Intracellular localization of integrin-like protein and its roles in osmotic stress-induced abscisic acid biosynthesis in Zea mays

Summary. Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, and this is believed to be a major mechanism through which plants enhance drought tolerance. In this study, we explore the possible mechanisms of osmotic stress perception by plant cells and the consequent induction of ABA biosynthesis. Immunoblotting and immunofluorescence localization experiments, using a polyclonal antibody against human integrin β1, revealed the presence of a protein in Zea mays roots that is similar to the integrin proteins of animals and mainly localized in the plasma membrane. Treatment with GRGDS, a synthetic pentapeptide containing an RGD domain, which interacted specifically with the integrin protein and thus blocked the cell wall–plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, and the GRGDS analog which does not contain the RGD domain had no effect. Our results show that a strong interaction exists between the cell wall and plasma membrane and that this interaction is largely mediated by integrin-like proteins. They also imply that the cell wall and/or cell wall–plasma membrane interaction plays important roles in the perception of osmotic stress. Accordingly, we conclude that the cell wall and/or cell wall–plasma membrane interaction mediated by the integrin-like protein plays important roles in osmotic stress-induced ABA biosynthesis in Zea mays. Correspondence: J. S. Liang, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, People’s Republic of China.     

Extensive accumulation of influenza virus NS1 protein in the nuclei causes effective viral growth in vero cells

We previously showed that modified A/Puerto Rico/8/34 (PR8) influenza master strain had improved viral rescue and growth properties in African green monkey kidney (Vero) cell line by introducing NS gene of Vero-adapted A/England/1/53 (vaEng53). In the present study, it was found that the NS1 protein derived from vaEng53 was extensively accumulated in the nuclei than that of PR8. This accumulation was caused by 7 amino acid differences in C-terminal region of NS1 protein. These results suggest that specific accumulation of NS1 protein may contribute to efficient viral replication in Vero cells.     

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.     

Identification of cellular isoform of oviduct-specific glycoprotein: role in oviduct tissue remodeling?

The oviduct is known to secrete mucins (MUC1 and MUC9) under the influence of ovarian steroids. The secreted form of MUC1 binds gametes in the oviduct, whereas the cellular form seen in breast cancers has been implicated in cell adhesion and morphogenesis. The secreted MUC9 or oviduct-specific glycoprotein (OGP), in addition to being a mucin, belongs to family 18 glycosylhydrolases and is known to bind gametes and embryos in the oviduct. Studies in our laboratory have identified non-muscle myosin IIA (involved in cell shape, polarity, and morphogenesis) as the protein partner to OGP in gametes. In view of the crucial role of the cortical cytoskeleton in the selective internalization of tight junctions (TJs) /adherent junctions (AJs) or apical junctional complex (AJC) in simple epithelial cells during tissue remodeling, the present study has been undertaken to evaluate the existence of a cellular form of OGP in oviductal tissue, which itself undergoes cyclic tissue remodeling. In silico analysis of the deduced amino-acid sequence of OGP has revealed the presence of several conserved motifs; these imply that OGP is a component of multi-protein complexes such as TJs. Corroborative immunoelectron-microscopic analysis in peri-ovulatory oviduct epithelia in the bonnet monkey has revealed the presence of OGP at the TJ. Co-localization studies of OGP and cadherin demonstrate that, whereas OGP is localized at the tonofilaments of the TJs, cadherin is localized at the intercellular space of the AJ. The possible role of OGP in oviductal tissue remodeling is discussed in light of the present findings and those reported in the literature. This study was supported by funds from the Indian Council for Medical Research and by senior research fellowships (to K. Kadam) from the Council for Scientific and Industrial Research.