In situ molecular identification of the Ntf4 MAPK expression sites in maturing and germinating pollen

Background information. MAPKs (mitogen-activated protein kinases) are involved in the transduction of different signals in eukaryotes. They regulate different processes, such as differentiation, proliferation and stress response. MAPKs act through the phosphorylation cascade, being the last element that phosphorylates the final effector of the cell response. They are activated when their threonine and tyrosine residues are phosphorylated. Ntf4, a MAPK with a molecular mass of 45 kDa, has been reported to be expressed in pollen and seeds. Biochemical studies have indicated that the expression and the activation of Ntf4 is regulated during pollen maturation, although an increase of the activation is observed when the pollen is hydrated, just at the beginning of the germination. However, nothing is known about its subcellular localization. Results. In the present study, the in situ expression and subcellular localization of Ntf4 have been analysed during the tobacco pollen developmental pathway. Cryosections, freeze-substitution and cryo-embedding in Lowicryl K4M were used as processing techniques for subsequent immunofluorescence, immunogold labelling and in situ hybridization assays. During pollen maturation, Ntf4 showed an increase in expression, as demonstrated by in situ hybridization, and specific subcellular distributions. We found that the protein was expressed from mid bicellular pollen stage until the pollen was mature. In germinating pollen, the protein increased after the initiation of germination. Translocation of the protein to the nucleus was found at specific stages; the presence of Ntf4 in the nucleus was found in the last stage of the pollen maturation and in germinating pollen. Double immunofluorescence and immunogold labelling with anti-Ntf4 (AbC4) and anti-P-MAPK (phosphorylated MAPK) antibodies revealed the co-localization of both epitopes in the nucleus at late developmental stages. Conclusions. The temporal and spatial pattern of the expression sites of Ntf4 has been characterized during pollen development, indicating that Ntf4 is a 'late gene' that is upregulated during maturation and germination, with a possible role in the gametophytic function. The translocation of the Ntf4 protein from the cytoplasm to the nucleus at late pollen developmental stages, and its co-localization with the P-MAPK epitope in several nuclear sites, indicates a relationship between the Ntf4 nuclear translocation and its active state.