myo-Inositol Metabolism in Lilium longiflorum Pollen: Uptake and Incorporation of myo-Inositol-2-H

Germinating Lilium longiflorum pollen absorbs and metabolizes myo-inositol-2-(3)H (MI-2-(3)H) with a pronounced lag when label is supplied from the beginning of germination. If MI-2-(3)H is given after 3 hours of germination, incorporation of labeled metabolic products into pollen tube polysaccharides is constant over a range of 0.56 mm to 2.78 mm MI. When MI-2-(3)H is supplied as a 0.5-hour pulse 3 hours after germination, the proper precursor-product relationship to tube wall polysaccharides is observed. Replacing 10% of the germination media with sigmatic exudate from a compatible cultivar hastens germination and tube elongation. Enhanced MI metabolism accompanies tube growth in this exudate-enriched media.     

Post‐pollination process in a partially self‐compatible distylous plant,Primula sieboldii (Primulaceae)

Pollen germination and pollen‐tube growth under natural conditions were observed in a population of a distylous species, Primula sieboldii, in which partial self‐compatibility has been demonstrated in some long‐styled genets. We observed post‐pollination processes microscopically in styles collected after self‐morph and inter‐morph hand pollination (with standardized pollen load on the stigmas) in four genets each from the following three ‘genet types’: self‐incompatible long‐styled (SI), partially self‐compatible long‐styled (SC) and self‐incompatible short‐styled morph genets. Irrespective of the genet type, pollen germination began within 24 h after pollination and tubes of pollen reached to the style base with 48–96 h after inter‐morph pollination. Although pollen tubes germinated after self‐pollination in the SC genets, the number of germinated pollen tubes was significantly lower than in the case of inter‐morph pollination. Few pollen tubes germinated after self‐pollination of the SI or short‐styled genets. In SC genets, the rate of pollen‐tube growth did not differ between self‐morph and inter‐morph pollination (～1.9 mm/day). Therefore, differences in self‐compatibility between SC and SI genets in P. sieboldii are likely to be attributable to differential pollen germination rates rather than to differential pollen‐tube growth rates.     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

Latrunculin B has different effects on pollen germination and tube growth

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.     

Azetidine-2-carboxylic Acid and Lily Pollen Tube Elongation

Azetidine-2-carboxylic acid (AZC), which occurs naturally inLiliaceous plants, is reported to be a proline (pro) analoguePlant cell walls contain ‘extensin’, which is richin hydroxyproline (hyp). Peptidyl hyp arises through hydroxylationof peptidyl pro followed by glycosylation (arabinose attachment)of hyp Because AZC replaces peptidyl prolyl residues, it maybe a useful tool for evaluating the significance of hyp-o-arabinoselinkages in cell elongation. Therefore, we determined the effectof AZC on [¹⁴C]pro uptake, incorporation and conversion to wall-bound[¹⁴C]hyp in relation to elongation of lily pollen tubes whosewalls consist, in part, of hyp-containing glycopeptides TheAZC suppressed pollen germination 9–42 per cent (1–10mM) and subsequent tube elongation 40–54 per cent (0·1–1mM without affecting respiration In contrast, similar hyp concentrationswere without effect on tube elongation Whereas uptake of [¹⁴C]prowas 16·5–6·2 per cent of the control at0·1–1 mM AZC, [¹⁴C]leucine uptake was 85–25per cent of the control. Light microscope radioautography revealedfewer silver grains over tubes elongated in 0·1–1mM AZC than in its absence. Incorporation of [¹⁴C]pro into tnchloroaceticacid (TCA)-precipitable cytoplasm was reduced by only 10 percent at 0·01–1 mM but 43 per cent at 10 mM AZCGel filtration of cytoplasm from pollen germinated without AZCbut with [¹⁴C]pro resulted in labelled void volume (V) and threeretarded peaks (RI–III) Incorporation into V and RI wasinhibited at both 0·01 and 1 mM AZC These AZC concentrationsreduced conversion of [¹⁴C]pro to wall-bound hyp by 20 percent However, total incorporation of [¹⁴C]pro into salt-water-purifiedwall fractions was suppressed 47–53 per cent (0·1–1mM AZC). Lilium longiflorum, lily, hydroxyproline, proline, azetidine-2-carboxylic acid, pollen, pollen tube elongation     

Inhibition of RNA and protein synthesis in pollen tube development of Pinus bungeana by actinomycin D and cycloheximide

* The effects of actinomycin D and cycloheximide on RNA and protein synthesis were investigated during pollen tube development of Pinus bungeana. * RNA and protein contents, protein expression patterns, cell wall components and ultrastructural changes of pollen tubes were studied using spectrophotometry, SDS-PAGE electrophoresis, Fourier transformed infrared (FTIR) microspectroscopy and transmission electron microscopy (TEM). * Pollen grains germinated in the presence of actinomycin D, but tube elongation and RNA synthesis were inhibited. By contrast, cycloheximide inhibited pollen germination and protein synthesis, induced abnormal tube morphology, and retarded the tube growth rate. SDS-PAGE analysis showed that protein expression patterns changed distinctly, with some proteins being specific for each phase. FTIR microspectroscopy established significant changes in the chemical composition of pollen tube walls. TEM analysis revealed the inhibitors caused disintegration of organelles involved in the secretory system. * These results suggested RNA necessary for pollen germination and early tube growth were present already in the pollen grains before germination, while the initiation of germination and the maintenance of pollen tube elongation depended on continuous protein synthesis.     

In vitro pollen germination in Hypericum perforatum L. and Hypericum rumeliacum Boiss

In vitro pollen germination and pollen tube growth investigations are valuable tools used in identification of the effects of environmental factors and genotypic differences on pollen viability, pollen germination and tube elongation. In this study pollen viability, in vitro pollen germination capacity, abnormality ratios and tube length in germinated pollens of Hypericum perforatum L. and H. rumeliacum Boiss. were investigated. Both of these species has spheroid-shaped and tricolporate pollen grains. The diameters of Hypericum perforatum and H. rumeliacum pollens were found as 24 +/- 3 microm and 19 +/- 2 microm, respectively. Pollen viability of H. perforatum and H. rumeliacum was found as 83% and 72%, respectively. The germination percentages were found as 12.85% for H. perforatum and 64.42% for H. rumeliacurm. Tube lengths in germinated pollens of both taxa were measured approximately as 95.25 +/- 38 microm in H. perforatum and 165.92 +/- 53 microm in H. rumeliacium 4 h after inoculation. In germinated pollen grains of H. perlbratum and H. rumeliacumn abnormality percentages were determined as 13.23% and 43.97%, respectively. In germinated pollens of these two species, highly significant (P < 0.00001) differences in in vitro germination percents and abnormality percents were observed. Abnormalities such as swollen tube tip, branched tube, spiralled tube and excessive tube formation were observed in pollen tubes. The results of this study showed that there were obvious differences in pollen germinability between these two species growing under the same environmental conditions.     

Regulation der Nucleinsäuren-Synthese durch Polyamine in keimendem Pollen vonPetunia

Summary Putrescine, spermine, spermidine, and agmatine in concentrations between 5–15 g/ml inhibit pollen germination. Whereas spermine reduces pollen tube length, putrecine and agmatine do not affect pollen tube growth. Spermidine effects a small increase (about 5%) of pollen tube elongation. Spermine and spermidine can be found in pollen. Addition of spermine (7 or 10 g/ml) depresses protein synthesis, whilst spermidine does not affect protein synthesis. On the basis of uridine-5-T incorporation it could be shown that both spermine and spermidine increase RNA synthesis. On tho basis of thymidine-T incorporation in the first hpurs of germination it seems that DNA synthesis is also stimulated by spermine and spermidine present in the medium. A net increase of nucleic acids was found when spermidine was added to the germination substrate.These results are interpreted as suggesting that, in the pollen tubes investigated, polyamine concentration may be a factor in the regulation of nucleic acid synthesis, resulting in a prolonged synthesis of specific proteins and in this way influencing growth and the developmental pattern of pollen tubes.     

应用水稻花粉离体萌发体系观察花粉管内微丝分布

采用非固定、DMSO渗透和异硫氰酸标记的鬼笔环肽（FITC—Ph）染色方法,观察水稻花粉离体萌发过程中花粉管内肌动蛋白微丝的形态和分布。结果表明：（1）水稻花粉水合2min后即可萌发,花粉管生长速度在600～1500μm／h之间。（2）水合而未萌发的花粉粒中,大量较短的梭形微丝束构成微丝网络结构,萌发过程中花粉粒内的梭形微丝束松解,部分微丝转移至萌发的花粉管内沿花粉管纵轴呈束状结构;随着花粉管的伸长,微丝束主要分布在花粉管中前端,但在花粉管顶端区域始终未见明显的微丝束。（3）水合后不能正常萌发的花粉粒内肌动蛋白微丝呈弥散不规则分布,在相同萌发时间生长迟缓的花粉管中,微丝束较少,且主要位于花粉管近萌发孔的部位。表明微丝骨架的形态和分布影响水稻花粉管的萌发和生长。     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> calmodulin-like protein CML24 regulates pollen tube growth by modulating the actin cytoskeleton and controlling the cytosolic Ca<Superscript>2+</Superscript> concentration

Cytosolic free calcium ([Ca²⁺]_cyt), which is essential during pollen germination and pollen tube growth, can be sensed by calmodulin-like proteins (CMLs). The Arabidopsis thaliana genome encodes over 50 CMLs, the physiological role(s) of most of which are unknown. Here we show that the gene AtCML24 acts as a regulator of pollen germination and pollen tube extension, since the pollen produced by loss-of-function mutants germinated less rapidly than that of wild-type (WT) plants, the rate of pollen tube extension was slower, and the final length of the pollen tube was shorter. The [Ca²⁺]_cyt within germinated pollen and extending pollen tubes produced by the cml24 mutant were higher than their equivalents in WT plants, and pollen tube extension was less sensitive to changes in external [K⁺] and [Ca²⁺]. The pollen and pollen tubes produced by cml24 mutants were characterized by a disorganized actin cytoskeleton and lowered sensitivity to the action of latrunculin B. The observations support an interaction between CML24 and [Ca²⁺]_cyt and an involvement of CML24 in actin organization, thereby affecting pollen germination and pollen tube elongation.     

Greater ability of pollen tube growth in rye plants with 2B chromosomes

Summary To contribute to knowledge on the significance of B chromosomes in rye populations, a component of fertility has been estimated by a comparative study of germination and growth of pollen tubes in plants with and without B chromosomes. The highest percentage germination of pollen and mean speed of pollen tube growth was shown by 14 + 2B plants, followed by normal plants.Observations of first pollen mitosis indicated that in most cases the chromatid constitution of the tube nucleus was the same in 14, 14 + 1 and 14 + 2B plants. In contrast, the mean growth rate of pollen tubes differed significantly among the three cases. From these data it seems that the determination of germination ability and pollen tube growth depends on the sporophyte and is related to the number of Bs carried by the plant.     

Heterostyly inPrimula. 2. Sites of pollen inhibition,and effects of pistil constituents on compatible and incompatible pollen-tube growth

Summary In incompatible (intramorph) pollinations of the heterostylousPrimula vulgaris, pollen germination or tube growth may be partially inhibited in several sites associated with the stigma or style. Blockage may occur, a) on the stigma surface through the failure of germination or of pollen tube penetration after germination, b) in the stigma head during the passage of the tube through the specialized transmitting tissue of the head, or c) in the transmitting tract of the style. None of the barriers is complete, and the prohibition of selfing or intramorph crossing depends upon the cumulative screening effect of one following upon the other. In both morphs, the germination of incompatible pollen on the stigma is enhanced in high ambient relative humidity, but many tubes still fail to penetrate the stigma. Those that do are retarded or blocked in their growth in the transmitting tissues of the stigma head and style. Crude extracts from the tissues of the stigma head and style show some differential effect on the growth of pollen tubesin vitro, and dialysates of extracts containing high molecular weight fractions show a consistent differential effect, those from thrum tissues retarding thrum tubes while having a lesser effect on pin tubes, and those from pin tissues retarding pin tubes while having lesser effect on thrum. It is suggested that the factors influencing tube growth are present in the intercellular secretions of the transmitting tract.     

Differences in in vitro pollen germination and pollen tube growth of cotton cultivars in response to high temperature

* BACKGROUND AND AIMS: High-temperature environments with >30 degrees C during flowering reduce boll retention and yield in cotton. Therefore, identification of cotton cultivars with high-temperature tolerance would be beneficial in both current and future climates. * METHODS: Response to temperature (10-45 degrees C at 5 degrees C intervals) of pollen germination and pollen tube growth was quantified, and their relationship to cell membrane thermostability was studied in 12 cultivars. A principal component analysis was carried out to classify the genotypes for temperature tolerance. * KEY RESULTS: Pollen germination and pollen tube length of the cultivars ranged from 20 to 60 % and 411 to 903 microm, respectively. A modified bilinear model best described the response to temperature of pollen germination and pollen tube length. Cultivar variation existed for cardinal temperatures (T(min), T(opt) and T(max)) of pollen germination percentage and pollen tube growth. Mean cardinal temperatures calculated from the bilinear model for the 12 cultivars were 15.0, 31.8 and 43.3 degrees C for pollen germination and 11.9, 28.6 and 42.9 degrees C for pollen tube length. No significant correlations were found between pollen parameters and leaf membrane thermostability. Cultivars were classified into four groups based on principal component analysis. * CONCLUSIONS: Based on principal component analysis, it is concluded that higher pollen germination percentages and longer pollen tubes under optimum conditions and with optimum temperatures above 32 degrees C for pollen germination would indicate tolerance to high temperature.     

Pollen germination, tube growth and morphology, and microtubule organization after exposure to benomyl

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0,480, 600 or 720 mg 1⁻¹. Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.     

Effects of Nifedipine on Pollen Germination,Pollen Tube Growth and Division of Generative Nucleus in Nicotiana tabacum

The deals with the effects of nifedipine (Nif), a Ca2+ channel blocker of rather high specificity, on pollen germination, pollen tube growth and division of generative nucleus (GN) in experimentlly germinated pollen tubes of Nicotiana tabacurn L. Pollen germination was inhibited by the addition of 10-4 mol/L Nif whereas no significant inhibition by 10-7~10-5 mol/L Nif was observed. The effects of Nif on pollen tube growth were related to its concentration and duration of treatment. At the earlier stage, tube growth was promoted at the lower concentrations (10-7~10-5 mol/L), but was significantly inhibited at a concentration of 10 4 mol/L Nif. With increasing time of culture, even the lower concentrations also became harmful; the stronger the concentration, the earlier the transition from promotion to inhibition. Generally, inhibition of tube growth occurred within 24 hours of culture with different extent in various concentrations. Moreover, higher concentrations also tended to disturb tube morphology and cytoplasmic streaming. Nif was observed to perturb GN division at various concentrations, either blocked it completely at 10-4 mol/L, or only delayed it at 10-7~10-5 mol/L. The dynamics of membrane-associated calcium in pollen tubes was tested with chlorotetracycline (CTC). With increasing time of culture and escalating Nif concentration, CTC fluorescence weakened gradually, indicating that the physiological effects of Nif is mediated by its in hibition on Ca2+ channel activities.     

沙田柚花柱蛋白对花粉管生长的影响

本文以沙田柚及其授粉树种酸柚为材料,对其花粉在不同培养介质中的萌发率进行测定,筛选了０２～０４ｍｏｌ／Ｌ聚乙二醇－４００（ＰＥＧ－４００）＋００１％硼酸作为花粉萌发介质。用授粉后３ｄ的花柱提取液进行沙田柚、酸柚花粉萌发抑制实验,花柱提取液对自交花粉萌发无抑制作用,明显地抑制花粉管的生长。其中以花柱提取液的３５％饱和度硫酸铵级分对花粉管伸长的抑制最明显。模拟了花粉在雌蕊中的生长方式,进行花粉萌发生长,能收集到较多的花粉管,为研究花粉管的Ｓ－糖蛋白提供了材料。     

Isolation and Cultural Behavior of Pollen Tube Subprotoplasts in,Antirrhinum majus L.

Large numbers of subprotoplasts were isolated enzymatically from pollen tubes of Antirrhinum majus L. When these subI)rotoplasts, either nucleate or enucleate, were cultured in D2 liquid eulture medium, each formed a thick cell wall and germinated a pollen tube like strueture which also deposited a thick wall, except at the tip of the tube. Tube growth was accomparied by a continuous movement of the mass of cell inelusion in this tube to the tip. Rupture of the naked tip oeeurred within one to six days releasing the mass of cell inelusion in the tube into the culture medium. The faet that both nucleate and enneleate subprotoplasts show the same cultural behavior eharaeteristie of the gene expression of a normal pollen tube demonstrates the presence of presynthesized mRNA in the germinated tubes.     

Localization of pectins in the pollen tube wall of Ornithogalum virens L. Does the pattern of pectin distribution depend on the growth rate of the pollen tube?

Monoclonal antibodies that recognize pectins were used for the localization of esterified (JIM7) and acidic, unesterified (JIM5) forms of pectin in pollen tube walls of Ornithogalum virens L. (x = n = 3). The results indicated that the distribution of the two forms of pectin in the pollen tube wall depended on the medium (liquid or solid) used for pollen germination. In pollen tubes grown in the liquid medium, the localization of JIM7 was limited to the very tip of the pollen tube, whereas the localization of JIM5 indicated a uniform distribution of unesterified pectins in the very tip of the tube and along the subapical parts of the tube wall. In tubes germinated on the medium stabilized with agar (1–2%) the localization of JIM7 and JIM5 indicated the presence of both forms of pectin in the tube tip and along the whole length of the pollen tube wall in a ring-like pattern. Thus, the localization of esterified pectins in the sub-apical part of the pollen tube wall, below the apex of the tube, is described for the first time. Measurements of the growth rates of pollen tubes growing on the two types of medium indicated that oscillations in tube growth rate occur but these do not coincide with the pattern of pectin distribution in the tube wall. Our results complement the previous data obtained for the localization of JIM5 and JIM7 in pollen tube walls of other plant species. (Y.-Q. Li et al. 1994, Sex Plant Reprod 7: 145–150) and provide new insight into an understanding of the construction of the pollen tube wall and the physiology of pollen grain germination. Received: 25 January 1999 / Accepted: 23 June 1999     

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

 We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. Received: 28 October 1996 / Revision accepted: 24 January 1997