André A. M. van Lammeren

Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth

The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.

ABA Inhibits Embryo Cell Expansion and Early Cell Division Events During Coffee (Coffea arabica ‘Rubi’) Seed Germination

BACKGROUND AND AIMS Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes. METHODS Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against beta-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy. KEY RESULTS The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of beta-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of beta-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion. CONCLUSIONS Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiosperms within the surrounding maternal and filial tissues has hampered studies on early plant embryogenesis. Somatic and gametophytic embryo cultures are often used as alternative systems for molecular and biochemical studies on early embryogenesis, but are not widely used in developmental studies due to differences in the early cell division patterns with seed embryos. A new Brassica napus microspore embryo culture system, wherein embryogenesis highly mimics zygotic embryo development, is reported here. In this new system, the donor microspore first divides transversely to form a filamentous structure, from which the distal cell forms the embryo proper, while the lower part resembles the suspensor. In conventional microspore embryogenesis, the microspore divides randomly to form an embryonic mass that after a while establishes a protoderm and subsequently shows delayed histodifferentiation. In contrast, the embryo proper of filament-bearing microspore-derived embryos undergoes the same ordered pattern of cell division and early histodifferentiation as in the zygotic embryo. This observation suggests an important role for the suspensor in early zygotic embryo patterning and histodifferentiation. This is the first in vitro system wherein single differentiated cells in culture can efficiently regenerate embryos that are morphologically comparable to zygotic embryos. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during the early stages of plant embryogenesis.

Desiccation sensitivity and cell cycle aspects in seeds of Inga vera subsp. affinis

The desiccation sensitivity of seeds of Inga vera Willd. subsp. affinis, a recalcitrant-seeded tree from Brazil, was analysed, focusing on water relations and cell-cycle aspects, including DNA content and the microtubular cytoskeleton. Seeds were collected at four developmental stages, dried to different moisture contents (MCs), assessed regarding water activity and set to germinate. Samples of fresh (non-dried) developing and mature seeds were used for assessment of DNA content by flow cytometry. Immunohistochemical detection of microtubules (MTs) was done in mature seeds at different MCs. Slight desiccation of immature seeds increased germination, but further drying resulted in a quick decline of germinability. During seed development the desiccation sensitivity decreased slightly, but DNA content of the embryonic axis cells remained constant, suggesting no relation between those two parameters. Embryonic axis cells of mature seeds showed abundant cortical microtubule arrays, which were not affected by mild desiccation, but broken down by further drying. It appeared that, upon rehydration, damaged cells were not able to reconstitute the microtubular cytoskeleton. The failure of germination of Inga vera seeds after drying could not be attributed to cellular damage to DNA synthesis and mitosis, since the radicle protruded by means of cell elongation, without a need for cell division. However, the breakdown of MTs during desiccation, and their subsequent inability to reassemble upon rehydration, may be related to the decreased germination, since MTs are required for cell elongation.

AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana

The Arabidopsis thaliana primordia timing (pt) mutant was transformed with an AtSERK1::GUS construct. Liquid cultures of this line were used to study the relationship between somatic embryogenesis and the expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (AtSERK1) as a marker for cells competent to form embryos. In order to search for the expression of AtSERK1::GUS during early stages of somatic embryogenesis, histochemical as well as immunochemical approaches were used for the detection of beta-glucuronidase (GUS). Four sites of AtSERK1 expression were found in the embryogenic cultures: in embryogenic callus, where primary somatic embryos developed; in the basal parts of primary somatic embryos; in the outer layers of cotyledons of primary somatic embryos where secondary embryos were formed; and in provascular and vascular strands of developing somatic embryos. The in vitro expression of AtSERK1::GUS coincides with embryogenic development up to the heart-shaped stage. Prior to the expression in embryos, AtSERK1 was expressed in single cells and small cell clusters, indicating that AtSERK1 indeed marks embryogenic competence. Its expression in (pro)vascular strands, suggests that embryogenic cells in tissue culture retain at least in part their original identity.

ps-2, the gene responsible for functional sterility in tomato, due to non-dehiscent anthers, is the result of a mutation in a novel polygalacturonase gene.

The recessive mutation ps-2, which appeared spontaneously in tomato, confers functional male sterility due to non-dehiscent anthers. In this study, we isolated and characterized the PS-2 gene. A single nucleotide mutation in a novel tomato polygalacturonase gene is responsible for the ps-2 phenotype. The mutation in ps-2 is responsible for an alternative splicing during maturation of the pre-mRNA, which leads to an aberrant mRNA. Differentiation between ps-2 and wild type (PS-2) anthers only appears in the final developmental stage in which the stomium remains closed in the mutant. To our knowledge, this is the first functional sterility gene isolated in the Solanaceae family. The specific expression of the Arabidopsis homolog of PS-2 in the anther dehiscence zone suggests a conserved mode of action over the plant kingdom, which means that the repression of PS-2 homologs may be a potential way to introduce functional sterility in other species.

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.

Physiological and cytological aspects of Inga vera subsp. affinis embryos during storage

The remarkably short storability of recalcitrant seeds has prevented their inclusion in programs of ex situ conservation. The causes of their desiccation sensitivity and rapid decline in viability during storage are not fully elucidated yet. In this study the highly recalcitrant, fully viable embryos of the tree species Inga vera subsp. affinis were stored under various conditions and analyzed physiologically and cytologically at intervals, in order to obtain more insights in their loss of viability during storage. Embryos stored fully hydrated at 5oC fully lost viability in 18 d. Sealed storage of partially dehydrated embryos slightly prolonged their viability, with 95% germination being attained after 14 d of storage. However, after 30 d of storage, viability was completely lost. Storage of hydrated embryos in a solution of polyethylene glycol (PEG) at -1.7 MPa water potential was capable of maintaining high germinability until 30 d of storage. When added to the PEG solution, abscisic acid showed a strong temperature-dependent interaction, with a positive effect on the longevity of embryos stored at 20oC and a negative effect on embryos stored at 5oC. In any case, embryo viability could be maintained no longer than 50 d. Embryos from seeds collected from the same trees in the following year showed better storability and still attained 45% germination after 62 d of storage. Analysis of cellular alterations during storage and viability loss of the embryos showed disappearance of starch granules and various damages to the cells, such as cell wall folding and cytoplasm fragmentation.

Gametophytic development of Brassica napus pollen in vitroenables examination of cytoskeleton and nuclear movements

Isolated microspores and pollen suspension of Brassica napus “Topas” cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a “comet-tail” form. These GC “tail” MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a “whole-mount” immunodetection technique and confocal laser-scanning microscopy was essential.