A workflow for peptide-based proteomics in a poorly sequenced plant: a case study on the plasma membrane proteome of banana.

Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.

Aspects of the barley seed proteome during development and germination.

Analysis of the water-soluble barley seed proteome has led to the identification of proteins by MS in the major spots on two-dimensional gels covering the pI ranges 4-7 and 6-11. This provides the basis for in-depth studies of proteome changes during seed development and germination, tissue-specific proteomes, cultivar differences related to quality parameters, analysis of the genetic basis for spot variations and targeted investigations of specific proteins.

Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component.

Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS(+) strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn of R. leguminosarum. This indicates that the surface of A. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

14-3-3 proteins: eukaryotic regulatory proteins with many functions.

The enigmatically named 14-3-3 proteins have been the subject of considerable attention in recent years since they have been implicated in the regulation of diverse physiological processes, in eukaryotes ranging from slime moulds to higher plants. In plants they have roles in the regulation of the plasma membrane H+-ATPase and nitrate reductase, among others. Regulation of target proteins is achieved through binding of 14-3-3 to short, often phosphorylated motifs in the target, resulting either in its activation (e.g. H+-ATPase), inactivation (e.g. nitrate reductase) or translocation (although this function of 14-3-3 proteins has yet to be demonstrated in plants). The native 14-3-3 proteins are homo- or heterodimers and, as each monomer has a binding site, a dimer can potentially bind two targets, promoting their association. Alternatively, target proteins may have more than one 14-3-3-binding site. In this mini review, we present a synthesis of recent results from plant 14-3-3 research and, with reference to known 14-3-3-binding motifs, suggest further subjects for research.

Characterization of Rhizobium leguminosarum exopolysaccharide glycanases that are secreted via a type I exporter and have a novel heptapeptide repeat motif.

The prsDE genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from Rhizobium leguminosarum bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (plyA and plyB) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a prsD (protein secretion) mutant. The plyA gene is upstream of prsD but appears to be expressed at a very low level (if at all) in cultured bacteria. A plyB::Tn5 mutant has a very large reduction in degradation of EPS and CMC. Cultures of plyB mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a plyA plyB double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca2+-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS.

The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification.

NodO is a secreted protein from Rhizobium leguminosarum bv. viciae with a role in signalling during legume nodulation. A Tn5-induced mutant was identified that was defective in NodO secretion. As predicted, the secretion defect decreased pea and vetch nodulation but only when the nodE gene was also mutated. This confirms earlier observations that NodO plays a particularly important role in nodulation when Nod factors carrying C18:1 (but not C18:4) acyl groups are the primary signalling molecules. In addition to NodO secretion and nodulation, the secretion mutant had a number of other characteristics. Several additional proteins including at least three Ca2+-binding proteins were not secreted by the mutant and this is thought to have caused the pleiotropic phenotype. The nodules formed by the secretion mutant were unable to fix nitrogen efficiently; this was not due to a defect in invasion because the nodule structures appeared normal and nodule cells contained many bacteroids. The mutant formed sticky colonies and viscous liquid cultures; analysis of the acidic exopolysaccharide revealed a decrease in the ratio of reducing sugars to total sugar content, indicating a longer chain length. The use of a plate assay showed that the mutant was defective in an extracellular glycanase activity. DNA sequencing identified the prsDE genes, which are homologous to genes encoding protease export systems in Erwinia chrysanthemi and Pseudomonas aeruginosa. An endoglycanase (Egl) from Azorhizobium caulinodans may be secreted from R. leguminosarum bv. viciae in a prsD-dependent manner. We conclude that the prsDE genes encode a Type I secretion complex that is required for the secretion of NodO, a glycanase and probably a number of other proteins, at least one of which is necessary for symbiotic nitrogen fixation.