Comparison of polypeptide expression in anthers from gametophytic sterile-2 and wild type maize by 2D-electrophoresis.

Pollen development is of great interest for fundamental research in cell biology, as well as for plant breeding programs and genetic manipulation of haploid cell lines. The present research has been focused on the isolation and characterization of pollen-specific genes which mutations may lead to pollen development defects . Gametophytic male sterile-2 (gaMS-2 ), is a maize mutant obtained by transposon insertion mutagenesis. It is characterized by lack of differentiation between the vegetative and the generative nuclei in pollen grains, which leads to various nuclear abnormalities and to approximately 50% of mature fertile pollen. Although the cytological events characterizing sterile pollen grains are well described, little is known about the biochemical and molecular processes at the basis of the mutant phenotype. Here, we describe a comparative study of anther proteins of gaMS-2 and wild type (wt) by 2-D electrophoresis, carried out in order to enlighten proteins selectively expressed between the two pollen types. Protein extracts from anthers for 2-D electrophoresis were prepared by use of liquid phenol (85%), followed by precipitation with 0.1 M ammonium acetate in methanol. Phenol extraction can minimize protein degradation that may occur during sample preparation. Our results revealed that in 2-D gels, approximately 300 protein spots could be resolved efficiently after Commassie blue staining. These spots range from 10 to 116 kDa (pI 3-10) and the majority of them are identical in the mutant and wt. The difference in anther proteins appears to be limited to a few spots. In particular in the wt anthers, a protein having a MW larger than 31 kDa (pI 10), is expressed at high levels and it seems to be absent in the mutant. In addition, even some homologue proteins of the mutant and wt exhibit differential levels of expression. Apparently, no visible differences in the proteomic patterns were found in the anthers of either wt or the mutant, at different developmental stages. The herein described anther-specific/-enriched proteins of wild type, deserve further investigation. It will be crucial to determine if some of these proteins are involved in eliciting the genetic mechanisms regulating pollen development. Protein micro-sequencing is under way and subsequently the expression analysis will be carried out.