Characterisation of rice anther proteins expressed at the young microspore stage

In combination with two-dimensional polyacrylamide gel electrophoresis (2-DE) protein mapping and mass spectrometry analysis, the pattern of gene expression in specific tissues at a specific stage can be displayed and characterised. We used this approach for rice (Oryza sativa L. cultivar Doongara) to display and assign identity to proteins in the anthers at the young microspore stage. Over 4000 anther proteins in the pI range of 4-11 and molecular mass range of 6-122 kDa were reproducibly resolved after silver staining, representing about 10% of the estimated total genomic output of rice. Two hundred and seventy-three protein spots have been extracted either from polyninylidene diffluoride membrane blots or from colloidal Coomassie blue stained 2-DE gels and analysed by N-terminal sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS) analysis or tandem MS sequencing. This enabled identification of 53 anther protein spots representing 43 different proteins. Using the publicly available rice expressed sequence tag (EST) database at the National Centre for Biotechnology Information, a further 37 protein spots were matched to ESTs. After BLAST searching with these ESTs, we were able to predict the identity of 22 of these protein spots. Proteome reference maps of rice anthers have been constructed according to the SWISS-2DPAGE standards and are available for public access at http://semele.anu.edu.au/2d/2d.html.     

Flavonoids:New Roles for Old Molecules

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions including defense, UV protection, auxin transport inhibition, allelopathy, and flower coloring. Interestingly, these compounds also have considerable biological activity in plant, animal and bacterial systems – such broad activity is accomplished by few compounds. Yet, for all the research over the last three decades, many of the cellular targets of these secondary metabolites are unknown. The many mutants available in model plant species such as Arabidopsis thaliana and Medicago truncatula are enabling the intricacies of the physiology of these compounds to be deduced. In the present review, we cover recent advances in flavonoid research, discuss deficiencies in our understanding of the physiological processes, and suggest approaches to identify the cellular targets of flavonoids.     

microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root development. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5′ RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/target relationships for miR397 and miR160 to be conserved in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species.     

Effect of early cold stress on the maturation of rice anthers

Male reproductive development in rice (Oryza sativa Linnaeus is very sensitive to various forms of environmental stresses including low temperature. Here, we present our findings on the proteomic analysis of the later developmental consequences of low temperature treatment on rice anthers. Anther proteins at the trinucleate stage, with or without cold treatment for four days at 12 degrees C at the young microspore stage, were extracted, separated by two-dimensional gel electrophoresis (2-DE) and compared. More than 3000 rice anther proteins of cold-sensitive cultivar Doongara plants at the trinucleate stage were resolved on 2-DE gels over a pH range of 4-7 and detected by silver-staining. Seventy protein spots were differentially displayed after four days of cold treatment at the young microspore stage. Of these, 12 protein spots were newly-induced, 47 were up-regulated, and 11 were down-regulated by cold treatment at the early microspore stage. We identified 18 by matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI-TOF) analysis. Of the identified proteins, seven were observed as breakdown (cleavage) products by a combination of 2-DE and MALDI-TOF analysis, thus demonstrating for the first time that cold temperature stress at the young microspore stage enhances and induces partial degradation of proteins in the rice anthers at the trinucleate stage.     

Flavonoids:New Roles for Old Molecules

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions including defense, UV protection, auxin transport inhibition, allelopathy, and flower coloring. Interestingly, these compounds also have considerable biological activity in plant, animal and bacterial systems-such broad activity is accomplished by few compounds. Yet, for all the research over the last three decades, many of the cellular targets of these secondary metabolites are unknown. The many mutants available in model plant ...     

Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting

We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing. While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.     

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.