Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development

Here we report for the first time the ultrastructural localization of DNA r eplication sites in the nucleus of plant cells and the timing of replicatio n through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by l abelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin sub-domain in th e interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoe lectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscop y, revealing two peaks of DNA synthesis, at the beginning (early tetrad), a nd the end (late vacuolate), of microspore interphase. High-resolution auto radiography after [H-3]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellul ar pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. Th e use of anti-PCNA antibodies at the ultrastructural level is an easier, fa ster and more feasible method than the detection of in vivo-incorporated nu cleotides, especially in plant systems with long cell cycles. PCNA immunogo ld labelling is, there fore, proposed as an efficient marker for mapping th e sites and timing of replication at the electron microscopy level.