H-ATPase in the plasma membrane of Arabidopsis pollen cells is involved in extracellular calmodulin-promoted pollen germination

In this paper, the role of the plasma membrane (PM) H⁺-ATPase in extracellular calmodulin (CaM)-promoted pollen germination and in tube growth of Arabidopsis thaliana was investigated. Pollen germination, pollen tube growth rate, free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and Ca²⁺ channel activity in the PM of pollen cells were measured. In response to fusicoccin or CaM treatment, in vitro pollen germination and pollen tube growth rate, [Ca²⁺]_cyt and activity of a hyperpolarization-activated Ca²⁺-permeable channel increased. Sodium vanadate inhibited the promotion of these parameters by extracellular CaM. The results suggest that H⁺-ATPase may be involved in extracellular CaM-regulated pollen germination and in tube growth by modulation of the hyperpolarization-activated Ca²⁺ channel in the PM of A. thaliana pollen cells.     

Identification and function analysis of spectrin-like protein in pollen tubes of lily （Lilium davidii Duch）

The elongation of pollen tube is an important process of sexual reproduction in higher plant. Cytoskeleton plays a major regulatory role in the elongation of pollen tubes. But whether membrane skeleton is involved in the pollen tube elongation is not clear. In this study, immunochemical detection of spectrin-like protein has been carried out in pollen tubes. By use of 2-dimensional electrophoresis(2DE) and western blotting, two spectrin-like proteins are found, one is 150 kD, and the other is 105 kD, with pl being 4.54 and 4.39, respectively. 150 kD spectrin-like protein is located in plasma membrane of pollen tube and 105 kD spectrin-like protein is located in cytoplasm, probably functioning as a subunit to form a dimmer (210 kD) in vivo. The elongation of pollen tubes is inhibited after spectrin antibody was injected into a growing pollen tube. These results suggest that spectrin-like proteins exist in pollen tube and play an important regulating role in the elongation process of pollen tubes from lily.     

Effects of extracellular calmodulin on pollen germination and tube growth

The effects of calmoddin (CaM) antagonist W7-agarose, anti-CaM serum and exogenous purified CaM on pollen germination and tube growth ofForsythia suspensu were studied. The pollen germination and tube growth were inhibited or completely stopped by CaM antagonist W7-agarose. The addition of exogenous purified CaM stimulated pollen germination and tube growth, whereas the same amount of bovine serum albumin (BSA) had no effect. The inhibitory effects caused by W7-agarose and anti-CaM serum could be reversed completely by the addition of exogenous purified CaM. The tube growth of germinated pollen was also inhibited or completely stopped by W7-agarose. The results suggest that endogenous extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes.     

Actin and myosin during pollen germination

Actin and myosin from pollen tubes of Lilium davidii were studied by using immunoblotting, Dot_Blot and myosin Ca 2+_ATPase analysis. On immunoblotting of the total soluble pollen tube proteins, anti_α_actin antibody labelled a polypeptide approximately 43 ku, which is considered to be the actin of lily. The mRNA encoding actin in ungerminated pollen and germinated pollen were both undetectable in our experiments. A myosin exhibited Ca 2+_ATPase activity, with a native molecular weight of 460 ku has been identified by using immunoblotting. A polypeptide of about 205 ku and a polypeptide of about 20 ku were the heavy chain and a set of light chain of the myosin, which can crossreact with anti_skeletal muscle myosin heavy chain monoclonal antibody and anti_skeletal muscle myosin light chain (20 ku) monoclonal antibody, respectively. The Ca 2+_ATPase activities of myosin in crude extracts of germinated pollen were positively related to the growth rates of pollen tubes.     

S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility

Many flowering plants avoid inbreeding through a genetic mechanism termed self-incompatibility. An extremely polymorphic S-locus controls the gametophytic self-incompatibility system that causes pollen rejection (that is, active arrest of pollen tube growth inside the style) when an S-allele carried by haploid pollen matches one of the S-alleles present in the diploid style. The only known product of the S-locus is an S-RNase expressed in the mature style. The pollen component to this cell-cell recognition system is unknown and current models propose that it either acts as a gatekeeper allowing only its cognate S-RNase to enter the pollen tube, or as an inhibitor of non-cognate S-RNases. In the latter case, all S-RNases are presumed to enter pollen tubes; thus, the two models make diametrically opposed predictions concerning the entry of S-RNases into compatible pollen. Here we use immunocytochemical labelling of pollen tubes growing in styles to show accumulation of an S-RNase in the cytoplasm of all pollen-tube haplotypes, thus providing experimental support for the inhibitor model.     

Cryopreservation of pollen by vitrification inBrassica

The key factors affecting pollen cryopreservation by vitrification inBrassica campestris var.purpurea were investigated. The factors involved the pollen maturity, the pollen resistance to dehydration, the components of vitrification solution (PVS), and the concentration of diluent. Thus, a suitable procedure was established for pollen vitrification, the maximum relative survival rates of mature (at the day of anthesis) and nearly mature (3 days before anthesis) pollen were about 80%, 63% respectively. This procedure has been also successfully applied to two other species (Brassica napus, Brassica chinensis). The advantage of cryopreservation of pollen by vitrification was discussed. Xu Bingfang: born in Sep. 6. 1970, Ph. D graduate student     

慈姑雌花在自然居群中的受粉过程--花粉粒落置、萌芽及花粉管生长的荧光显微观察

用荧光显微术观察了慈姑（Ｓａｇｉｔｔａｒｉａ ｔｒｉｆｏｌｉａ Ｌｉｎｎ）不同时期的雌花柱头上花粉粒的落置、萌发及花粉管的生长过程与途径。结果表明：该种雌花开花２ｈ 柱头受粉率为０,开花４ｈ 和８ｈ受粉率分别为５３８％ 和９８３％ ,柱头上花粉萌发率达１００％ ;花粉管沿雌蕊之向心一侧的组织中穿行,至子房基部后部分花粉管转向胚珠,由珠孔进入珠心。本研究发现该种花粉管常穿过子房基部至花托组织,并可进入其他雌蕊中,作者认为这种行为可能对保证雌花的结实率有一定意义     

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.     

Effects of nitrogen ion implantation on lily pollen germination and the distribution of the actin cytoskeleton during pollen germination

The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results showed that the ratio of pollen germination increased from (16.0± 1.6)% to (27.0±2.1)% when implanted with nitrogen ions by 100 keV and a dose of 10¹³ ions/cm². Further experiments were performed by staining the actin filaments in pollen with rhodamine-phalloidin and detected by using laser confocol microscopy. After hydration for 10 h, the actin filaments in ion implanted pollen grains tended to form thick bundles oriented in parallel or ring shape at the germinal furrow, indicating that the effect of nitrogen ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.     

Preparation of exine-detached pollen inNicotiana tabacum

A method of preparing exine-detached pollen inNicotiana tabacum was established. Anthers containing early-middle binucleate pollen were cold-pretreated at 4–6°C for 7–14 days, and were suspended in 0.3 mol/L sucrose solution for 2 days. During this process, the exine of most pollen grains dehisced. Then they were transferred into an enzyme solution containing 1% cellulase, 1% pectinase, 0.1% pectolyase, 1 mol/L mannitol, 0.3 mol/L sorbitol, 0.5% potassium dextran sulphate and K3 medium macro elements. After 15–20 min enzymatic maceration, the exine was detached resulting in the release of exine-detached pollen. Factors affecting preparation of exine-detached pollen were investigated, including cold-pretreatment, osmoticum concentration and enzymes used. Xia Huijun: born in Oct. 1963, Ph.D. Current research interest is in plant reproductive biology Supported by the National Natural Science Foundation of China     

Atomic force microscopic observation on substructure of pollen exine inCedrus deodara andMetasequoia glyptostroboides

The substructure of pollen exine inCedrus deodara (Roxb.) Loud. andMetasequoia glyptostroboides Hu et Cheng has been examined with an atomic force microscope (AFM). The results indicate that the exine substructure units containing sporopollenin in two species are similar in shape, which are granular, but slightly different in size. InCedrus the substructure unit of pollen exine appears to be 56–99 nm long and 42–74 nm wide, while inMetasequoia it appears to be 81–118 nm long and 43–98 nm wide. It has been observed that the subunits of pollen exine inCedrus arranged tightly to form short-rod-like or spheroidal pollen exine units, several or more than ten of which formed an island-like structure. There are various spaces among these island-like structures which are interconnected to occupy the entire pollen exine. InMetasequoia, the subunits of pollen exine also arranged tightly with a distribution tendency of cluster of 3–10, however, no obvious boundary exists among these clusters. From our results, it is concluded that there is no tendency of helical arrangement for the subunits of pollen exine inCedrus andMetasequoia, and the results support Southworth’ view that subunits of pollen exine are granular shape in lattice structure.     

Characterization of an endo-1,4-β-glucanase from Lilium longiflorum that functioned in pollen germination and pollen tube growth

Endo-β-glucanases play vital roles in the regulation of pollen tube growth. Here, a previously identified endo-l,4-β-glucanase from Lilium Iongiflorum （lily）, named LlpCell, was expressed in Escherichia coli, purified, and further investigated for its physiological function. The recombinant LlpCell protein hydrolyzed carboxy-methylcellulose （CMC） and exhibited activity towards laminarin from Eisenia. arborea and 1,3：l,4-β-glucan of barley. The pH for the optimum activity was 6.0 and the value of Km calculated from CMC was 5.0 mg/mL. Adding EDTA resulted in the total loss of the enzymatic activity, and this effect could be restored by the addition of Ca^2＋. Western blotting analysis showed that LlpCell protein was present in pollen grains and rehydrated pollen grains, and the amount of the protein was increased during pollen germinating, but not in the pollen tube. Consistently, the immunofluorescence labeling study with the antibody against LIpCell also indicated the presence of LIpCell at the begin-ning of germination, but not in the elongating pollen tube. Furthermore, incubation of LlpCell with pollen at the beginning of pollen germination increased the germination percentage and the length of pollen tube. All of these results suggested that LlpCell could play an important role in the regulation of lily pollen germination and the initiation of pollen tube growth.     

Surface pollen distribution patterns in Beibu Gulf and corresponding sediment dynamics environment

The Beibu Gulf is a semi-enclosed gulf in the northwest of the South China Sea.We palynologically analyzed 306 surface sediment samples from the eastern Beibu Gulf to improve bioclimatic interpretation of fossil pollen records there.Surface pollen assemblages could be classified into five pollen regions based on the distributions of total,arboreal,herbaceous and fern pollen concentrations.Four high-concentration and three low-concentration subregions could be distinguished within these regions.The distribution patterns of surface pollen assemblages were consistent with those of grain sizes of surface sediments.Sediments from regions with high pollen concentrations were very fine and fine silts (>7.0,<0.008 mm),whereas those with low pollen concentrations were fine sand (<3.5,>0.088 mm).Sedimentary heterogeneity of surface pollen assemblages was closely related to pollen source,transportation and sedimentation controlled by ocean currents,tides and waves,and oceanic bottom topography.Fern spores exhibited higher percentages along the east margins of the region,while arboreal types like Pinus increased in concentration towards the center.Herbaceous pollen appeared in high percentages around seacoasts near their source areas.Dacrydium and mangrove pollen were distributed near their source regions at low percentages.We discuss the sediment dynamic environments in the eastern Beibu Gulf based on surface pollen distributions.The estuary region is an important access to the sea basin and a depositional site for terrestrial pollen grains.Coastal regions can accumulate pollen due to the back-and-forth movements of tides there.Although ocean currents on a gulf scale tend to spread pollen grains,the interaction of multiple currents could lead to pollen accumulation and deposition.Low surface pollen concentrations in the northeast shallow-water regions of the eastern Beibu Gulf could be attributed to repeated washing and sediment floatation caused by severe wave activities during the summer.Strong tides in Qiongzhou Strait frequently wash the seabed and disadvantage pollen deposition,leading to low surface pollen concentrations there.     

Surface pollen assemblages as indicators of human impact in the warm temperate hilly areas of eastern China

Investigation of 78 surface pollen samples from warm temperate hilly areas of eastern China shows that pollen assemblages in areas of different land use are significantly different. Pollen concentrations in wastelands are higher than in plantations; these, in turn, are higher than in farmlands; implying that pollen concentration decreases with increasing human impact. Arboreal pollen dominated by Pinus and Quercus is common in all samples. Herbaceous pollen percentages are higher while shrub pollen, fern spores and fern allies are lower in farmlands than in wastelands. Crop pollen is only detectable in and near farmlands; its percentages and concentrations decrease in wastelands. Cereal and Cruciferae pollen percentages average 16.7% and 6.7% in farmland respectively, but Cereal reduces to less than 3% and Cruciferae to less than 0.5% in nearby wastelands. Principle coordinates analysis and clustering analysis indicate that pollen assemblages from farmlands are distinguishable from those under other vegetation types. Occurrence of Poaceae, Chenopodiaceae, Artemisia, and Compositae pollen and Selaginella sinensis spores are closely related to human activities, and their types and frequencies indicate intensity of human impact. The spatial distribution of crops, Chenopodiaceae, and Artemisia reflects changes in both natural environments and human activities. Percentages of cereal and Cruciferae pollen, for example, increase with decreasing altitude, but decrease with increasing latitude. Understanding pollen assemblages under artificial and human-disturbed vegetation in hilly areas may aid understanding of human impacts on the plains during the early-middle Holocene.     

Primary measurement of pollen flow inLiriodendron chinense using RAPD markers

Techniques of tracking movements of pollen grains are essential for many topics in pollination biology. Pollen transfer ofLiriodendron chinense (Hemsl.) Sarg., a hermaphroditic plant, may be both interfloral and intrafloral pollination. Observations on pollinator movements and flower emasculated experiments in natural populations showed that most of pollen grains deposited on stigmas in a flower are foreign. But it is not certain whether cross pollination or long distance pollen transfer occurs in this insect-pollinated plant. The authors attempt to use random amplified polymorphic DNA (RAPD) loci for paternity analysis to mark pollen dispersal. The results indicated pollen via gene transportation reached at least 100 m away from the pollen source in a selected population. The primary study suggests a larger effective population size in this endangered plant. With its rapid and simple characters, RAPD methods can be a suitable technique for marking pollen flow and able to be widely used in various taxa.     

Quantitative relationships between vegetation and several pollen taxa in surface soil from North China

According to the vegetation investigation and pollen analysis of surface samples sampled along a precipitation gradient of the Northeast China Transect (NECT), several pollen taxa, includingPinus, Betula, Quercus, Tilia, Acer, Ulmus, Artemisia, Chenopodiaceae, Gramineae and Cyperaceae, were chosen to make the regression and correlation analyses. The results indicated that there exists a close relationship between vegetation and pollen taxa in surface samples. The regression parameters for ten taxa in the forests in the eastern part of NECT were different from those in the steppes in the western part.Pinus, Betula, Artemisia and Chenopodiaceae, which have large slope and y-intercept terms, were over-representative taxa.Acer, Gramineae and Cyperaceae, which have small slope andy-intercept terms, were under-representative taxa.Quercus, Tilia andUlmus whose slope terms have negative correlation withy-intercept terms were equi-representative taxa. The pollen taxa with large slope or largey-intercept terms have small variability coefficients, implying that the slope andy-intercept terms for these pollen taxa are of high accuracy in the estimation of plant abundance from pollen frequencies.     

Surface pollen assemblages of human-disturbed vegetation and their relationship with vegetation and climate in Northeast China

Pollen assemblages of 53 surface pollen samples from farmlands and wastelands in Northeast China were analyzed. Tree pollen percentages were usually higher than 30%, with Pinus (26.9%), Quercus (0.9%), Betula (0.9%) and Populus (0.7%) as the major types, and herb pollen percentages were usually higher than 50%, with weedy Poaceae (8.7%), Chenopodiaceae (7.1%), Artemisia (1.9%) and Compositae (3.5%) as the major taxa. Thus, the pollen assemblages were consistent with the regional vegetation compositions. However, there were differences in pollen assemblages among regions, especially among different geomorphological units. For example, in the mountains, there were more types of tree pollen and higher total percentages (average 42%) than in other areas, while cereal pollen percentages were lowest (11.2%). In the hills and high plains, herbs made up more than 60% of the pollens, with cereals (average 53.6%) the dominant type. In the low plains, pollen types were similar to those in the hills and high plains, but total pollen concentrations and the proportion of Concentricystes were much higher, while cereal pollen percentages were slightly lower (average 41.6%). Pollen assemblages in different land use types also differed. For example, in farmland, cereal pollen percentages were more than 40% and Chenopodiaceae was usually less than 2.5%, while in wasteland, weedy Poaceae was usually less than 10% and Chenopodiaceae was usually higher than 25%. Total pollen concentrations in farmland (average 3909 grains/g) were much lower than in wasteland (average 15074 grains/g). Redundancy analysis revealed that pollen assemblages were significantly negatively correlated with mean annual temperature (-0.73) and July mean temperature (-0.81) and significantly positively correlated with mean annual precipitation (0.48), indicating that pollen assemblages in the artificial or human-disturbed vegetation reflect regional climate well. Comparison of pollen assemblages in different areas of northern China showed that pollen concentrations reflect the intensities of human impact to some degree. For example, pollen concentrations decrease as human impacts increase in intensity. The cereal pollen proportions in farmland differed by area. In Northeast China, cereal pollen proportions were distinctly higher than in most other areas of northern China, suggesting differences in planting habits and climate.     

芍药花粉离体萌发培养基组分的优化

以芍药花粉为材料,采用正交设计L^9（34）法研究蔗糖、硝酸钙、硼酸对芍药花粉离体萌发和花粉管生长的影响,筛选芍药花粉离体萌发最适宜的培养基,并研究了不同pH值对芍药花粉萌发的影响。结果表明,蔗糖对芍药花粉萌发率和花粉管生长影响最大,硼酸次之,硝酸钙影响最小;芍药花粉离体萌发最适宜培养基为,蔗糖20g·L^-1＋硼酸20mg·L^-1＋硝酸钙60mg·L^-1;其萌发率和花粉管长度分别为63．12％和183．33μm;适合芍药花粉萌发的pH为6～7。     

Immunochemical identification of gelsolin in maize pollen

Gelsolin is a representative of a type of actin-binding proteins (ABPs) universally found in eukaryotes. It plays role in nucleation, capping and severing of actin filamentsin vitro. In our experiment, gelsolin was purified from pig plasma and the polyclonal antibodies against it were prepared. The crude extracts of maize pollen were immunodetected by Western-blotting with polyclonal antibody and monoclonal antibody respectively. The immunodetection results show that gelsolin exists in maize pollen and its molecular weight is about 91 ku, similar to that of gelsolin found in animal tissues.