Showing papers in "Sexual Plant Reproduction in 1998"

Biogenesis and function of the lipidic structures of pollen grains

Abstract: Pollen grains contain several lipidic structures, which play a key role in their development as male gametophytes. The elaborate extracellular pollen wall, the exine, is largely formed from acyl lipid and phenylpropanoid precursors, which together form the exceptionally stable biopolymer sporopollenin. An additional extracellular lipidic matrix, the pollen coat, which is particularly prominent in entomophilous plants, covers the interstices of the exine and has many important functions in pollen dispersal and pollen-stigma recognition. The sporopollenin and pollen coat precursors are both synthesised in the tapetum under the control of the sporophytic genome, but at different stages of development. Pollen grains also contain two major intracellular lipidic structures, namely storage oil bodies and an extensive membrane network. These intracellular lipids are synthesised in the vegetative cell of the pollen grain under the control of the gametophytic genome. Over the past few years there has been significant progress in elucidating the composition, biogenesis and function of these important pollen structures. The purpose of this review is to describe these recent advances within the historical context of research into pollen development.

A rapid and simple procedure to determine stigma receptivity

Abstract: We describe a new method to determine stigma receptivity by using a Peroxtesmo esterase indicator paper liquid (one paper+1 ml water). This technique enables the researcher to check instantly the receptivity of various types of stigmas and to locate the receptive area.

The Arabidopsis AGL9 MADS box gene is expressed in young flower primordia.

Abstract: MADS box genes are likely involved in many different steps of plant development, since their RNAs accumulate in a wide variety of tissues, including roots, stems, leaves, flowers and embryos. In flowers, MADS box genes regulate the early step of specifying floral meristem identity as well as the later step of determining the fate of floral organ primordia. Here we describe the isolation and characterization of a new MADS box gene from Arabidopsis, AGL9. Sequence analyses indicate that AGL9 represents the putative ortholog of the FBP2 and TM5 genes from petunia and tomato, respectively. In situ hybridization analyses show that AGL9 RNA begins to accumulate after the onset of expression of the floral meristem identity genes, but before the activation of the organ identity genes. These data indicate that AGL9 functions early in flower development to mediate between the interaction of these two classes of genes. Later in flower development, AGL9 RNA accumulates in petals, stamens, and carpels, suggesting a role for AGL9 in controlling the development of these organs.

Sexual and apomictic development in Hieracium

Abstract: Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis.

The structure of the transmitting tissue of Arabidopsis thaliana (L.) and the path of pollen tube growth

Abstract: The compatible response in flowering plants is a complex process involving a series of cell communication events leading the pollen tube through the gynoecium to the ovule. We provide the first description of pollen tube growth in vivo in Arabidopsis thaliana. The structure and composition of the transmitting tissue of the septum are examined at the light microscope and transmission electron microscope levels. We demonstrate that once pollen tubes leave the extracellular matrix (ECM) of the central septum, they adhere to and travel along the ECM of the septum epidermis. This ECM, which surrounds the pollen tubes, is secreted through breaks in the cuticle of the septum surface. The transmitting tract ECM stains strongly for acidic polysaccharides. Monoclonal antibodies to esterified and low-esterified homogalacturonans (pectins) do not localize to this ECM. Yariv phenylglycoside and monoclonal antibodies to arabinogalactan proteins (AGPs) provide evidence that AGPs are a component of the septum epidermal ECM.

The evolution of double fertilization and endosperm: an ”historical” perspective

Abstract: One hundred years ago, the developmental origin of endosperm from double fertilization was discovered independently by Navashin and Guignard. For much of the twentieth century, specific events related to the evolutionary origin of the endosperm of flowering plants remained a mystery. However, during the past 20 years, advances in phylogenetic reconstruction of seed plants, genetic theory associated with kin selection, and comparative study of the reproductive biology of the closest living relatives of angiosperms (Gnetales) have advanced our understanding of the evolutionary events associated with the origin of double fertilization and endosperm. Recent developmental analyses of Ephedra and Gnetum (members of Gnetales) indicate that these nonflowering seed plants undergo a regular process of double fertilization that yields two diploid zygotes. Use of explicit genetic and developmental criteria for analysis of evolutionary homology demonstrates congruence with the hypothesis that double fertilization processes in Gnetales and angiosperms were inherited from a common ancestor of the two lineages. In its rudimentary form, the second fertilization event in the ancestors of flowering plants yielded a supernumerary diploid embryo that was genetically identical to the normal embryo, a process most similar to what occurs in extant Ephedra. Subsequent to the divergence of the angiosperm stem lineage, the supernumerary embryo derived from double fertilization was developmentally modified into an embryo-nourishing structure, endosperm, that now characterizes angiosperms.

Influence of intraovular reserves on ovule fate in apricot (Prunus armeniaca L.)

Abstract: In many plant species with multiovulate ovaries, a considerable reduction in the number of ovules takes place. However, the underlying physiological causes are not clear. In Prunus spp., although flowers present two ovules, usually only one seed is produced. We have followed the development and degeneration of the two ovules in apricot (Prunus armeniaca L.) and examined the extent to which carbohydrates within the ovule might be involved in determining the fate of the ovule. While the primary ovule grows in the days following anthesis, growth of the secondary ovule is arrested. Starch distribution along the different ovular tissues exhibits several changes that are different in the two ovules. Primary ovule growth is inversely related to starch content and this growth takes place independently of pollination since it occurs in the same way in pollinated and unpollinated flowers. In the secondary ovule, starch disappears simultaneously from all ovular structures and callose is layered at the chalazal end of the nucellus. The size of the secondary ovule does not change significantly from anthesis to degeneration, and callose starts to accumulate 5 days after anthesis. Likewise, this process occurs independently of pollination. These results are discussed in terms of the implications of the starch content of ovules in fertilization success and ovule fate.

Role of 1,4,5-inositol triphosphate-induced Ca2+ release in pollen tube orientation

Abstract: Pollen tube reorientation is a dynamic cellular event crucial for successful fertilization. Previously, it was shown that reorientation is preceded by an asymmetric increase of cytosolic free calcium ([Ca2+]c) in the side of the apex to which the cell will bend. In order to find the targets for this signal transduction pathway, the effects of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the reorientation process were analyzed. Ins(1,4,5)P3 was artificially increased in different cell domains by localized photoactivation of caged Ins(1,4,5)P3 and its effects on [Ca2+]c monitored by ion confocal microscopy. It was found that photolysis of caged Ins(1,4,5)P3 in the nuclear or subapical region resulted in a transient increase in [Ca2+]c and reorientation of the growth axis, while photolysis in the apex frequently resulted in disturbed growth or tip bursting. Perfusion of the cells with the Ins(1,4,5)P3 receptor blocker heparin prior to photoactivation inhibited the increase in [Ca2+]c and no reorientation was observed. Ca2+ release from Ins(1,4,5)P3-dependent stores localized in the shank of the tube thus seems to be part of the signal transduction pathway that controls tube guidance, although not the primary stimulus leading to reorientation.

Detection and inheritance of stylar ribonucleases associated with incompatibility alleles in apricot

Abstract: Stylar proteins were surveyed by non-equilibrium pH gradient electrofocusing to identify S-RNases associated with gametophytic self-incompatibility in nine apricot cultivars. RNase activities associated with the alleles of incompatibility S 1 , S 2 , S 5 , and S 6 and with the allele of compatibility Sc were clearly identified. Two other bands that we considered related to the alleles S 3 and S 4 were unique to cultivars Sunglo and Harcot, respectively. Two generations of 17 seedlings from the cross Moniqui× Pepito and 38 from Gitano × Pepito were used to determine the inheritance of the S-RNases. Inheritance of these RNase bands followed the expected segregation ratios and the band combinations correlated perfectly with the known self-incompatibility status of the seedlings determined after self-pollination and observation of pollen tube growth. All evidence presented in this study strongly suggests that RNases are associated with gametophytic self-incompatibility of apricot and that RNases may be the S-gene products. This is the first report identifying S-RNases and describing the inheritance of these S-RNases in apricot.

Cell cycle regulatory genes from maize are differentially controlled during fertilization and first embryonic cell division

Abstract: Regulation of early embryo development in higher plants is not understood. As one approach to gain insight into the mechanisms that govern zygotic embryogenesis, we studied the regulation of zygotic cell division by analyzing expression of cell cycle regulatory genes. Transcripts of cdc2 and cyclin genes as well as of a histone gene from maize were detected in single cells by employing recently developed techniques which allow fertilization of isolated gametes and zygote culture in vitro. We analyzed gene expression in sperm cells, in the egg cell and in other cells present in the embryo sac, i.e. synergids, central cell and antipodal cells. Moreover, in vitro gamete fusion enabled us to examine gene expression in the zygote at defined time points from fertilization to the two-celled embryo stage. Whereas the cdc2 gene is expressed constitutively in all cells of the embryo sac, in sperm cells, and throughout zygote development, the cyclin genes display cell-specific expression in the embryo sac and differential expression during zygote development. All three cyclins examined are transcribed de novo after fertilization, indicating a different level of embryonic cell cycle regulation in plants than what is commonly found in animals where early embryonic cell divisions are driven by maternally supplied cyclin mRNA.

The loculus content and tapetum during pollen development in Lilium.

Abstract: The ratio of loculus volume to the volume of the entire anther began to increase from the microspore mother cell stage and reached 32.3% at anthesis. The content of the loculus was examined in Lilium during pollen development and two waves could be distinguished. From the premeiotic stage until the vacuolated microspore stage, the loculus consisted of neutral polysaccharides, pectins and proteins. These substances originated from tapetal activity from the premeiotic stage until the young microspore stage. Dictyosomes and rough endoplasmic reticulum seemed to be involved in tapetal secretion, although, in some mitochondria, vesicles progressively developed as early as premeiosis and increased until the young microspore stage, which could reveal their involvement in the secretion process. At this stage, numerous cytoplasmic vesticles containing material similar to the locular material fused with the plasma membrane of the tapetum so that vesicle content was in contact with the loculus. It seems that tapetal and callose wall degradation at the late tetrad stage may also have contributed to the production of material in the loculus. From pollen mitosis to anthesis, the anther loculus contained mainly the pollenkitt which was synthesized in the tapetum between the young microspore stage and the vacuolated microspore stage. At the young microspore stage, proplastids divided and developed into elaioplasts and smooth endoplasmic reticulum (SER) increased dramatically. Pollenkitt had a double origin: some droplets were extruded directly from the plastid stroma through the plastid envelopes; the others were unsaturated lipid globules, which presumably derived from the interaction between SER saccules and plastids.

Distribution of calmodulin protein and mRNA in growing pollen tubes

Abstract: Pollen tube growth is a vital process for angiosperm fertilisation and is dependent on the presence of a tip-focused gradient of cytosolic free calcium ([Ca2+]c). In order to clarify some of the target molecules which convey the Ca2+ signal information, we investigated calmodulin distribution during tube growth. Fluorescently labelled calmodulin was pressure microinjected into pollen tubes and its distribution monitored by confocal microscopy. Calmodulin distributes evenly throughout the cell, but some of its binding sites form a V-shaped collar behind the apical region. This specific association dissipates upon growth arrest, and suggests an interaction of calmodulin with cytoskeletal-bound target proteins. The distribution of calmodulin mRNA was also analysed by microinjection of fluorescently labelled mRNA. No specific pattern was observed, with an even localisation in the body of tube and a lower concentration in the cell apex. Studies with localised application of inhibitors/activators indicate that calmodulin plays a crucial role in tip elongation but does not direct tube orientation.

Identification and cloning of related self-incompatibility S-genes in Papaver rhoeas and Papaver nudicaule

Abstract: The primary goal of this study was to identify, clone and analyse new S-gene sequences in order to provide a basis for identifying amino acid residues that confer S-allele specificity. Three new putative S-alleles from Papaver rhoeas and Papaver nudicaule were identified using immunological and PCR methods. cDNAs encoding full-length open reading frames of the P. rhoeas S 8 and P. nudicaule Sn 1 genes were isolated. Nucleotide sequencing of these cDNAs, together with the partial S 7 sequence obtained by PCR, was used to derive the corresponding amino acid sequences. It is of interest that the P. nudicaule Sn1 sequence, which is the first S-allele isolated from another species of Papaver, shares a closer sequence identity to the P. rhoeas S3 amino acid sequence than S3 does to S1 from P. rhoeas. The identity of the S8 allele was confirmed by expressing the coding region in Escherichia coli and demonstrating that the recombinant protein, designated S8e, specifically inhibited S 8 pollen in an in vitro bioassay. Information from sequence analysis of the S8, Sn1 and partial S7 amino acid sequences revealed important information about Papaver S-proteins. It confirmed previous observations based on only two S-alleles, that whilst exhibiting a high degree of amino acid sequence polymorphism ranging from 51.3% to 63.7%, these molecules probably share very similar secondary structures. These studies also revealed that, in contrast to the S-proteins from the Solanaceae and Brassica, amino acid sequence variation is not found in hypervariable blocks, but instead, is found throughout the S-proteins, interspersed with numerous short strictly conserved segments.

Reproductive biology of a rare cactus, Opuntia spinosissima (Cactaceae), in the Florida Keys: why is seed set very low?

Abstract: Opuntia spinosissima (Martyn) Mill. (Cactaceae) is an extremely rare taxon with a single remaining wild population of 13 plants located on Little Torch Key, Florida. The plants rarely set viable seeds and most ovaries abscise without initiation of fruits. Pollination and breeding system were examined in Little Torch Key and in plant accessions located at Fairchild Tropical Garden. Four pollination treatments were carried out to test for apomixis, self-pollination, outcrossing, and autogamy; a control treatment was also monitored. Additionally, pollen viability, pollinators, and seed set and viability were investigated. On selfed, crossed, and open (control) stigmas pollen grains germinated, but the tubes usually did not reach the base of the style. This suggests that O. spinosissima has pre-zygotic self-incompatibility. None of the pairwise crosses set seed, so the extant plants were apparently not inter-compatible. Out of 173 manipulated and control flowers, only one set fruit. Although this flower was outcrossed, no pollen tubes germinated on the stigma; this suggests agamospermy, a process common in the Cactaceae. In those flowers where pollen tubes did reach the ovary they failed to penetrate ovules, suggesting ovarian inhibition or that this taxon has lost the ability to be fertilized. Most field-collected seeds were viable, but there is no seedling recruitment under natural conditions, and vegetative reproduction is common. Based on these findings, I hypothesize that O. spinosissima is a sterile polyploid and that the 13 extant plants are asexually derived from a single lineage.

A novel thaumatin-like protein gene of tobacco is specifically expressed in the transmitting tissue of stigma and style

Abstract: A cDNA clone that encodes a novel thaumatin-like glycosylated protein, SE39b, which constitutes one of the major proteins of the stigmatic exudate of tobacco, was isolated and sequenced. The deduced amino acid sequence of SE39b shows 37% identity with the pathogenesis-related protein of tobacco, PR-R1, and 52% identity with the thaumatin-like protein (TLP) of Arabidopsis. All 16 cysteine residues are conserved in SE39b as they are in all TLPs. Three potential glycosylation sites found in SE39b were consistent with a previous finding that concanavalin A has an affinity for SE39b. Northern blot and in situ analyses demonstrated that the gene was specifically expressed in the transmitting tissue of the stigma and style, and the transcript amounts reached maximum levels at anthesis. mRNA from orthologs of the gene of SE39b was detected in species of Cestreae, but not in species of Solaneae. SE39b should be categorized as a PR-like glycoprotein which is developmentally regulated and specifically expressed in the transmitting tissue of the stigma and style, such as Sp41 [(1,3)-β-glucanase], SK2 and Chi2;1 (chitinase).

Dynein heavy chain-related polypeptides are associated with organelles in pollen tubes of Nicotiana tabacum

Abstract: It is known that pollen tubes contain two high molecular weight polypeptides which share some biochemical and immunological properties with dynein heavy chains. This paper reports data on the subcellular localization of the two dynein heavy chain-related polypeptides during pollen tube growth. Immunofluoresence studies using a purified antibody (Dy-1) raised against a synthetic peptide reproducting the P-loop conserved sequence of dynein heavy chains showed spot-like structures, with a characteristic distribution pattern that depended on the tube length. Biochemical evidence confirmed the presence of dynein heavy chain-related bands in the pollen tube membrane fraction. The association of proteins carrying dynein heavy chain-related polypeptides to cell membranes was affected by detergent (Triton×100), whereas other stripping agents, like NaCl and Na2CO3, did not significantly influence the interaction of dynein heavy chain-related doublet with their cytoplasmic targets. These data suggest that dynein heavy chain-related polypeptides associate with membranous organelles within the vegetative cell of Nicotiana tabacum pollen tubes, implying their involvement in the cytoplasmic distribution of these organelles.

MEI1, an Arabidopsis gene required for male meiosis: isolation and characterization.

Abstract: The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces after meiosis an abnormal tetrad, consisting of five to eight microspores of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-type genomic library, using the plant flanking DNA as a probe. Using RT-PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic sequence in this region indicated a gene which contained two introns. The 5′ and 3′ splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. The deduced sequence of the MEI1 wild-type gene, which contains 89 amino acids, shows possible similarity with the human acrosin-trypsin inhibitor, HUSI-II, and is about the same size. Two wild-type DNA fragments, both extending over the T-DNA insertion site, were introduced into mutant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several independent male-fertile transformants were obtained with one of the DNA fragments. The fragment showing complementation of the mutant phenotype indicated that the sequence with similarity to the acrosin-trypsin inhibitor is MEI1. Within the 16-kb genomic fragment two other genes were identified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Arabidopsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characterization of the mei1 phenotype because the individual microspores continued to be held together after callose dissolution.

Unexpected DNA content in the endosperm of Cupressus dupreziana A. Camus seeds and its implications in the reproductive process

Abstract: Nuclear DNA content of embryo and endosperm from mature and immature Cupressus dupreziana A. Camus seeds was estimated using laser flow cytometry. Relative DNA content of endosperm nuclei corresponded to four ploidy levels: 2C, 4C, 6C and 8C. The embryo nuclei invariably exhibited a diploid pattern. In all endosperm tissue analyzed no haploid nucleus was found. This is problematic since, in gymnosperms, endosperm and female gametes originate from one functional haploid megaspore produced by meiosis. The possible origin and derivation of C. dupreziana endosperm are discussed in light of previous results concerning the two other Mediterranean cypresses, C. sempervirens and C. atlantica.

Actin coronas in normal and indeterminate gametophyte1 embryo sacs of maize

Abstract: The actin cytoskeletal organization and nuclear behavior of normal and indeterminate gametophyte1 (ig1) embryo sacs of maize were examined during fertilization. After pollination, during degeneration of one of the synergids and before arrival of the pollen tube, the cytoskeletal elements undergo dramatic changes including formation of the actin coronas at the chalazal end of the degenerating synergid and at the interface between the egg cell and central cell. The actin coronas are present only for a limited period of time and their presence is coordinated with pollen tube arrival and fusion of the gametes; they disappear before the zygote divides. This allows us to estimate the frequency of fertilized ovules along the ear. Up to 88% of the ovules on an ear contain actin coronas in the embryo sacs when observed 16–19 h after pollination, indicating the high frequency of fertilizing kernels along the ear at this stage. In the ig embryo sacs, two or more degenerated synergids containing actin coronas at their chalazal ends receive multiple pollen tubes for gametic fusion and can consequently give rise to twin or polyembryos. These findings with the monocot maize are consistent with previous reports on the dicots Plumbago and Nicotiana, suggesting that the formation of actin coronas in the embryo sac during fertilization is a universal phenomenon in angiosperms and is part of a mechanism of interaction between gametic signaling and actin cytoskeleton behavior which appears to precisely position and facilitate the access of male gametes to the egg cell and central cell for fusion.

Identification of pollination-induced genes from the ovary of apple (Malus domestica)

Abstract: A set of fifteen cDNA clones from apple (Malus domestica Borkh) corresponding to fruit genes induced or enhanced by pollination have been identified by differential hybridization. Expression of corresponding mRNAs was induced in apple flowers by pollination, and in six clones mRNA levels also showed induction by gibberellin treatment of flowers. Sequence analysis and database searches showed that these cDNAs correspond to genes involved in defence responses, transport, protein and flavonoid synthesis, as well as cell division. One of the pollination-enhanced cDNAs was found to be similar to plant and animal genes encoding histone H2B. This mRNA was very highly expressed in flower buds and in fruit at early stages of development, but transcript levels were relatively low in young leaves and shoot tips. RNA in situ hybridization showed histone H2B mRNA detectable at high levels in the nucellus tissue of ovules in unopened flower buds. Five days after pollination, transcript levels decreased in the nucellus; however, weak signals were observed in the fleshy cortex tissue.

The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacum L)

Abstract: Sperm cells released from in vivo-in vitro grown pollen tubes of tobacco are associated in pairs and initially enclosed by the plasma membrane of the pollen tube. When the sperm cells are placed together, using glass microinjector needles, in an enzymatic solution, up to half undergo cellular fusion with subsequent nuclear fusion. The frequency of sperm cell fusion decreases with time during the elongation of the pollen tube, suggesting that mechanisms inhibiting self-fusion of sperm cells may develop as the pollen tube elongates through the style toward the ovule. This tendency may play an important role in inhibiting fusion of the two sperm cells inside the calcium-rich synergid where the male germ unit dissociates and sperm cells are transported to their target cells - the egg and central cell.

Chromosome elimination in Paspalum subciliatum (Notata group)

Abstract: This paper reports the occurrence of chromosome elimination during microsporogenesis in a Brazilian accession of Paspalum subciliatum. The accession was tetraploid (2n=4x=40) and meiosis was normal until diakinesis, with 20 regularly distributed bivalents. Starting at metaphase I, meiosis was very peculiar. In this phase, while ten bivalents were clustered in the equatorial plate, the other ten were still dispersed in the cytoplasm. In anaphase I the chromosomes showed different abilities to migrate to the poles. While one genome reached the poles in telophase I, the laggard was in metaphase or anaphase and was engulfed by extra nuclei. In the second division, behavior was the same. Our results show clear asynchrony in cell cycle, especially in some meiotic phases. Unfortunately we cannot explain the causes of the phenomenon, but this event shows once more that chromosome elimination serves as an incompatibility barrier preventing divergent genomes from coexisting in the same cellular system. The chromosome elimination affected pollen fertility but did not impair seed viability.

Differences in the initiation of the zygotic and parthenogenetic pathway in the Salmon lines of wheat: ultrastructural studies

Abstract: The ultrastructure of the egg apparatus of the sexual (aestivum)-Salmon line (aS) and the isogenic but alloplasmic (kotschyi)-Salmon line (kS) of the Salmon system of wheat was studied by transmission electron microscopy 3 days before and during anthesis. Additionally, the zygotic stage of aS, 17 h after pollination, was included. Metabolic activity of egg cells from the sexual line aS was low 3 days before anthesis and increased dramatically after pollination and fertilization. This timing of increased activity was evident because of changes occurring in the egg cell nucleus and nucleolus, polysomes, endoplasmic reticulum and Golgi apparatus, and the completion of the cell wall around the zygote. In contrast to the sexual line, the egg cell of the parthenogenetic line showed high activity 3 days before anthesis. The metabolic and ultrastructural characters observed in the nucleus and cytoplasm of the kS line 3 days before and during anthesis corresponded with those of the isogenic sexual line aS during anthesis and 17 h after pollination, respectively. High metabolic activity observed in the persistent synergid of kS may be connected with the occurrence of additional embryos in seeds (twins) of this line.

A newly identified chemotactic sexual pheromone from Closterium ehrenbergii

Abstract: In sexual reproduction of Closterium ehrenbergii, pairing with the sexual partner cells is the first process observed. A cell migration-inducing activity, specific for mating-type plus (mt+; NIES-228) cells, was detected in the culture medium of mating-type minus (mt–; NIES-229) cells. Light was necessary for production of the active substance by mt– cells and for migration of mt+ cells. The active substance was heat-labile and had an apparent molecular mass of 20 kDa, as determined by gel filtration. A protein of 20 kDa was detected in the active fraction of gel filtration after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on these results, it is proposed that a chemotactic sexual pheromone involved in the formation of sexual pairs of cells is secreted by mt– cells of C. ehrenbergii and is proteinaceous, like other sexual pheromones secreted by Closterium species.

Pollen fertility and intraspecific and interspecific compatibility in mangroves of Fiji

Abstract: Pollen fertility and compatibility status were investigated in three species of mangroves: Bruguiera gymnorhiza, Rhizophora samoensis, R. stylosa and a hybrid between the two Rhizophora species. Pollen fertility was greater than 80% in the three species but less than 10% in the hybrid. Also, all three species were intraspecifically compatible, while the hybrid showed very low compatibility which may be due to its low pollen fertility. The genera Bruguiera and Rhizophora were found to be completely cross incompatible, while a low reciprocal cross compatibility was observed in interspecific crosses between Rhizophora stylosa and R. samoensis.

Oligomerization of profilins from birch, man and yeast. Profilin, a ligand for itself?

Abstract: Profilins are structurally well conserved low molecular weight (12–15 kDa) eukaryotic proteins which interact with a variety of physiological ligands: (1) cytoskeletal components, e.g., actin; (2) polyphosphoinositides, e.g., phosphatidylinositol-4,5-bisphosphate; (3) proline-rich proteins, e.g., formin homology proteins and vasodilatator-stimulated phosphoprotein. Profilins may thus link the microfilament system with signal transduction pathways. Plant profilins have recently been shown to be highly crossreactive allergens which bind to IgE antibodies of allergic patients and thus cause symptoms of type I allergy. We expressed and purified from Escherichia coli profilins from birch pollen (Betula verrucosa), humans (Homo sapiens) and yeast (Schizosaccharomyces pombe) and demonstrated that each of these profilins is able to form stable homo- and heteropolymers via disulphide bonds in vitro. Circular dichroism analysis of oxidized (polymeric) and reduced (monomeric) birch pollen profilin indicates that the two states have similar secondary structures. Using 125I-labeled birch pollen, yeast and human profilin in overlay experiments, we showed that disulphide bond formation between profilins can be disrupted under reducing conditions, while reduced as well as oxidized profilin states bind to actin and profilin-specific antibodies. Exposure of profilin to oxidizing conditions, such as when pollen profilins are liberated on the surface of the mucosa of atopic patients, may lead to profilin polymerization and thus contribute to the sensitization capacity of profilin as an allergen.

Double fertilization: a personal view

Abstract: This year marks the 100th anniversary of the discovery of double fertilization by Nawaschin in St. Petersburg, Russia and, independently, Guignard in France. This discovery came at the end of a period of controversy about fertilization in angiosperms and ushered in a new period of intense research. Still, by 1950, there were many unanswered questions about double fertilization because of limitations of the light microscope. The introduction of the electron microscope stimulated new research and helped resolve some of the questions. My own research with the electron microscope and that of people who worked in my laboratory is recounted and some of the still unanswered questions raised.