Localization of Phosphorus and Cation Reserves in Lilium longiflorum Pollen

Transmission electron microscopy of pollen from Lilium longiflorum Thunb. reveals electron-dense inclusions in storage body organelles ubiquitous in the cytosol. In ungerminated pollen, these inclusions are rounded in appearance and appressed to the inner surface of the smooth membrane of the storage body. During pollen germination, these inclusions become less rounded, smaller, and enclosed in storage bodies that have developed crenated membranes. Energy dispersive x-ray analysis reveals high levels of P, Mg, K, and Ca in the inclusions relative to other regions of the cytosol in which elemental signals can be obtained. The elemental composition and the degradation of inclusions during germination are offered as evidence for storage of phytin in these structures which are thus analogous to phytin storage globoids of seed tissues.     

A Calcium-Activated Phytase from Pollen of Lilium longiflorum

A phytase was isolated and partially purified from the pollen of Lilium longiflorum Thumb. Optimum activity was at pH 8.0. The phytase was activated by Ca²⁺ and Sr²⁺ but not by the other divalent cations tested. Activity was inhibited by ethylenediaminetetraacetate. The phytase had a temperature optimum of 55 to 60°C and an activation energy of about 12,700 calories/mole. Extraction of L. longiflorum pollen with 0.1% Triton X-100 increased recovery of the phytase by nearly 4-fold. The phytase had a molecular weight of about 88,000 as determined by gel filtration chromatography and a K_m value of 7.2 micromolar for phytic acid in the presence of Ca²⁺.     

Metabolism of myo-Inositol by Germinating Lilium longiflorum Pollen

Lilium Iongiflorum pollen tubes absorbed myo-[2-³H]inositol produced labeled metabolites which were separated into acid-soluble and -insoluble fractions. The soluble fraction contained labeled myo-inositol, d-glucuronic acid, myo-inositol 1-phosphate, and at least three other unidentified compounds. The acid-insoluble fraction contained considerable chloroformsoluble radioactivity and a labeled residue. Labeled myo-inositol was also absorbed by germinating pollen prior to the time of pollen tube initiation; however, there was a marked reduction in amounts of myo-inositol 1-phosphate and glucuronic acid produced by this pollen in comparison with growing pollen tubes.     

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb

A Mg²⁺-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and β-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg²⁺. At 4 millimolar, Co²⁺, Fe²⁺ or Mn²⁺ are less effective. Substantial inhibition is obtained with 0.25 molar Li⁺. With β-glycerophosphate as substrate the K_m is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, β-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.     

beta-1, 3-Glucan Synthase from Lilium longiflorum Pollen

A particulate fraction from pollen tubes and ungerminated pollen of Lilium longiflorum incorporated ¹⁴C-glucose from UDP-glucose-¹⁴C into a lipid fraction and into β-1, 3-glucan. Partial hydrolysis of the glucan yielded laminaribiose as the only radioactive disaccharide. The preferred substrate was UDP-glucose, and enzyme activity was stimulated by glucose and by β-linked di- and trisaccharides. Enzyme from growing pollen tubes synthesized β-1, 3-glucan more rapidly and produced a higher proportion of alkali-insoluble glucan than did enzyme from ungerminated pollen. The onset of pollen tube growth may be dependent on altered activity of β-1, 3-glucan synthase.     

Evidence for a Functional myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen

Addition of myo-inositol to pentaerythritol-based germination media repressed the conversion of d-[1-¹⁴C]glucose to labeled uronosyl and pentosyl units of tube wall pectic substance in lily pollen (Lilium longiflorum Thunb.). Conversion of d-[1-¹⁴C]glucose to labeled glucosyl, galactosyl, and rhamnosyl units was unaffected. The reverse experiment, addition of d-glucose to pentaerythritol-based media, failed to affect the conversion of myo-[2-³H]inositol to uronosyl and pentosyl units although the flow of label into products of myo-inositol-linked glucogenesis was blocked. Results of these experiments are discussed in terms of a functional myo-inositol oxidation pathway.     

Phytic Acid Metabolism in Lily (Lilium longiflorum Thunb.) Pollen

The accumulation of phytic acid during development of lily (Lilium longiflorum Thunb.) pollen and its degradation during germination have been studied. A substantial amount of phytic acid accumulates in lily pollen by 5 days before anthesis, and little change occurs during subsequent maturation. Mature lily pollen contains 7 to 8 micrograms phytic acid per milligram pollen. Considerable degradation of phytic acid occurs by 15 minutes of incubation in glucose culture medium, and very little is left by 3 hours. No partially phosphorylated myo-inositol accumulates during germination. The breakdown of phytic acid proceeds at a constant rate during this time period. The rate is calculated to be 0.037 microgram phytic acid/milligram pollen/minute. Two phytases are detected in germinated lily pollen extract using high performance liquid chromatography with an anion exchange column (diethylaminoethyl-5PW). The results suggest that one of the phytases is already present in mature ungerminated lily pollen and the other one is newly synthesized during germination from a long-lived, pre-existing mRNA.     

Pollen tube penetration and fertilization in Lilium longiflorum (Liliaceae)

The ultrastructure of the embryo sac, nucellus, and parts of the micropyle of Lilium longiflorum were studied both before and after pollen tube penetration to examine the interactions between ovule and pollen tube, using transmission electron microscopy and light microscopy. Before pollen tube penetration the egg cell and two synergids are similar. No filiform apparatus was detected and no synergid degeneration occurs prior to pollen tube penetration. The polar nuclei do not fuse until fertilization. No differences in embryo sac ultrastructure were detected between pollinated ovules unpenetrated by pollen tubes and unpollinated flowers of a comparable age. Shortly after the discharge of the pollen tube two enucleated cytoplasmic bodies with different ribosome densities were observed in the degenerated cytoplasm. These structures border both on the central cell and the egg cell as well as each other and are interpreted as remains of sperm cytoplasm after transmission of sperm nuclei. In the central cell both the sperm nucleus and the polar nuclei are associated with endoplasmic reticulum (ER). ER is thought to be a transport mechanism to achieve contact between the haploid polar nuclei and the sperm nucleus. In the egg cell sperm nucleus alignment is not visibly achieved by ER. The persistent cells of the egg apparatus and the central cell appear to become more metabolically active after pollen tube penetration. Pollen tube penetration already occurs despite the absence of a filiform apparatus and a low level of differences between the cells of the egg apparatus.     

Pistil Exudate Production and Pollen Tube Growth in Lilium longiflorum Thunb.

Exudate production in the pistil of Lilium longiflorum was studiedin relation to pollen tube growth, using scanning electron microscopy(SEM), transmission electron microscopy and light microscopy.In contrast with conventional fixation for SEM, during whichthe exudate of L. longiflorum largely washes away, the exudateremains present through freezing in case of cryo-SEM. Usingthe latter method we observed that exudate production on thestigma and in the style started before anthesis. Just underneaththe stigma the exudate was first accumulated at the top of eachsecretory cell, followed by a merging of those accumulationsas exudate production proceeded. Exudate is also produced bythe placenta. It was however not possible to determine whetherany of this fluid originated from the micropyle. Apart fromthe cell shape and the cuticle present in between the secretorycells, the ultrastructure of the secretory cells covering theplacenta was comparable to those of the stylar canal. The transferwall of the secretory cells of the placenta originated fromfusing Golgi vesicles but the endoplasmic reticulum seemed tohave an important role as well. After pollination the pollen tubes grew across the stigma andentered the style through one of the slits in the three stigmalobes. The pollen tubes grew straight downward through the styleand were covered by exudate. As the pollen tubes approachedthe ovary their growth was restricted to the areas with secretorycells. In the cavity the pollen tubes formed a bundle and theybent from this bundle in between the ovules towards the micropylarside. There they bent again to stay close to the secretory cells.After bud pollination the pollen tube growth was retarded. Laterarriving pollen tubes had a tendency to grow close to the secretorycells of the style, which resulted in a growth between thesecells and preceding pollen tubes. If there was still a littleexudate produced, it resulted in a lifting up of the pollentubes, out of the exudate. The relationship between exudateproduction and pollen tube growth is discussed. Both the speedand the guidance of the pollen tube seemed determined by theproperties of the exudate.Copyright 1994, 1999 Academic Press Cryo-scanning electron microscopy, exudate, Lilium longiflorum, lily, ovary, pollination, pollen tube growth, secretory cell, stigma, style     

Incorporation of Label into Pollen Tube Walls from Myoinositol-labeled Lilium longiflorum Pistils

Compatible and incompatible pollen tubes growing on detached Lilium longiflorum pistils which had been prelabeled with myoinositol-U-(14)C take up a portion of the label and utilize it for biosynthesis of tube wall substance. The label is transferred from pistil to pollen tubes apparently via the secretion products (exudate) of the pistil. The exudate thus appears to have a major nutritional role in pollen tube growth in vivo.     

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.     

Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb

myo-Inositol-1-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to 1l-and 1d-myo-inositol-1-phosphate, it shows high specificity for 1l-chiro-inositol-3-phosphate. As observed earlier with other primary phosphate esters, d-glucitol-6-phosphate and d-mannitol-6-phosphate are hydrolyzed very slowly. No activity is observed with inorganic pyrophosphate or myo-inositol pentaphosphate as substrate. The enzyme is inhibited by fluoride, sulfate, molybdate, and thiol-directed reagents. Partial protection against N-ethylmaleimide inhibition by substrate and Mg²⁺ together suggests sulfhydryl involvement at the active site.     

Growth of anthers in Lilium longiflorum

The post-initiation growth of 64 anthers (1.1–17.4 mm long) in Lilium longiflorum Thumb. was examined by time-lapse marking experiments in combination with serial sections and the scanning electron microscope. Each anther was characterized by spatial and temporal variation in growth rate. Larger anthers had two, and occasionally three, series of peaks and troughs in local growth rate. Regions of negative growth rate were frequently encountered. When observed over several days, the growth maxima and minima were found to move basipetally as a waveform down the length of the anther. The wavelength was longer in taller anthers; amplitude and frequency were variable, and anthers of the same size were not always synchronous. Distribution patterns of cell division (and elongation, once division has ceased) recapitulate the growth data. Anther growth is a non-steady system, therefore, with growth centers constantly shifting. Implications for future studies in organ growth patterns are discussed.Abbreviation SEM scanning electron microscope     

Microprojectile bombardment-mediated transformation of Lilium longiflorum

We have obtained transgenic lily (Lilium longiflorum) plants after microprojectile bombardment, using the Biolistics PDS 1000/He system, of morphogenic calli derived from bulblet scales, followed by bialaphos selection. Parameters which gave the highest transient uidA expression were used: a bombardment pressure of 1100 psi, a target distance of 6 cm and a 48-h preculture on medium with 3% sucrose. A total of 1800 morphogenic calli were co-bombarded with plasmids containing either the uidA reporter or PAT selectable marker genes. After bombardment, the calli were exposed to 2 mg/l bialaphos. Only 72 of the shoot-forming calli (4%) survived. The 72 shoot clusters produced 342 shoots on elongation medium containing 0.5 mg/l bialaphos. Only 55 plantlets survived subsequent exposure to 2.0 mg/l bialaphos. PCR analysis indicated that 19 of these plantlets contained the PAT transgene. Southern analysis of 3 of the plants indicated that all contained the PAT gene. Received: 21 March 1997 / Revision received: 8 July 1997 / Accepted: 7 August 1997     

Uridine Diphosphate Glucose Metabolism and Callose Synthesis in Cultured Pollen Tubes of Nicotiana alata Link et Otto

Membrane preparations from cultured pollen tubes of Nicotiana alata Link et Otto contain a Ca2+ -independent (1-3)-[beta]-D-glucan (callose) synthase activity that has a low affinity for UDP-glucose, even when activated by treatment with trypsin (H. Schlupmann, A. Basic, S.M. Read [1993] Planta 191: 470-481). Therefore, we investigated whether UDP-glucose was a likely substrate for callose synthesis in actively growing pollen tubes. Deposition of (1-3)-[beta]-glucan occurred at a constant rate, 1.4 to 1.7 nmol glucose min-1, in tubes from 1 mg of pollen from 3 h after germination; however, the rate of incorporation of radioactivity from exogenous [14C]-sucrose into wall polymers was not constant, but increased until at least 8 h after germination, probably due to decreasing use of internal reserves. UDP-glucose was a prominent ultraviolet-absorbing metabolite in pollen-tube extracts, with 1.6 nmol present in tubes from 1 mg of pollen, giving a calculated cytoplasmic concentration of approximately 3.5 mM. Radioactivity from [14C]-sucrose was rapidly incorporated into sugar monophosphates and UDP-glucose by the growing tubes, consistent with a turnover time for UDP-glucose of less than 1 min; the specific radioactivity of extracted UDP-[14C]glucose was equal to that calculated from the rate of incorporation of [14C]sucrose into wall glucans. Large amounts of less metabolically active neutral sugars were also present. The rate of synthesis of (1-3)-[beta]-glucan by nontrypsin-treated pollen-tube membrane preparations incubated with 3.5 mM UDP-glucose and a [beta]-glucoside activator was slightly greater than the rate of deposition of (1-3)-[beta]-glucan by intact pollen tubes. These data are used to assess the physiological significance of proteolytic activation of pollen-tube callose synthase.     

西瓜花粉壁超微结构与花粉ATP酶细胞化学定位

研究了西瓜花粉壁超微结构以及单核花粉液泡化时期ATP酶活性超微细胞化学定位。花粉壁的外壁分为外层和内层 ,外层包括覆盖层、基粒棒和基足层等三层 ,内层只包含一层。外层电子密度相对较小 ,内层电子密度相对较大 ;外层与内层之间有缝隙。ATP酶活性反应产物主要分布在细胞质基质、质体、内质网和花粉内壁中     

Electrorotation of Isolated Generative and Vegetative Cells, and of Intact Pollen Grains of Lilium longiflorum

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m⁻¹. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques. Received: 9 June 1997/Revised: 4 August 1997