Comparative proteomic analysis of pollen of Trifolium pratense, T-alexandrinum and T-repens

Clovers are widely distributed, but their pollen proteome still has not been completely elucidated. In this study we performed a comprehensive comparative proteomic analysis of red, berseem and white clover pollen. Hand-collected pollen of different clover species were used for total protein extraction. Proteins isolated from red clover cvs. 'Kirsiniai' and 'Vyciai', berseem clover cv. 'Faraon' and white clover cv. 'Medunai, populations Nos. 2295 4n and 2196 4n, were subjected to fractionation by one-dimensional electrophoresis. Some quantitatively different protein groups were characterized in protein maps typical of analyzed clover pollens. For the detailed proteomic analysis we chose pollen of red clover cv. Vyciai, berseem clover cv. 'Faraon' and white clover cv. 'Medunai'. Proteins isolated from the pollen were fractionated by two-dimensional electrophoresis (2DE) and stained for visualisation by using Coomassie blue. Each of the 2DE images indicated over 200 protein spots. Computational methods developed by us were applied for characterization and comparison of proteins isolated from the pollen of the three clover species. The computational methods enabled the evaluation of protein expression variations in red, berseem and white clover pollen proteome maps. By using computer-assisted image analysis of the gels, the expression levels of the proteins were evaluated and their molecular weight and isoelectric point were precisely characterized. We detected over 30 protein spots whose quantitative levels were most divergent in investigated clover pollen proteome map. They were analyzed by mass spectrometry and identified. The fold changes of identified proteins representing red clover cv. 'Vyciai, berseem clover cv. 'Faraon' and white clover cv. 'Medunai' were calculated using computer-assisted methods.