Genes driving potato tuber initiation and growth: identification based on transcriptional changes using the POCI array

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database (http://pgrc.ipk-gatersleben.de/poci).

Analysis of tyrosine phosphorylation and phosphotyrosine-binding proteins in germinating seeds from Scots pine

Protein tyrosine phosphorylation in angiosperms has been implicated in various physiological processes, including seed development and germination. In conifers, the role of tyrosine phosphorylation and the mechanisms of its regulation are yet to be investigated. In this study, we examined the profile of protein tyrosine phosphorylation in Scots pine seeds at different stages of germination. We detected extensive protein tyrosine phosphorylation in extracts from Scots pine (Pinus sylvestris L.) dormant seeds. In addition, the pattern of tyrosine phosphorylation was found to change significantly during seed germination, especially at earlier stages of post-imbibition which coincides with the initiation of cell division, and during the period of intensive elongation of hypocotyls. To better understand the molecular mechanisms of phosphotyrosine signaling, we employed affinity purification and mass spectrometry for the identification of pTyr-binding proteins from the extracts of Scots pine seedlings. Using this approach, we purified two proteins of 10 and 43 kDa, which interacted specifically with pTyr-Sepharose and were identified by mass spectrometry as P. sylvestris defensin 1 (PsDef1) and aldose 1-epimerase (EC:5.1.3.3), respectively. Additionally, we demonstrated that both endogenous and recombinant PsDef1 specifically interact with pTyr-Sepharose, but not Tyr-beads. As the affinity purification approach did not reveal the presence of proteins with known pTyr binding domains (SH2, PTB and C2), we suggest that plants may have evolved a different mode of pTyr recognition, which yet remains to be uncovered.

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.