Juxtaposition of the generative cell and vegetative nucleus in the mature pollen grain of amaryllis (Hippeastrum vitatum)

Summary Computer-generated, three-dimensional reconstructions from serial ultrathin sections were used to investigate the spatial organization and extent of association between the generative cell and vegetative nucleus within the mature pollen grain of amaryllis. In all cases examined, the highly lobed vegetative nucleus was found in close proximity and positioned laterally to the elongated, oval shaped generative cell. Numerous projections of the vegetative nucleus come to within 53 nm of the inner vegetative cell plasma membrane which surrounds the generative cell. These areas of close association may continue transversely around the generative cell for a distance of up to 4 m. Although an association exists between the generative cell and vegetative nucleus of the mature pollen grain, it is apparent that several changes must take place after pollination in order to achieve the high amount of close contact that occurs between the vegetative nucleus and the numerous terminal cell extensions of the leading sperm in the pollen tube of amaryllis (Mogensen 1986). Thus, this study demonstrates that the spatial organization among components of the male germ unit in the mature pollen grain does not necessarily reflect relationships that ultimately exist among these components within the pollen tube.     

Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.     

Developmental accumulation of hydroxyproline and hydroxyproline-containing proteins in Zea mays pollen

The hydroxyproline content of developing Zea mays (maize) pollen was determined. The level of hydroxyproline in uninucleate microspores early in pollen development was low (0.004% of dry weight). In contrast, mature pollen is enriched for this amino acid (0.1% of dry weight). In mature pollen, 90% of the hydroxyproline is in the soluble fraction. Upon in vitro pollen germination, hydroxyproline associated with the insoluble fraction increased from 10% to 26% of the total hydroxyproline. Antibodies specific to extensins and arabinogalactan proteins (AGPs), two major classes of hydroxyproline-containing proteins, recognized two distinct groups of proteins in maize pollen by Western analysis. The two types of pollen hydroxyproline-containing proteins could also be distinguished based on their behavior upon anion exchange chromatography.     

Isolation of sperm cells from mature pollen grains of Spinacia oleracea L.

Summary When mature pollen grains of Spinacia oleracea were squashed in a 25% sucrose solution and subsequently centrifuged on a percoll layer, sperm cells were isolated in high numbers. All steps were carried out at 4° C. Isolated sperm cells could be kept alive for several hours.     

RNA and protein synthesis in sperm cells isolated from Zea mays L. pollen

Summary Sperm cells are thought to be quiescent in pollen and activated upon pollen germination. To test this hypothesis, protein, RNA and DNA synthesis were assessed in Zea mays sperm cells at different times after isolation from pollen. Protein synthesis changed with time; while some proteins were found to be constitutive in both 0 and 24 h cells, others were synthesized and some disappeared. Overall, the number of proteins detected at 24 h doubled compared with freshly isolated cells. Incorporation of [³H]leucine in 24 h cells was about 50 times that in freshly isolated cells, and that of [5, 6-³H]uridine, about 7 times. Very low incorporation of [6-³H]thymidine into the cells was detected; there was no difference between freshly isolated and 24 h cells. It is possible that the differences in synthetic activity between freshly isolated and 24-h-old cells might correspond to sperm cell activation during pollen tube growth. If so, these metabolic changes may play an important role in fertilization.Supported by funds from a Strategic Grant (D.D.C.) and an Operating Grant (D.J.G.) from the Natural Sciences and Engineering Research Council of Canada     

Ultraviolet irradiation of maize (Zea Mays L.) pollen grains

Summary Mature pollen grains from two single cross (F₁) hybrids, Wf9 X H55 and K64 X K55, were exposed to eleven levels (0 to 6.80 erg/cm² × 10⁵ at 0.68 intervals) of ultraviolet irradiation and then were used to pollinate their source. Height and kernel characteristics (kernel weight, weight/100 kernels, kernel number) of individual F₂ plants produced by the normal F₂ kernels obtained from these pollinations were measured within each level and population. Highly significant exposure X population interactions were found for all characters, indicating that the effect of irradiation depended on the genetic source of the pollen grains. Increasing exposure increased or did not change the mean of Wf9 X H55 and decreased the mean in K64 X K55 for all characters. For coefficient of variation values, the interaction, exposure X population, was not significant for any character measured, indicating that irradiation-induced variability was unrelated to pollen source. The results indicate that pollen source strongly influenced the effect of ultraviolet irradiation on plant means but had no influence on variability.     

Quantitative ultrastructural analysis of barley sperm

Summary Complete serial ultrathin sections of seven sperm pairs, computer-assisted measurements of cell, nuclear and organelle surface areas and volumes, and three-dimensional imagery were used to demonstrate that a process of cytoplasm and organelle elimination occurs during sperm maturation in barley. The number of mitochondria per sperm cell is reduced by 50%; sperm cell surface area and volume are reduced by 30% and 51% respectively. Mean volume and surface area per mitochondrion are significantly less in mature sperms. No examples of mitochondrial fusion or degeneration were observed within sperm cells. These data, along with observations of plasma membrane apposition and vesiculation within cytoplasmic extensions containing mitochondria, support the proposition that cytoplasm and organelle loss results primarily from the formation of cytoplasmic projections that are subsequently discarded from the sperm cell body. Comparisons of the quantitative data, including the number of mitochondria, indicate that differences between sperm cells of a pair are absent to very slight. Spatial organization within the pollen grain is such that the mature sperms, as well as the sperms and vegetative nucleus, are not in close proximity.     

Genotypic effects on the amino acid relationships in maize (Zea mays L.) pollen and style

Summary The free amino acid content of the pollen grains and the style from three single cross hybrids (Wf9 x H55, Ky49 x Ky27, K64 x K55) and two inbred lines (Oh43, H55) was determined. No tyrosine was detected in the pollen grains of any genotype. Significant differences between pollen genotypes were found for aspartic acid, threonine, serine, lysine and histidine with no effect resulting from the vigor of the pollen source. No proline was found in the style of any genotype. Significant differences between style genotypes were obtained for threonine, serine, glutamic acid, leucine, tyrosine, ethanolanine, aminobutyric acid and histidine with a relationship between vigor of the style genotype present for tyrosine, ethanolanine and aminobutyric acid. The relationship between the pollen and style level for each amino acid as influenced by genotype was analyzed. A significant negative correlation was found only for threonine. Apparently, a complementary relationship between the pollen and style exists for some amino acids. Proline and tyrosine are available only from the pollen and style respectively. Threonine levels are balanced with varying contributions from both pollen and style depending on the genotype.     

B chromosomes of maize (Zea mays L.) are positioned nonrandomly within sperm nuclei

We used fluorescence in situ hybridization to identify and map the position of B chromosomes (supernumerary chromosomes) within maize sperm cells. Observations on over 1,000 sperm cells from several genotypes show that, on average, the B chromosomes are positioned in the tip one-fourth of the sperm nucleus two-thirds of the time. In contrast, the centromeres and knobs of the A chromosomes (the normal set) are not restricted to the tip portion of the nucleus. To our knowledge, this is the first example of specific chromosome positioning within a plant gamete. Studies on nuclear architecture of somatic cells in both plants and animals suggest that chromosome behavior and gene expression may correlate with chromosome position within the nucleus. The functional significance of nonrandom positioning of the B chromosomes within maize sperm is as yet unclear. Received: 10 May 2000 / Revision accepted: 6 September 2000     

Effects of calcium,magnesium, potassium and boron on sperm cells isolated from pollen of Zea mays L.

Our previous studies showed that Brewbaker and Kwack salts, which have been widely used in pollen germination and sperm isolation, are not appropriate for the maintenance of isolated maize (Zea mays L.) sperm cells. In the present study, we have characterized the effects of each BKS component salt on the integrity of isolated sperm cells using hemacytometry. At 0.01 and 0.1 mM, there were no differences in cell number between control and any salt-treated cells except a 22% decrease with 0.1 mM MgSO₄ at 48 h. At the 1 mM level, cell number decreased with time in the presence of Ca(NO₃)₂ and MgSO₄, with loss of integrity of most cells at 48 h, while KNO₃ and H₃BO₃ had little or no effect. Further characterization of calcium-induced reduction in cell integrity using flow cytometry showed that depletion of possible residual free calcium by addition of EGTA to the suspension medium improved cell longevity and viability. Exposure of isolated sperm cells to 1 mM calcium had no effect on cell integrity and viability in 5 h; however, only 12% of cells remained intact at 24 h. The reduction in cell integrity was hastened when cells were pretreated with the calcium ionophore A23187 prior to exposure to 1 mM calcium, with a 54% reduction in cell number at 1 h and complete cell lysis at 24 h. However, depletion of cytosolic free calcium by pretreatment of cells with the calcium ionophore followed by resuspension in the presence of EGTA resulted in rapid reduction of cell integrity as well. These results collectively suggest that maize sperm cells are sensitive to exogenous free calcium; however, a certain level of cytosolic free calcium is necessary for maintenance of integrity. Mechanisms of calcium-induced reduction in cell integrity are discussed along with possible roles of the sensitivity of sperm cells to calcium in fertilization.     

Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts

Summary Isolated egg cell protoplasts ofZea mays L., inbred line A 188, have been studied at the transmission electron microscope level. Their preparation for electron microscopy has been performed by embedding in ultra-low gelling agarose as a preliminary step. Five isolated egg cell protoplasts were serially ultrathin sectioned and studied in detail. One of these protoplasts was reconstructed in three dimensions to provide additional information on its structure. After enzymatic digestion and microdissection, isolated egg cells are true, highly vacuolized protoplasts. The structure of their organelles agrees with in situ observations, indicating an ultrastructural intactness after isolation: the mitochondria are polymorphic, form reticulate networks, and have well developed cristae; the plastids contain starch grains; and the spherical nucleus is euchromatic. As in situ, the organelles of the isolated egg cell protoplasts are aggregated near the nucleus. The complete picture provided by this work should serve as a comparative base for studies on in vitro fertilization products.     

Survival at 20° C and cryopreservation of isolated sperm cells from Zea mays pollen grains

Summary The survival of isolated sperm cells from maize pollen grains at 20° C and their cryopreservation were studied by means of the fluorochromatic reaction (FCR) test. An osmotic pressure of 500 mOsmol, a pH of 7 adjusted with MOPS buffer, and the ten-fold dilution of Brewbacker and Kwack salts enabled isolated sperm cells to survive for a maximum of 35 h at 20° C. The addition of 3 mM of calcium chloride or 0.1 % glucose also slightly improved survival. The addition of 1% glutamine gave the best percentage of FCR-positive cells at 20 h. The addition of all of these substances in a unique medium provoked the formation of spermcell aggregates that were FCR positive. With respect to cryopreservaton, 70% of the isolated sperm cells remained FCR positive after freezing at -80° C and quick thawing at 37° C in the selected survival medium.     

Treatment of maize pollen to reduce nuclease activity

Recently it has been reported that maize (Zea mays L.) pollen can be stored up to several hours in a hypertonic aqueous medium at 0°C without losing its germinability (Broglia and Brunori 1994). We found that both release and activity of pollen nucleases are diminished in a cold hypertonic aqueous medium. Nucleases can be washed off while preserving germination ability and thus preserving the possibility of passive uptake of exogeneous DNA into germinating pollen grains. Alternatively, active DNA transfer into non-germinating pollen grains in the storage medium itself may be facilitated, due to the very reduced nuclease activity in this medium.     

Associations among components of the male germ unit followingin vivo pollination in barley

Summary The male gametophyte of barley has been studied during post-pollination stages from the time of pollen activation through pollen tube growth to the level of the ovule. Techniques included the use of serial thick sectioning, serial ultrathin sectioning of re-embedded thick sections, and computer generation as well as manual production of three-dimensional reconstructions. It was found that after pollination, but before the sperms exit the pollen grain, an intimate association is formed between the vegetative nucleus and the two sperms, and between the sperms themselves. The latter association is maintained through the duration of pollen tube growth observed in this study. These results indicate that the male germ unit of barley, a grass, is ultimately similar to that of other angiosperms studied thus far at the electron microscope level and that the concept of the male germ unit may have even greater significance and application within flowering plants than previously thought.     

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.     

The spatial association of the sperm cells and vegetative nucleus in the pollen grain ofBrassica

Summary In mature viable pollen ofBrassica oleracea, the pair of sperm cells and the nucleus of the vegetative cell are linked to form a structured unit we term the male germ unit. The sperm cells are held within a common periplasm and have no cell walls. Each sperm cell has a central globular body containing the nucleus surrounded by several evaginations which provide the means for linkage between them. One sperm cell, usually that closest to the nucleus of the vegetative cell contains most of mitochondria profiles (plastids are absent). This sperm cell appears to be linked by its protoplasmic evaginations to the envelope of the vegetative nucleus. The role of this unit in interactions with the female gametic complex is considered.     

An analysis of the quantity and diversity of messenger RNAs from pollen and shoots of Zea mays

Summary The mRNAs of the mature pollen grain of Zea mays and of vegetative shoots were analyzed by comparing the kinetics of hydridization between homologous and heterologous reactions of cDNA to poly-(A)RNA in excess. The total complexity of pollen poly-(A)RNA i s 2.4×10⁷ nucleotides, whereas in the seedling shoot it is 4.0×10⁷ nucleotides. This corresponds to 24,000 different sequences in pollen and about 31,000 diverse mRNAs in shoots. The mRNAs in pollen can be divided into three abundance classes that constitute 35%, 49% and 15% of the total mRNAs having complexities of 2.1×10⁵, 6.4×10⁶ and 1.8×10⁷ nucleotides, respectively. Estimates have been made of the number of copies of each sequence per pollen grain or shoot cell, and also of the pollen mRNA sequences shared with those of the shoot.     

Biochemical composition of maize (Zea mays L.) pollen

Summary Mean percentages of sugars, water-soluble polysaccharides, starch, total carbohydrates and lipids were 40.1, 7.4, 28.6, 76.1, and 1.8 respectively. Differences among the mutants were found only for water-soluble polysaccharides with both wx and sh ₂ decreasing the percentage a small but significant amount. In terms of the various carbohydrates measured, no correlation was found between the expression of these mutants in the pollen and the kernel.

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Zusammenfassung Die Untersuchung des Kohlenhydrat- und Lipid-Gehaltes im Pollen der Endosperm-Mutanten waxy, shrunken und sugary vom Mais ergab einen gemittelten Gehalt an Zucker von 40,1%, wasserlöslichen Polysacchariden 7,4%, Stärke 28,6, Gesamtkohlenhydraten 76,1% und Lipiden 1,8%.Unterschiede zwischen den Mutanten wurden lediglich hinsichtlich der wasserlöslichen Polysaccharide gefunden, und zwar bei wx und sh ₂, die einen signifikant niedrigeren Gehalt aufwiesen.Hinsichtlich der verschiedenen analysierten Kohlenhydrate wurde keine Korrelation zwischen der Expression der Mutanten im Pollen und in den Karyopsen gefunden.

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Herrn Professor Dr. J. Straub (Köln-Vogelsang) zu seinem 60. Geburtstag gewidmet.

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Journal Series Paper No. 3621, Florida Agricultural Experiment Station.     

Genome-wide association analysis of seedling root development in maize (Zea mays L.)

Background

Plants rely on the root system for anchorage to the ground and the acquisition and absorption of nutrients critical to sustaining productivity. A genome wide association analysis enables one to analyze allelic diversity of complex traits and identify superior alleles. 384 inbred lines from the Ames panel were genotyped with 681,257 single nucleotide polymorphism markers using Genotyping-by-Sequencing technology and 22 seedling root architecture traits were phenotyped.

Results

Utilizing both a general linear model and mixed linear model, a GWAS study was conducted identifying 268 marker trait associations (p ≤ 5.3×10^-7). Analysis of significant SNP markers for multiple traits showed that several were located within gene models with some SNP markers localized within regions of previously identified root quantitative trait loci. Gene model GRMZM2G153722 located on chromosome 4 contained nine significant markers. This predicted gene is expressed in roots and shoots.

Conclusion

This study identifies putatively associated SNP markers associated with root traits at the seedling stage. Some SNPs were located within or near (<1 kb) gene models. These gene models identify possible candidate genes involved in root development at the seedling stage. These and respective linked or functional markers could be targets for breeders for marker assisted selection of seedling root traits.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1226-9) contains supplementary material, which is available to authorized users.     

Analysis of gene expression in microspores,pollen, and silks of Zea mays L.

Summary During pollen formation within anthers of Zea mays, post-meiotic expression of the cat-3 gene was observed from soon after microspore release from the tetrads until microspore mitosis; from the binucleate stage to maturity the cat-1 gene was expressed instead. Gene expression during pollen function, i.e., germination and tube growth, was determined by means of a new approach based on the in vivo comparative analysis of pollinated and non-pollinated silks. In the early autotrophic stage of germinating pollen ADH-1, CAT-1, and GOT-1 were expressed, whereas during further tube elongation only GOT-1 was detected. Pollination did not give rise to pollen-style hybrid enzymatic molecules nor to the induction of new enzymes on either partner. Silks, even when non-pollinated, showed the expression of additional alcohol dehydrogenase and catalase enzymatic activities, specifically ADH-2 and CAT-3. These data support the view that expression of the catalase system in the male gametophyte is stage specific, and suggest that the style may provide support to the elongating pollen tube at the functional as well as at the nutritional level.