Embryogenesis in plants is a unique process in the sense that it can be initiated from a wide range of cells other than the zygote. Upon stress, microspores or young pollen grains can be switched from their normal pollen development towards an embryogenic pathway, a process called androgenesis. Androgenesis represents an important tool for research in plant genetics and breeding, since androgenic embryos can germinate into completely homozygous, double haploid plants. From a developmental point of view, androgenesis is a rewarding system for understanding the process of embryo formation from single, haploid microspores. Androgenic development can be divided into three main characteristic phases: acquisition of embryogenic potential, initiation of cell divisions, and pattern formation. The aim of this review is to provide an overview of the main cellular and molecular events that characterize these three commitment phases. Molecular approaches such as differential screening and cDNA array have been successfully employed in the characterization of the spatiotemporal changes in gene expression during androgenesis. These results suggest that the activation of key regulators of embryogenesis, such as the BABY BOOM transcription factor, is preceded by the stress-induced reprogramming of cellular metabolism. Reprogramming of cellular metabolism includes the repression of gene expression related to starch biosynthesis and the induction of proteolytic genes (e.g. components of the 26S proteasome, metalloprotease, cysteine, and aspartic proteases) and stress-related proteins (e.g. GST, HSP, BI-1, ADH). The combination of cell tracking systems with biochemical markers has allowed the key switches in the developmental pathway of microspores to be determined, as well as programmed cell death to be identified as a feature of successful androgenic embryo development. The mechanisms of androgenesis induction and embryo formation are discussed, in relation to other biological systems, in special zygotic and somatic embryogenesis.

In previous studies we have shown that, after stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbilical vein endothelial cells (HUVEC). However, the mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules.By density gradient centrifugation of rat lung homogenates, a particle was identified that contained nearly all tPA activity and antigen. This particle had an average density of 1.11–1.12 g/ml, both in Nycodenz density gradients and in sucrose density gradients. A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC. After thrombin stimulation of HUVEC to induce tPA secretion, the amount of tPA present in high-density fractions decreased, concomitant with the release of tPA into the culture medium and a shift in the density distribution of P-selectin.vWf, known to be stored in Weibel-Palade bodies, showed an identical distribution to tPA in Nycodenz gradients. In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, suggesting that tPA and vWf were not present in the same particle.Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing particles showed a different distribution by confocal microscopy. The distribution of tPA also differed from the distribution of tissue factor pathway inhibitor, endothelin-1, and caveolin. By immunoelectronmicroscopy, immunoreactive tPA could be demonstrated in small vesicles morphologically different from the larger Weibel-Palade bodies. It is concluded that tPA in endothelial cells is stored in a not-previously-described, small and dense (d = 1.11– 1.12 g/ml) vesicle, which is different from a Weibel-Palade body.

To study colonization of the tomato root system, we previously have described a gnotobiotic quartz sand system, in which seedlings inoculated with one or two bacterial strains were allowed to grow. Here we present a scanning electron microscope description of the colonization of the tomato root system by Pseudomonas fluorescens biocontrol strain WCS365, with emphasis on spatial-temporal colonization patterns, based on an improved scanning electron microscopy procedure. Upon inoculation of the germinated seed, proliferation on the seed coat was observed for 2 to 3 days. Within 1 to 3 days, micro-colonies developed, mainly at the root base. Most micro-colonies were localized in junctions between epidermal root cells, whereas others were found in indented parts of the epidermal surface. Downward to the root tip, only single bacterial cells were found. Colonization progressed down the root, initially as single cells. A semi-transparent film appeared to enclose the root surface and micro-colonies present on the root. After 7 days, micro-colonies had developed at positions where only single cells were observed previously and distribution of the bacteria along the root varied from ≈106 CFU per cm of root near the root base to ≈102 to 103 CFU per cm of root near the root tip. Similar colonization patterns were found for the P. fluorescens biocontrol strains CHA0 and F113, and P. putida strain WCS358, as well as for four species that have repeatedly been isolated from tomato roots from a commercial tomato field near Granada, Spain. In contrast, four Rhizobium strains and one Acinetobacter radioresistens strain showed poor colonization and micro-colonies were not observed. Based on the described data, we present a model for colonization of the deeper root parts after seed inoculation by P. fluorescens biocontrol strains, in which single cells occasionally establish on a deeper root section where they sometimes develop into micro-colonies. We hypothesize that micro-colonies are the sites where the intracellular N-acyl-L-homoserine lactone concentration is sufficiently high to cause maximal production of biocontrol factors such as antibiotics and exoenzymes and that micro-colonies explain the relatively high conjugation frequency observed between pseudomonads in the rhizosphere.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre-treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.

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