbohydrate-specific antibodies: the major xyloglucan oligosaccharides found in N. alata pollen grains and tubes were fucosylated, an unusual structure for the Solanaceae, the family to which Nicotiana belongs. Finally, carbohydrate linkages consistent with xyloglucan were identified chemically in the walls of N. alata pollen grains and pollen tubes grown in culture. The presence of a fucosylated xyloglucan in Nicotiana pollen tube walls was thus confirmed. The consequences of this discovery to models of pollen tube growth dynamics and more generally to polarised tip-growing cells in plants are discussed

Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates

The pellicles of alveolates (ciliates, apicomplexans, and dinoflagellates) share a common organization, yet perform very divergent functions, including motility, host cell invasion, and armor. The alveolate pellicle consists of a system of flattened membrane sacs (alveoli, which are the defining feature of the group) below the plasma membrane that is supported by a membrane skeleton as well as a network of microtubules and other filamentous elements. We recently showed that a family of proteins, alveolins, are common and unique to this pellicular structure in alveolates. To identify additional proteins that contribute to this structure, a pellicle proteome study was conducted for the ciliate Tetrahymena thermophila. We found 1,173 proteins associated with this structure, 45% (529 proteins) of which represented novel proteins without matches to other functionally characterized proteins. Expression of four newly identified T. thermophila pellicular proteins as green fluorescent protein-fusion constructs confirmed pellicular location, and one new protein located in the oral apparatus. Bioinformatic analysis revealed that 21% of the putative pellicular proteins, predominantly the novel proteins, contained highly repetitive regions with strong amino acid biases for particular residues (K, E, Q, L, I, and V). When the T. thermophila novel proteins were compared with apicomplexan genomic data, 278 proteins with high sequence similarity were identified, suggesting that many of these putative pellicular components are shared between the alveolates. Of these shared proteins, 126 contained the distinctive repeat regions. Localization of two such proteins in Toxoplasma gondii confirmed their role in the pellicle and in doing so identified two new proteins of the apicomplexan invasive structure - the apical complex. Screening broadly for these repetitive domains in genomic data revealed large and actively evolving families of such proteins in alveolates, suggesting that these proteins might underpin the diversity and utility of their unique pellicular structure

Expression profiles of XyG-related genes in various <i>N. alata</i> tissues.

<p>RT–PCR was carried out using the indicated cDNA template and primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077140#pone.0077140.s003" target="_blank">Table S3</a>) specific for each of the XyG-related gene listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077140#pone-0077140-t001" target="_blank">Table 1</a>. RT-PCR for each template using actin-specific primers (positive control) is also shown.</p