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).     

Messenger-RNA and protein changes associated with induction ofBrassica microspore embryogenesis

Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

A short severe heat shock is required to induce embryogenesis in late bicellular pollen of Brassica napus L.

 Until now it has been considered that in rape seed (Brassica napus) only late uninucleate microspores and early bicellular pollen are competent for induction of in vitro embryogenesis. Here we describe that pollen isolated at the late bicellular stage can also be induced to undergo embryogenesis. By the application of an additional short and more severe heat stress treatment, DNA synthesis was initiated in both generative and vegetative nuclei, but only vegetative cells were able to complete the cell cycle and to divide further. The ability of late bicellular pollen to respond to embryogenic induction treatment was accompanied by rearrangements of the microtubulular cytoskeleton and by the nuclear localization of 70 kDa heat shock proteins (HSP70). These findings confirm earlier observations that there is a strong correlation between the induction of embryogenesis and the synthesis and nuclear localization of HSP70. Received: 9 January 1997 / Revision accepted: 23 May 1997     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

A CYTOLOGICAL ANALYSIS OF MICROSPORES OF TRITICUM AESTIVUM (POACEAE) DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Uninucleate microspores of Triticum aestivum cv. Pavon can be induced in vitro to alter their development to produce embryoids rather than pollen. Microspores expressed their embryogenic capacity through one of two division pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen grain. Here the generative cell detached from the intine, migrated to a central position in the pollen grain, and underwent a second haploid mitosis as the vegetative cell divided to give rise to the embryoid. In the second pathway, the first division was symmetric and both nuclei divided repeatedly to form the embryoid. This comparative analysis of normal pollen ontogeny and induced embryogenesis provided no evidence for the existence of predetermined embryogenic microspores in vitro or in vivo. Instead, microspores are induced at the time of culture, and embryogenesis involves continued metabolic activity associated with the gradual cessation of the gametophytic pathway and a redifferentiation into the sporophytic pathway. In conjunction with a previous study, it appears that embryogenic induction of wheat microspores involves switching off gametophytic genes and derepressing sporophytic genes.     

Induction of embryogenesis with colchicine instead of heat in microspores ofBrassica napus L. cv. Topas

Prior to this report, heat treatment (32.5°C, 24 h) was the method used to induce embryogenesis fromBrassica napus microspores. Continuous culture at 25°C results in pollen development. This study shows that colchicine alone, at the non-inductive temperature of 25°C, can induce embryogenesis, thus demonstrating that heat shock is not required for embryogenic induction inB. napus cv. Topas. Embryogenic frequencies of over 15% were obtained by culturing isolated microspores with 25 M colchicine for 42 h at 25°C. The microspore developmental stages responsive to colchicine were unicellular vacuolate and late unicellular, somewhat earlier stages than the population responsive to heat induction. Other groups have reported that heat-shock proteins are essential to the induction of embryogenesis. The present study offers a method of embryogenic induction without the use of heat which will allow discrimination between the factors associated with response to heat shock and those involved with changing cell development.Abbreviations LU Late-unicellular - PPB Preprophase band - UV unicellular-vacuolate The authors wish to thank C. Bornman for his interest and encouragement. We gratefully acknowledge support from the School of Graduate Studies and Research, Queen's University to J.-P. Z., from Hilleshog AB, Sweden to D.H.S., and from the Natural Sciences and Engineering Research Council of Canada to D.H.S. and W.N. Plant Research Centre contribution No. 1595.     

Cell cycle dependent distribution of phosphorylated proteins in microspores and pollen ofBrassica napus L., detected by the monoclonal antibody MPM-2

Summary The monoclonal antibody MPM-2, which interacts with a mitosis-specific phosphorylated epitope, has been used to study phosphorylation of proteins in microspores and pollen ofBrassica napus. One- (1-D) and two-dimensional (2-D) immunoblots revealed that MPM-2 recognized a family of phosphorylated proteins in freshly isolated microspores and pollen. The same set of phosphorylated proteins was found after 8 h of culture at embryogenie (32 °C) and non-embryogenic (18 °C) conditions. Two major spots were observed on 2-D immunoblots, one of which (M_r75 kDa, pI5.1) co-localized with the 70 kDa heat shock protein. Immunolabelling of sectioned microspores and pollen showed that MPM-2 reactive epitopes were predominantly observed in the nucleoplasm from G₁ until G2-phase, and in the cytoplasm during mitosis. This may be due to a cell cycle related translocation of phosphoproteins from the nucleus to the cytoplasm, or alternate phosphorylation and dephosphorylation in nucleus and cytoplasm. Detectability of epitopes on sections depended on the embedding procedure. Cryo processing revealed epitope reactivity in all stages of the cell cycle whereas polyethylene glycol embedded material showed no labelling in the cytoplasm during mitosis. Processing might reduce the antigenicity of cytoplasmic MPM-2 detectable proteins, probably due to dephosphorylation. The MPM-2 detectable epitope was observed in all cells investigated, irrespective of culture conditions, and its intracellular distribution depended on the cell cycle stage and was not related to the developmental fate of the microspores and pollen.     

Isolated pollen culture: plant reproductive development in a nutshell

Abstract

Isolated pollen can develop into two different directions when cultured in vitro. In a rich medium, microspores and young pollen grains develop into mature pollen that is fertile. When pollen is treated by a stress treatment such as a hunger treatment in a sucrose - and/or nitrogen - free medium, embryogenic pollen is formed that, after transfer to a rich medium, develops into embryos and haploid plants. This system of isolated pollen culture offers an opportunity to study two developmental processes, i.e. pollen development and embryogenesis, as well as a basic regulatory event, i.e. the transition from the gametophytic to the sporophytic phase in the alternation of generations in higher plants. In addition, both systems offer various application-oriented possibilities, such as production of doubled haploids, to overcome self-incompatibility, to rescue sterile pollen, pollen selection and pollen transformation. An understanding of the cell biological and molecular events during embryogenic induction may promote a wider application of doubled haploid breeding and the use of such plants for gene transfer.     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.     

The 35S-CaMV promoter is silent during early embryogenesis but activated during nonembryogenic sporophytic development in microspore culture

Summary The cauliflower mosaic virus 35S (35S-CaMV) promoter, which is generally used as a constitutive promoter in plants, is known to be silent during microspore and pollen development. Here we analyzed whether the 35S-CaMV promoter fused to thegus (-glucuronidase) gene can be used as a marker for early sporophytic development in embryogenic microspore cultures of tobacco andBrassica napus. In microspore culture ofB. napus, the 35S-CaMV promoter remained off from the start of embryogenic culture up to the mid-cotyledonary embryo stage. 35S-CaMV promoter activity was only present in those microspores that initiated sporophytic development, but failed to enter embryogenic development. Similar results were also obtained with shed-microspore cultures of tobacco, in which rapid, direct embryogenesis takes place. In isolated-microspore cultures, in which embryogenesis is delayed, an intermitting period of sporophytic development was observed, characterized by extensive 35S-CaMV promoter activity. Therefore, the 35S-CaMV promoter discriminates between two classes of sporophytic development: it is activated in microspores which change fate from gametophytic into (temporarily) nonembryogenic sporophytic development, whereas the promoter is silent in sporophytic microspores that enter embryogenic development directly. This mirrors our observation that the 35S-CaMV promoter is also silent in young zygotic embryos.     

Microspore culture in wheat (Triticum aestivum) – doubled haploid production via induced embryogenesis

The inherent potential to produce plants from microspores or immature pollen exists naturally in many plant species. Some genotypes in hexaploid wheat (Triticum aestivum L.) also exhibit the trait for androgenesis. Under most circumstances, however, an artificial manipulation, in the form of physical, physiological and/or chemical treatment, need to be employed to switch microspores from gametophytic development to a sporophytic pathway. Induced embryogenic microspores, characterized by unique morphological features, undergo organized cell divisions and differentiation that lead to a direct formation of embryoids. Embryoids `germinate' to give rise to haploid or doubled haploid plants. The switch from terminal differentiation of pollen grain formation to sporophytic development of embryoid production involves a treatment that halts gametogenesis and initiates sporogenesis showing predictable cellular and molecular events. In principle, the inductive treatments may act to release microspores from cell cycle control that ensures mature pollen formation hence overcome a developmental block to embryogenesis. Isolated microspore culture, genetic analyses, and studies of cellular and molecular mechanisms related to microspore embryogenesis have yielded useful information for both understanding androgenesis and improving the efficiency of doubled haploid production. The precise mechanisms for microspore embryogenesis, however, must await more research.     

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.     

Complexity and Genetic Variability of Heat-Shock Protein Expression in Isolated Maize Microspores

The expression of heat-shock proteins (HSPs) in isolated maize (Zea mays L.) microspores has been investigated using high-resolution two-dimensional electrophoresis coupled to immunodetection and fluorography of in vivo synthesized proteins. To this end, homogeneous and viable populations of microspores have been purified in sufficient amounts for molecular analysis from plants grown in controlled conditions. Appropriate conditions for thermal stress application have been defined. The analysis revealed that isolated microspores from maize display a classical heat-shock response characterized by the repression of the normal protein synthesis and the expression of a set of HSPs. A high complexity of the response was demonstrated, with numerous different HSPs being resolved in each known major HSP molecular weight class. However, the extent of this heat-shock response is limited in that some of these HSPs do not accumulate at high levels following temperature elevation. Comparative analysis of the heat-shock responses of microspores isolated from five genotypes demonstrated high levels of genetic variability. Furthermore, many HSPs were detected in microspores at control temperature, indicating a possible involvement of these proteins in pollen development at stages close to first pollen mitosis.