PubMed Health⌕ Search

Biomedical subjects

V E Franklin-Tong

Publications and source records attributed to V E Franklin-Tong.

13 recordsLinked to original sources
PubMed2004

The self-incompatibility response in Papaver rhoeas pollen causes early and striking alterations to organelles.

Self-incompatibility (SI) in Papaver rhoeas is accompanied by a cascade of signalling events that result in the rapid arrest and eventual death of the pollen tube. We have used rapid freeze fixation, freeze substitution and transmission electron microscopy to provide the first description of changes to pollen at the ultrastructural level during SI in this species. Our studies reveal that dramatic alterations to the morphology of mitochondria, Golgi bodies and ER occur within 1 h of SI induction. Similar symptoms have also been observed during programmed cell death (PCD) in some cell types. These include: the conspicuous condensation of the vegetative and generative nuclei, the swelling and loss of cristae in mitochondria and the disappearance of Golgi bodies. Some of the early alterations to the mitochondria and Golgi bodies observed at 1 h, almost certainly occur when cells are still alive. Other events, such as nuclear condensation, occur later and coincide with DNA fragmentation and the loss of cell viability. Our observations suggest that the SI response in P. rhoeas pollen may potentially involve a type of PCD.

Apoptosis↗
PubMed2003

The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas.

The integration of signals received by a cell, and their transduction to targets, is essential for all cellular responses. The cytoskeleton has been identified as a major target of signalling cascades in both animal and plant cells. Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in the inhibition of incompatible pollen. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. It has been demonstrated recently that SI induces dramatic alterations in the organization of the pollen actin cytoskeleton. This implicates the actin cytoskeleton as a key target for the SI-stimulated signals. The cytological alterations to the actin cytoskeleton that are triggered in response to SI are described here and there seem to be several stages that are distinguishable temporally. Evidence was obtained that F-actin depolymerization is also stimulated. The current understanding that the actin cytoskeleton is a target for the signals triggered by the SI response is discussed. It is suggested that these F-actin alterations may be Ca(2+)-mediated and that this could be a mechanism whereby SI-induced tip growth inhibition is achieved. The potential for actin-binding proteins to act as key mediators of this response is discussed and the mechanisms that may be responsible for effecting these changes are described. In particular, the parallels between sustained actin rearrangements during SI and in apoptosis of animal cells are considered.

Actins↗
PubMed2003

Genomic organization of the Papaver rhoeas self-incompatibility S(1) locus.

The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S(1) locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S(1) gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.

Amino Acid Sequence↗
PubMed2000

Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas.

Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen.

Calcium Signaling↗
PubMed2000

Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas.

Self-incompatibility (SI) is a genetically controlled process used to prevent self-pollination. In Papaver rhoeas, the induction of SI is triggered by a Ca(2)+-dependent signaling pathway that results in the rapid and S allele-specific inhibition of pollen tube tip growth. Tip growth of cells is dependent on a functioning actin cytoskeleton. We have investigated the effect of self-incompatibility (S) proteins on the actin cytoskeleton in poppy pollen tubes. Here, we report that the actin cytoskeleton of incompatible pollen tubes is rapidly and dramatically rearranged during the SI response, not only in our in vitro SI system but also in vivo. We demonstrate that nonspecific inhibition of growth does not result in similar actin rearrangements. Because the SI-induced alterations are not observed if growth stops, this clearly demonstrates that these alterations are triggered by the SI signaling cascade rather than merely resulting from the consequent inhibition of growth. We establish a detailed time course of events and discuss the mechanisms that might be involved. Our data strongly implicate a role for the actin cytoskeleton as a target for signaling pathways involved in the SI response of P. rhoeas.

Actins↗
PubMed1999

Signaling in pollination.

The past year has seen considerable advances in our understanding of signaling in pollen tubes. Evidence suggesting that lipids are involved in pollen tube guidance has opened up new avenues. Major advances have been made in understanding the roles of Rho-like GTPases and protein kinases in regulating pollen tube growth. Light is being shed on how signals may be integrated. It is becoming clear that the role of Ca(2+) in pollen tube growth is perhaps more complex than originally anticipated.

Plant Cells↗
PubMed1999

S-protein mutants indicate a functional role for SBP in the self-incompatibility reaction of Papaver rhoeas.

The self-incompatibility response involves S-allele specific recognition between stigmatic S proteins and incompatible pollen, resulting in S-specific pollen inhibition. In Papaver rhoeas, the pollen S gene product is predicted to be a receptor that interacts with the stigmatic S protein in an S specific manner. We recently identified an S protein binding protein (SBP) in pollen that binds stigmatic S proteins, although apparently not in an S-allele-specific manner. In order to investigate the functional significance of the interaction between S proteins and SBP, we constructed mutant derivatives of the S1 protein and tested their SBP-binding activity and their biological activity. Here we present an evaluation of nine mutant derivatives of the S1 protein. Western ligand blotting was used to show that mutations to amino acid residues in predicted loops 2 and 6 of the S1 protein cause significant reductions in their SBP-binding activity. These same mutants show a concomitant reduction in their ability to inhibit incompatible pollen. This establishes a direct link between SBP binding and inhibition of incompatible pollen and implicates SBP as a pollen component playing a key role in the self-incompatibility reaction. We discuss the possible nature of the contribution of SBP in the S-specific rejection of incompatible pollen.

Alleles↗
PubMed1998

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction.

Alleles↗
PubMed1998

A potential signaling role for profilin in pollen of Papaver rhoeas.

Regulation of pollen tube growth is known to involve alterations in intracellular calcium levels and phosphoinositide signaling, although the mechanisms involved are unclear. However, it appears likely that pollination events involve a complex interplay between signaling pathways and components of the actin cytoskeleton in pollen. In many eukaryotic cells, actin binding proteins function as stimulus-response modulators, translating signals into alterations in the cytoplasmic architecture. In this study, we examined whether profilin, which is a member of this class of signaling intermediate, might play a similar role in pollen. We have analyzed the functional properties of native profilin from pollen of Papaver rhoeas and have investigated the effects of profilin on the phosphorylation of pollen proteins in vitro by adding a slight excess of profilin to cytosolic pollen extracts. We present clear evidence that profilin interacts with soluble pollen components, resulting in dramatic alterations in the phosphorylation of several proteins. We also show, albeit in vitro, the involvement of profilin in modulating the activity of a signaling component(s) affecting protein phosphorylation. Our data, which suggest that pollen profilin can regulate actin-based cytoskeletal protein assembly and protein kinase or phosphatase activity, indicate a possible role for the involvement of profilin in signaling pathways that may regulate pollen tube growth.

Actins↗
PubMed1997

Ca2+ oscillations in plant cells: initiation by rapid elevation in cytosolic free Ca2+ levels.

Temporal increases in intracellular [Ca2+] are now recognized to be key triggers for a wide range of important physiological events in eukaryotic cells. In mammalian cells, signal-induced Ca2+-elevations have been found to be of a pulsatile nature and Ca2+ spikes display a high degree of spatiotemporal complexity. In plant cells a similar picture is beginning to emerge. To investigate the occurrence of pulsatile Ca2+ signals in plant cells we studied alterations of [Ca2+] in the tip region of pollen tubes from poppy (Papaver rhoeas). Time-Resolved Laser Scanning Confocal Microscopy of pollen tubes microinjected with the Dextran-linked Ca2+-indicator dyes Calcium Green or Indo-1 revealed that highly regular Ca2+ oscillations occur in these cells. We further demonstrate that artificial elevation of cytosolic Ca2+ by photolysis of caged-Ca2+ (Nitr-5) can trigger the onset of oscillations.

Calcium↗
PubMed1996

Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations.

The S3 allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S1 allele, preceded by an 18 amino acid signal sequence. Expression of the S3 coding region in Escherichia coli produced a form of the protein, denoted S3e, which specifically inhibited S3 pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S1 protein. In contrast to other S proteins identified to date, S3 protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of Western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S3 allele with antiserum raised to S3e, revealed a protein (S3s) which was indistinguishable in pI and Mr from that in the Shirley population. A cDNA encoding S3s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.

Alleles↗
PubMed1994

Cloning and expression of a distinctive class of self-incompatibility (S) gene from Papaver rhoeas L.

We present the identification, cloning, and characterization of a self-incompatibility (S) gene from Papaver rhoeas that has no significant homology to any previously reported gene sequences, including S genes from other species. This result suggests that a different self-incompatibility mechanism may be operating in this species and has important implications for the evolutionary relationships between the S genes. The S1 cDNA was cloned by using an oligonucleotide based upon N-terminal amino acid sequence data from stigmatic proteins that show complete linkage with the S1 gene. The single-copy gene has been expressed in Escherichia coli to test biological activity. Although the recombinant S1 protein (S1e) is not processed in the same way as the protein produced in the plant, it exhibits, in vitro, the specific pollen inhibitory activity expected of an S gene product; pollen carrying the S1 allele is inhibited, whereas pollen not carrying S1 is not inhibited. These results provide definitive demonstration that the product of a cloned S gene has S-specific pollen inhibitory activity.

Alleles↗