A change of developmental program induces the remodeling of the interchromatin domain during microspore embryogenesis in Brassica napus L.

After a stress treatment, in vitro-cultured pollen changes its normal gametophytic developmental pathway towards embryogenesis producing multicellular embryos from which, finally, haploid and double haploid plants develop. The architecture of the well-organized nuclear functional domains changes in response to DNA replication, RNA transcription, processing and transport dynamics. A number of subnuclear structures present in the interchromatin region (IR, the nuclear domain between chromosome territories) have been shown as involved, either directly or indirectly, in transcriptional regulation. These structures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies (CBs) and perichromatin granules (PGs). In this work, we present a cytochemical, immunocytochemical, quantitative and morphometric analysis at the light, confocal and electron microscopy levels to characterize the changes in the functional architecture of the nuclear interchromatin domain during two developmental programs followed by the microspore: differentiation to mature pollen grains (transcriptionally inactive), and microspore embryogenesis involving proliferation in the first stages (highly engaged in transcription). Our results revealed characteristic changes in size, shape and distribution of the different interchromatin structures as a consequence of the reprogramming of the microspore, allowing us to relate the remodeling of the interchromatin domain to the variations in transcriptional activities during proliferation and differentiation events, and suggesting that RNA-associated structures could be a regulatory mechanism in the process. In addition, we document the presence of two structurally different types of CBs, and of IGC and CB-associated regions, similar to those present in animal cells, and not yet described in plants.

Pathways to doubled haploidy: chromosome doubling during androgenesis.

Production of doubled haploid (DH) plants through androgenesis induction is a promising and convenient alternative to conventional selfing techniques for the generation of pure lines for breeding programs. This process comprises two main steps: induction of androgenesis and duplication of the haploid genome. Such duplication is sometimes indirectly induced by the treatments used to promote androgenic development. But usually, an additional step of direct chromosome doubling must be included in the protocol. Duplication of the haploid genome of androgenic individuals has been thought to occur through three mechanisms: endoreduplication, nuclear fusion and c-mitosis. In this review we will revise and analyze the evidences supporting each of the proposed mechanisms and their relevance during androgenesis induction, embryo/callus development and plant regeneration. Special attention will be devoted to nuclear fusion, whose evidences are accumulating in the last years.

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.

Differentiating plant cells switched to proliferation remodel the functional organization of nuclear domains.

The immature pollen grain, the microspore, under stress conditions can switch its developmental program towards proliferation and embryogenesis. The comparison between the gametophytic and sporophytic pathways followed by the microspore permitted us to analyse the nuclear changes in plant differentiating cells when switched to proliferation. The nucleus is highly dynamic, the architecture of its well organised functional domains--condensed chromatin, interchromatin region, nuclear bodies and nucleolus--changing in response to DNA replication, RNA transcription, processing and transport. In the present work, the rearrangements of the nuclear domains during the switch to proliferation have been determined by in situ molecular identification methods for the subcellular localization of chromatin at different functional states, rDNA, elements of the nuclear machinery (PCNA, splicing factors), signalling and stress proteins. The study of the changes in the nuclear domains was determined by a correlative approach at confocal and electron microscopy levels. The results showed that the switch of the developmental program and the activation of the proliferative activity affected the functional organization of the nuclear domains, which accordingly changed their architecture and functional state. A redistribution of components, among them various signalling molecules which targeted structures within the interchromatin region upon translocation from the cytoplasm, was also observed.

Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L.

A stress treatment of 32 degrees C for at least 8h was able to change the gametophytic program of the microspore, switching it to embryogenesis in Brassica napus, an interesting model for studying this process in vitro. After induction, some microspores started symmetric divisions and became haploid embryos after a few days, whereas other microspores, not sensitive to induction, followed their original gametophytic development. In this work the distribution and ultrastructural localization of two heat-shock proteins (Hsp70 and Hsp90) throughout key stages before and after embryogenesis induction were studied. Both Hsp proteins are rapidly induced, localizing in the nucleus and the cytoplasm. Immunogold labeling showed changes in the distribution patterns of these proteins, these changes being assessed by a quantitative analysis. Inside the nucleus, Hsp70 was found in association with RNP structures in the interchromatin region and in the nucleolus, whereas nuclear Hsp90 was mostly found in the interchromatin region. For Hsp70, the accumulation after the inductive treatment was accompanied by a reversible translocation from the cytoplasm to the nucleus, in both induced (embryogenic) and noninduced (gametophytic) microspores. However, the translocation was higher in embryogenic microspores, suggesting a possible additional role for Hsp70 in the switch to embryogenesis. In contrast, Hsp90 increase was similar in all microspores, occurring faster than for Hsp70 and suggesting a more specific role for Hsp90 in the stress response. Hsp70 and Hsp90 colocalized in clusters in the cytoplasm and the nucleus, but not in the nucleolus. Results indicated that stress proteins are involved in the process of microspore embryogenesis induction. The differential appearance and distribution of the two proteins and their association at specific stages have been determined between the two systems coexisting in the same culture: embryogenic development (induced cells) and development of gametes (noninduced cells).

The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division.

We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT-PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.