Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen

Summary The three-dimensional organization of microtubules in generative cells during their development in pollen grains of Hippeastrum vittatum and the dynamic changes that occur were studied by collecting large quantities of fixed and isolated generative cells for immunofluorescence microscopy. The framework configuration and the arrangement pattern of the microtubule organization was investigated. The microtubule framework changed in shape from being spherical at an early stage to being long spindle-shaped at maturity: various transitional forms were observed: ellipsoidal, pear-shaped and short spindle-shaped. The microtubule arrangement making up this framework changed correspondingly from the original network, which was random in distribution, to axially oriented long bundles via an intermediate pattern composed of a mixture of networks with long bundles. However, cells with the same framework configuration might be heterogeneous in microtubule arrangements.     

Heat-shock proteins during pollen development in maize

In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat-shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat-shock responses. One-dimensional Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis technique (SDS-PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat-shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat-stress conditions; however, the consti-tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.     

MAP kinase signaling during pollen development

The stereotypical events of pollen grain maturation and its coordinated development with other flower tissues requires the interplay of different signalling pathways in order to ensure efficient fertilisation and, eventually, seed set. In recent years evidence has accumulated that members of the mitogen-activated protein kinase (MAP kinase) family are expressed in pollen and may function as regulators of both pollen development and germination. In this review we describe what is known about MAP kinases in pollen and discuss their possible function(s) in pollen biology. Received: 14 December 2000 / Accepted: 6 June 2001     

Annulate lamellae in plant cells: Formation during microsporogenesis and pollen development in Canna generalis Bailey

Summary The occurrence of stacked annulate lamellae is documented for a plant cell system, namely for pollen mother cells and developing pollen grains of Canna generalis. Their structural subarchitecture and relationship to endoplasmic reticulum (ER) and nuclear envelope cisternae is described in detail. The results demonstrate structural homology between plant and animal annulate lamellae and are compatible with, though do not prove, the view that annulate lamellar cisternae may originate as a degenerative form of endoplasmic reticulum.     

Anther starch variations in Lilium during pollen development

Starch was cytologically localized and biochemically assayed in different anther cell layers of Lilium cv. Enchantment during pollen development and its presence was correlated with anther growth. Two phases could be distinguished: the first, the growth phase, extends from the beginning of meiosis to the vacuolated microspore stage and corresponds to maximum increase in anther size and weight. During this period, microspores lack amyloplasts and starch is degraded in the outer staminal wall layers. The tapetum does not contain starch reserves but accumulates a PAS-positive substance in its vacuole. The second phase, the maturation phase, begins with the late vacuolated microspore stage and lasts until pollen maturation. Anther growth is slowed during this phase. A wave of amylogenesis/ amylolysis occurs first in the late vacuolated-microspores and young pollen grains and, next, in the staminal envelopes. In the pollen grain, the cytoplasm of the vegetative cell is filled with starch, but amyloplasts are not detected in the generative cell. When pollen grains ripen, amylaceous reserves are replaced with lipids. In the staminal envelopes, the second amylogenesis is particularly evident in the endothecium and the middle layers; the peak of starch is reached at the young bicellular pollen grain stage; starch disappears from the anther wall early during the maturation phase. The wave of amylogenesis/amylolysis occurring in the staminal envelopes during the maturation phase is peculiar to Lilium. It is interpreted as a sudden increase in carbohydrate level caused by lower anther needs when the growth is completed. Staminal envelopes may act as a physiological buffer and regulate soluble sugar level in the anther. Stages of anther growth correlate with starch content variations and this suggests that during the growth phase, products of starch hydrolysis in the staminal envelopes may be consumed partly by anther cell layers and partly by microspores.     

The loculus content and tapetum during pollen development in Lilium

 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. Received: 2 September 1997 / Revision accepted: 12 March 1998     

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.Abbreviation pI isoelectric point     

Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana

Using the cryo-fixation/freeze-substitution method, we studied the ultrastructural changes and behavior of vacuoles and related organelles (rER and Golgi bodies) during microspore and pollen development, and pollen maturation of Arabidopsis thaliana. In young microspores forming exine (pollen outer cell wall), vacuoles looked like those of somatic cells. In microspores during the formation of intine (inner cell wall), a large vacuole appeared which was made by fusion of pre-existing vacuoles and probably absorption of solutions. In the young pollen grain after the first mitosis, a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally, but by the invagination of tonoplasts from one side to the opposite side of a vacuole. After the second mitosis, somatic type vacuoles disappeared. In mature pollen grains just before germination, membrane-bound structures containing fine fibrillar substances (MBFs) appeared. The MBFs were considered to be storage vacuoles. In pollen grains from flowers in bloom, MBFs changed to lysosomal structures with acid phosphatases (lytic vacuole). They gradually increased in number and volume, and decomposed the cytoplasm. The autolysis of pollen grains is the first finding in this study, which may contribute to the loss of ability of pollen germination after anthesis.     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

Floral organ growth and carbohydrate content during pollen development in Lilium

In order to understand floral sugar physiology, we correlated the growth of the organs with carbohydrate content in the flower of Lilium cv. “enchantment” during pollen development. In a previous work, we distinguished two phases in pollen ontogenesis: the anther growth phase, from the microspore mother cell until the vacuolated microspore, and the anther maturation phase, from the vacuolated microspore until anthesis. In the present work, we showed that during the growth phase, the anther underwent most of its size and dry weight growth, whereas the growth rate of nonanther organs was reduced. Anther and filament possessed the highest amounts of carbohydrates, which decreased progressively until the vacuolated microspore stage. During the maturation phase, sucrose and starch increased in all floral organs. Anther growth was completed at the Mi stage, whereas the nonanther organs began exponential growth. From these observations, we concluded that hierarchic nutritional correlations exist between the flower organs, in which the anther is the main actor: during the anther growth phase, the anther represents the highest sink strength floral organ, and mainly attracts assimilates through the filament. During the anther maturation phase, anther growth is achieved, its needs decrease, and assimilates are thus available for neighboring organs, which undergo intense growth until anthesis.     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000     

Characterization of a desiccation-related protein in lily pollen during development and stress

This work characterizes a lily (Lilium longiflorum Thunb. cv. Snow Queen) anther (LLA) protein associated with desiccation. Peptide mapping analysis revealed that the abundant LLA-23 doublet contained similar polypeptides, having an isoelectric point of 6.1. Immunoblots of pollen protein from developing anther/pollen confirmed that the LLA-23 protein accumulated only at the later stage of pollen maturation and that the levels remained steady in mature and vital pollen. The accumulation of LLA-23 proteins was correlated with desiccation that naturally occurred in pollen. Subcellular fractionation of pollen proteins revealed that the protein was located in the cytoplasmic fraction. Premature drying of developing pollen confirmed that the concomitant accumulation of LLA-23 was associated with desiccation. Peptide sequence analysis demonstrates similarities between the lily LLA-23 and a family of water-deficit/ripening-induced proteins including LP3 of pine, DS2 of potato, and Asr of tomato and pummelo. In addition, the concomitant accumulation of LLA-23 can be experimentally manipulated by methyl jasmonate (Me-JA) and salicylic acid (SA) as well as by mannitol and methyl viologen. The LLA-23 represents a novel member of the water-deficit/ripening-induced proteins.