Gender-specific selection on codon usage in plant genomes

<p>Abstract</p> <p>Background</p> <p>Currently, there is little data available regarding the role of gender-specific gene expression on synonymous codon usage (translational selection) in most organisms, and particularly plants. Using gender-specific EST libraries (with > 4000 ESTs) from <it>Zea mays </it>and <it>Triticum aestivum</it>, we assessed whether gender-specific gene expression <it>per se </it>and gender-specific gene expression level are associated with selection on codon usage.</p> <p>Results</p> <p>We found clear evidence of a greater bias in codon usage for genes expressed in female than in male organs and gametes, based on the variation in GC content at third codon positions and the frequency of species-preferred codons. This finding holds true for both highly and for lowly expressed genes. In addition, we found that highly expressed genes have greater codon bias than lowly expressed genes for both female- and male-specific genes. Moreover, in both species, genes with female-specific expression show a greater usage of species-specific preferred codons for each of the 18 amino acids having synonymous codons. A supplemental analysis of <it>Brassica napus </it>suggests that bias in codon usage could also be higher in genes expressed in male gametophytic tissues than in heterogeneous (flower) tissues.</p> <p>Conclusion</p> <p>This study reports gender-specific bias in codon usage in plants. The findings reported here, based on the analysis of 1 497 876 codons, are not caused either by differences in the biological functions of the genes or by differences in protein lengths, nor are they likely attributable to mutational bias. The data are best explained by gender-specific translational selection. Plausible explanations for these findings and the relevance to these and other organisms are discussed.</p

Isolation of an embryogenic line from non-embryogenic Brassica napus cv. Westar through microspore embryogenesis

Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7 d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10 642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered

Transcript Profiling Provides Evidence of Functional Divergence and Expression Networks among Ribosomal Protein Gene Paralogs in Brassica napus[W][OA]

The plant ribosome is composed of 80 distinct ribosomal (r)-proteins. In Arabidopsis thaliana, each r-protein is encoded by two or more highly similar paralogous genes, although only one copy of each r-protein is incorporated into the ribosome. Brassica napus is especially suited to the comparative study of r-protein gene paralogs due to its documented history of genome duplication as well as the recent availability of large EST data sets. We have identified 996 putative r-protein genes spanning 79 distinct r-proteins in B. napus using EST data from 16 tissue collections. A total of 23,408 tissue-specific r-protein ESTs are associated with this gene set. Comparative analysis of the transcript levels for these unigenes reveals that a large fraction of r-protein genes are differentially expressed and that the number of paralogs expressed for each r-protein varies extensively with tissue type in B. napus. In addition, in many cases the paralogous genes for a specific r-protein are not transcribed in concert and have highly contrasting expression patterns among tissues. Thus, each tissue examined has a novel r-protein transcript population. Furthermore, hierarchical clustering reveals that particular paralogs for nonhomologous r-protein genes cluster together, suggesting that r-protein paralog combinations are associated with specific tissues in B. napus and, thus, may contribute to tissue differentiation and/or specialization. Altogether, the data suggest that duplicated r-protein genes undergo functional divergence into highly specialized paralogs and coexpression networks and that, similar to recent reports for yeast, these are likely actively involved in differentiation, development, and/or tissue-specific processes

Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry

Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods due to the relatively low abundance of these proteins, and in plants this is further complicated by the high abundance of Rubisco protein in green tissues. We present a novel method for plant phosphoproteome analysis that firstly depletes Rubisco by polyethylene glycol fractionation, and then utilizes immobilized metal-ion affinity chromatography to enrich for phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO2 fixation, protein assembly and folding, stress response, redox regulation and cellular metabolism. Both the large and small subunits of Rubisco were found to be phosphorylated at multiple sites, while the depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discoveries of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal phosphoproteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of signal transduction during growth and development in plants.Peer reviewed: YesNRC publication: Ye

Enrichment and analysis of intact phosphoproteins in arabidopsis seedlings

Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P3DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.Peer reviewed: YesNRC publication: Ye