Nuclear bodies domain changes with microspore reprogramming to embryogenesis.

We analysed the presence of nuclear bodies and particularly Cajal bodies during representative stages of gametophytic and haploid embryogenic development in isolated microspore and anther cultures of a model system (Brassica napus cv. Topas) and a recalcitrant species (Capsicum annuum L. var. Yolo Wonder B). The nuclear bodies domain is involved on several important roles on nuclear metabolism, and Cajal bodies are specifically involved on the storage and maturation of both snRNPs and snoRNPs, as well as other splicing factors, necessary for mRNA and pre-rRNA processing, but not directly on the transcription. In this study, immunofluorescence and immunogold labelling with anti-trimethylguanosine antibodies against the specific cap of snRNAs, ultrastructural and cytochemical analysis were performed on cryoprocessed samples at confocal and electron microscopy respectively. Results showed that Cajal bodies increase during the early stages of microspore embryogenic development (young pro-embryos), compared to microspore and pollen development. Our results suggest that Cajal bodies may have a role in the transcriptionally active, proliferative stages that characterise early microspore embryogenic development.

New in situ approaches to study the induction of pollen embryogenesis in Capsicum annuum L.

The induction of pollen embryogenesis in Capsicum annuum L. has been studied at the cellular level using various in situ approaches with several molecular probes for DNA, RNA and proteins. The late vacuolated microspore and the young bicellular pollen grain are stages of gametophytic development in which embryogenesis can be induced. Our results show that the late vacuolated microspore stage is most responsive to embryogenesis induction. The proliferating cell nuclear antigen (PCNA) has been immunolocalized at the electron microscopy level, in order to map replication sites in relation to the fine structure of chromatin. It shows different patterns of labelling at both developmental stages studied, revealing that the late vacuolated microspore is in a period of replication. Other in situ studies have been performed to characterize the state of nuclear activity at the specific developmental stages in which the embryogenic induction can occur. The modern in situ terminal-deoxy-nucleotidyl transferase (TdT) reaction for DNA, the immunolocalization of various nuclear antigens (as snRNPs, fibrillarin, RNA) and the ultrastructural in situ hybridization using 18S and 25S ribosomal probes provided valuable data bout the specific features displayed by the functional nuclear compartments of the microspore, and the young vegetative and generative cells. They are related not only to the state of gene activity but also with probably the ability to switch to the sporophytic pathway at specific developmental times of their gametophytic program.

The immunolocalization of nuclear antigens during the pollen developmental program and the induction of pollen embryogenesis.

The immunolocalization of nuclear antigens, combined with cytochemical procedures as well as in situ hybridization and recent in situ molecular methods, has been applied at different steps of pollen development to characterize the functional organization of the nucleus during the formation of the male gametophyte in an agronomically interesting plant, Capsicum annuum L. Pollen embryogenesis has been induced in pepper and the first stages of the process have been studied at the cellular level. Low temperature processing methods including cryosections and Lowicryl sections were very convenient for performing the various in situ techniques used in the pollen grains. Different molecular probes for localizing DNA, RNA, snRNPs, specific nucleolar proteins, various rRNA species, and DNA/RNA hybrids provided positive results in the pollen nuclei. The data obtained, and the changes observed in the organization of the nuclear compartments during pollen development, are related to the variations in gene activity undergone by the male gametophyte. The methodology used is proposed as a very convenient approach to localize molecules and events involved in the nuclear function in both gametophytic and sporophytic pollen development.