The intracellular events triggered by the self-incompatibility response inPapaver rhoeas

Summary In recent years self-incompatibility (SI) has come to be recognised as an important model system for studying cell-cell interactions and signalling in flowering plants. In this article we discuss the intracellular events associated with the SI response in the field poppy,Papaver rhoeas. The SI response inP. rhoeas is known to involve a Ca²⁺-based signalling pathway, activated following molecular interactions on the surface of incompatible pollen tubes. Evidence demonstrates that, following a transient increase in the concentration of cytosolic free Ca²⁺ ([Ca²⁺];) initiated by the SI response, phosphorylation of certain cytosolic proteins occurs, followed by activation of pollen gene expression. The magnitude of this transient Ca²⁺ wave and the localisation of cytosolic [Ca²⁺]i following the SI response are discussed. We also describe the character of the proteins specifically phosphorylated in the SI response and the nature of the protein kinases involved in their phosphorylation. Finally, we consider the possibility that the end result of the SI response inP. rhoeas may be analogous to programmed-cell-death mechanisms such as those seen in developmental processes and defence responses in various plant cells.     

Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis

Locus At5g03555 encodes a nucleobase cation symporter 1 (AtNCS1) in the Arabidopsis genome. Arabidopsis insertion mutants, AtNcs1-1 and AtNcs1-3, were used for in planta toxic nucleobase analog growth studies and radio-labeled nucleobase uptake assays to characterize solute transport specificities. These results correlate with similar growth and uptake studies of AtNCS1 expressed in Saccharomyces cerevisiae. Both in planta and heterologous expression studies in yeast revealed a unique solute transport profile for AtNCS1 in moving adenine, guanine and uracil. This is in stark contrast to the canonical transport profiles determined for the well-characterized S. cerevisiae NCS1 proteins FUR4 (uracil transport) or FCY2 (adenine, guanine, and cytosine transport).     

A study of functionally active amino acids involved in the interaction of HLA-A2 or HLA-A3 molecules with cytolytic T lymphocytes

A large series of HLA-A2/HLA-A3 recombinant genes were generated by using the in vivo recombination technique. These genes have each been modified in the last two-thirds of the third exon such that one or several HLA-A2-specific substitutions have been made in the HLA-A3 gene and vice versa. The recombinant genes were transfected into the murine cell line P815 and the transfectants were used as targets for a series of 20 human CTL lines or clones specific for HLA-A2 or HLA-A3, or restricted by HLA-A2 and specific for influenza A. Several patterns of anti-HLA-A2, anti-HLA-A3, and HLA-A2-restricted anti-influenza CTL activity were observed and when uncloned cell lines were studied, a progressive selection of some clones with a similar pattern of activity was regularly found. From the comparison of these different patterns the following conclusions can be drawn: 1) In most but not all cases both domains of the class I molecule were essential for CTL recognition, but residue 152 was critically important for the majority of CTL tested; 2) amino acids 114/116 were also critical in most cases, and their position close to amino acid 152 in the tertiary structure of the molecule may have some functional significance; and 3) amino acid 161, although highly conserved, plays an unexpected but very important role in CTL function.     

Biosynthesis of calystegines: 15N NMR and kinetics of formation in root cultures of Calystegia sepium

Calystegines are nortropane alkaloids bearing between three and five hydroxyl groups in various positions. [15N]Tropinone was administered to root cultures of Calystegia sepium and the incorporation into calystegines was followed. Increase of label in calystegines was measured by one-dimensional 15N NMR and inverse-detected 2D NMR techniques. The results show that tropinone and pseudotropine are metabolites in the biosynthetic pathway of calystegines. The velocity of calystegine accumulation was followed kinetically by transfer of root cultures from 15N-enriched medium to 14N-medium and analysis by GC-MS. A constant calystegine formation with no interference by excretion or degradation was observed. A biosynthetic rate for individual calystegines at each time point was calculated, the maximum was 0.4 mg/day/g of biomass. This allowed the velocity of individual biosynthetic steps to be estimated.