Proteome analysis of male gametophyte development in rice anthers

In this thesis, two-dimensional electrophoresis (2-DE)-based comparative proteomics were applied to monitor the global changes in protein expression during the male gametophyte development in anthers of the Australian rice cultivar Doongara, with the aim of providing a protein-level insight into the molecular mechanism underlying this important reproductive developmental process. At the same time, this thesis also evaluates the potential application of 2-DE based proteomics to other aspects of plant developmental biology. In order to collect sufficient amount of homogenous anther populations which represent a number of discrete cellular events encompassing the process of microsporegenesis, the cytological examination of developing anthers was done to establish the allometric relationships between a number of growth parameters and anther developmental stages. This provided a base for the quick and nondestructive assessment of microspore developmental stages. From this study, a strategy for the collection of rice anthers for six developmental stages was established using a combination of anther length, auricle distance and days before heading. The findings of the cytological analysis opened up the possibility of establishing rice plants as a new model system for male gametophyte research of plants. Anther proteome maps were established for six microspore developmental stages within the pH ranges of 4 to 7 and 6 to 11. Over 3,500 protein spots were reproducibly resolved in the combined pH range of 4 to 11. Comparison of proteome maps of six developmental stages resulted in the detection of 150 differentially displayed protein spots at various stages. Putative identities were predicted for 49 out of 155 protein spots which were subjected to peptide mass fingerprinting (PMF) analysis. Eight low molecular weight protein spots were matched to putative translation products of rice expressed sequence tags (EST) by N-terminal terminal sequencing followed by homology searches. This verified the translation of these small open reading frames (ORF) and revealed the presence of some post translational modifications of these proteins. By integrating the information about the functions of identified proteins and their temporal regulation patterns, three developmentally regulated metabolic pathways Xl were identified and the significance of these pathways in relation to male gametophyte development was discussed. Based on the N-terminal sequencing data, three isoforms of rice homologues of grass group II pollen allergens (Ory s 2) were identified and further characterized using bioinformatics and immunochemical techniques. Polyclonal antibodies were produced against Ory s 2 isoforms using gel-separated proteins as the antigen. Immunoblot analysis revealed that Ory s 2 proteins are pollen specific and accumulated to high abundance at mature pollen, indicating their possible involvement in fertilization process. Immunochemical analysis also showed that rice group II allergens do not possess cross-reactivity with group II allergens of other grasses. This study produced valuable molecular data to provide some insight into the global changes of protein expression accompanying pollen development, and identified some developmentally regulated protein markers which have potential practical application to other research projects. From the promising results of this proteomic study it can be expected that our understanding of complex biological processes in plant development will be enhanced with the availability of a fully annotated rice genome and the application of integrated systems biology research approaches

Determination of Accase Inhibitor Herbicide Resistance of Wild Oats (Avena Spp.) in Wheat Planting Areas in Northern Districts of Kahramanmaraş

DergiPark: 798353tujesBu çalışma, Kahramanmaraş Göksun, Afşin ve Elbistan ilçelerindeki buğday ekim alanlarında Accase inhibitörü herbisitlere karşı dayanıklılık durumunun belirlenmesi amacıyla 2015-2016 yılları arasında yürütülmüştür. Bölgede 2015-2016 yıllarında yapılan surveylerde Avena spp. tohumları 25 farklı ekim alanından ve karşılaştırma amacıyla herbisit uygulanmayan tarla kenarlarından toplanmıştır. Tohumlar paçal yapılarak küvetlere ekilmiş ve dayanıklı (R) ve duyarlı (S) populasyonlarını tespit etmek amacıyla screen tarama testi ile testlenmiştir. 2-5 yapraklı dönemlerinde clodinafop-propargyl’in uygulama dozu olan 20 ml/da 4 tekerrürülü olarak uygulanmış ve 28. günde bitkilerin tamamının canlılıklarını devam ettiremediği gözlemlenmiş olup Clodinafop propargyl’e karşı duyarlı (S) olduğu belirlenmiştir

Comparison of the Effectiveness of Topical and Oral Beta Blockers in the Treatment of Childhood Hemangiomas

Purpose: Hemangiomas are the most common vascular tumors in childhood, and the treatment options have undergone profound changes in recent years. In this study, we aimed to compare the efficacy and safety of beta-blockers on hemangiomas, both topi-cal and oral, with non-pharmacological treatment in the pediatric age group. Material and Methods: We retrospectively reviewed the medical records of pediatric patients with hemangiomas. Results: Fifty-three patients (F/M=40/13) were enrolled in this study. Superficial hemangiomas were detected in 14 (26.4%) patients, and deep hemangiomas were detected in 39 (73.6%) patients. Seventeen patients were followed without medication, 19 were treated with a topical beta blocker, and 17 were treated with an oral beta blocker. Twelve patients with superficial hemangi-omas were followed without medication, while two received topical timolol treatment. A comparison of lesion progression in patients with superficial hemangiomas in the non-pharmacological treatment and topical treatment groups showed that the mean scores of success, in terms of mean fading and reduction in lesion depth, were significantly higher at the first month (7.0 vs. 1.66; p=0.049; 6.0 vs. 1.5; p=0.045). Among patients with deep hemangiomas, a comparison of mean fading scores showed no difference between the oral and topical treatment groups in the first and fourth months (p=0.551, p=0.551). Conclusion: We believe that oral beta-blockers can be used instead of topical treatment in the future, and they will be preferred more by clinicians and families due to less side effects

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°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

Proteome analysis of male gametophyte development in rice anthers

We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, β-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs

Low Temperature Treatment at the Young Microspore Stage Induces Protein Changes in Rice Anthers

Male reproductive development in rice is very sensitive to various forms of environmental stresses including low temperature. A few days of cold treatment (<20 °C) at the young microspore stage induce severe pollen sterility and thus large grain yield r

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 approac

Proteome analysis of male gametophyte development in rice anthers

We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, β-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs