Male gametic nucleus-specific H2B and H3 histones,designated gH2B and gH3, in Lilium longiflorum

Two proteins that resemble core histones and might be specific to the male gametic (generative) nucleus within the pollen of Lilium longiflorum Thumb, (originally designated p22.5 and p18.5; K. Ueda and I. Tanaka, 1994, Planta, 192, 446–452) were characterized biochemically and immunochemically. Patterns of digestion of p22.5 and p18.5 by Staphylococcus aureus V8 protease closely resembled those of somatic histones H2B and H3, respectively. However, peptide fragments that were unique to p22.5 or p18.5 were also detected. Antibodies raised against these proteins did not cross-react with any somatic histones. These results indicate that p22.5 and p18.5 are different from somatic histones in terms of primary structure. Analysis of their amino-acid compositions revealed that p22.5 is a moderately lysine-rich protein while p18.5 is an arginine-rich protein. From these results, we conclude that p22.5 is a variant of histone H2B and p18.5 is a variant of histone H3. Immunofluorescence staining of pollen grains using the specific antibodies revealed that both p22.5 and p18.5 are only present in the generative cell nucleus and are not to be found in the vegetative cell nucleus. This study demonstrates that (i) specific histone variants are present in the male gametic nucleus of a higher plant, as they are in the sperm nucleus of animals, and (ii) distinct differences in histone composition exist between the nuclei of generative and vegetative cells in pollen. These novel histones (p22.5 and p18.5), specific to male gametic nuclei, have been designated gH2B and gH3, respectively.Abbreviations DAPI 46-diamidino-2-phenylindole - FITC fluorescein isothiocyanate The authors thank Dr. Y. Sado (Shigei Medical Institute, Japan) for his helpful advice on immunization and Prof. T. Iguchi and Prof. K. Manabe (Yokohama City University, Japan) for providing facilities for experiments. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum

Chlorotetracyclin (10^-4M) has been used to observe the distribution of membrane-associated calcium during pollen germination of Lilium longiflorum. For comparison, the general membrane distribution has been determined with 4·10^-5 M fluorescamine. The pollen grains show a calcium gradient with either weak or strong chlorotetracycline-fluorescence intensity, but always increasing toward the germination colpus. This gradient intensifies during germination, reaching a maximum before the pollen tube emerges. The typical tip-to-base calcium gradient of the tube does not change during growth. Independent of the developmental stage, the pollen grains show a flat fluorescamine-fluorescence gradient with the highest intensity in one half of the grain. Pollen tubes reveal a tip-to-base membrane gradient, independent of their length. As an additional marker for membrane distribution, the distribution of phosphorus, measured by proton-induced X-ray emission in chemically fixed tubes, has been used. A tip-to-base phosphorus gradient, distinct from the calcium gradient measured with the same method, was detected.Abbreviation CTC chlorotetracycline     

Covalently bound wall proteins of pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination in Lilium longiflorum

Summary A method was worked out using trifluoromethanesulfonic acid (TFMS) as a reagent to split the covalently bound proteins, which are NaCl insoluble, from pollen tube walls of Lilium longiflorum, leaving the peptide bonds essentially intact. After electrophoretic separation, comparisons were made among these proteins from pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination. It was found that a) the patterns of covalently bound wall proteins were different between tubes grown in vitro and in vivo; b) fewer bands were found in covalently bound wall proteins than that in noncovalently bound proteins; c) the bands remained almost the same no matter whether the tubes had been cross pollinated or self pollinated, indicating that while the noncovalently bound proteins were involved in incompatibility as shown in the previous paper, the covalently bound proteins may only serve as a structural component, having little to do with incompatibility.     

Broad-range effects of ionophore X-537A on pollen tubes of Lilium longiflorum

The effects of the broad-range cationophore X-537A on pollen tubes of Lilium longiflorum were investigated, using both light and electron microscopy. Pollen tube growth is completely inhibited within 30 min after the application of 5·10^-5 M ionophore X-537A; cytoplasmic streaming is stopped only after 60 min of ionophore treatment. Ultrastructurally, X-537A effects are a vacuolation of Golgi cisternae and a general vacuolation. The wall is thickened at the very tip. Coated vesicles and coated regions are enriched close to and at the plasma membrane. The results indicate that pollen tube tip growth needs a specific ion distribution.Abbreviations CTC chlorotetracycline - DMSO dimethylsulfoxide     

Ultrastructure of freeze-substituted pollen tubes ofLilium longiflorum

Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial slow and fast lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.Abbrevations CF chemical fixation - ER endoplasmic reticulum - GC generative cell - MF microfilament - MT microtubule - PD plasmodesmata - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution - VN vegetative nucleus     

Isolation of generative cells and their protoplasts from pollen ofLilium longiflorum

Summary Methods are described for the isolation of large quantities of generative cells and their protoplasts from the pollen ofLilium longiflorum. First, large numbers of pollen protoplasts were enzymatically isolated from immature pollen grains. When they were gently disrupted mechanically, the pollen contents including spindle-shaped generative cells were released. The generative cells were separated from other structures by Percoll density gradient centrifugation. They were nearly spherical, but had a callosic cell wall. The isolated generative cells were then re-treated in enzyme solution to yield authentic protoplasts. The generative cell protoplasts, gametoplasts, were uniform in size and contained a condensed haploid nucleus with relatively little cytoplasm.     

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.     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

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     

Histone H3 variants in male gametic cells of lily and H3 methylation in mature pollen

Histones are vital structural proteins of chromatin that influence its dynamics and function. The tissue-specific expression of histone variants has been shown to regulate the expression of specific genes and genomic stability in animal systems. Here we report on the characterization of five histone H3 variants expressed in Lilium generative cell. The gcH3 and leH3 variants show unique sequence diversity by lacking a conserved lysine residue at position 9 (H3K9). The gH3 shares conserved structural features with centromeric H3 of Arabidopsis. The gH3 variant gene is strongly expressed in generative cells and gH3 histone is incorporated in to generative cell chromatin. The lysine residue of H3 at position 4 (H3K4) is highly methylated in the nuclei of generative cells of mature pollen, while methylation of H3K4 is low in vegetative cell nuclei. Taken together, these results suggest that male gametic cells of Lilium have unique chromatin state and histone H3 variants and their methylation might be involved in gene regulation of male gametic cells.Accession numbers for the sequence data The sequences reported in this paper have been deposited in the DDBJ database gcH3 GC1174 (accession no. AB195644), gH3 GC1008 (accession no. AB195646), leH3 GC1126 (accession no. AB195648), soH3-1 GC0075 (accession no. AB195650), soH3-2 GC1661 (accession no. AB195652), genomic sequence of gcH3 (accession no. AB195645), genomic sequence of gH3 (accession no. AB195647), genomic sequence of leH3 (accession no. AB195649), genomic sequence of soH3-2 (accession no. AB195651), genomic sequence of soH3-2 (accession no. AB195653).     

Calcium ionophore A 23187 affects localized wall secretion in the tip region of pollen tubes of Lilium longiflorum

The effects of the calcium inonophore A 23187 on growing pollen tubes of Lilium longiflorum Thunb. cv. Ace were investigated with the light and electron microscope. Tip growth is slowed down and stopped within 20 min after application of 5x10^-5 M ionophore A 23187. The main effects are the disappearance of the clear zone at the pollen tube tip and a thickening of the cell wall at the tip and at the pollen tube flanks. This effect on cell wall formation is confirmed under the electron microscope: The vesicular zone in treated pollen tubes is reduced, numerous vesicular contents are irregularly integrated in the pollen tube wall not only in the tip, but over a long distance of the pollen tube wall. In addition, effects on mitochondria and dictyosomes are observed. These results are interpreted as a disorientation of the Ca²⁺-based orientation mechanism of exocytosis after equilibration of the Ca²⁺-gradient     

Disoriented growth of pollen tubes of Lilium longiflorum Thunb. induced by prolonged treatment with the calcium-chelating antibiotic,chlorotetracycline

Pollen of Lilium longiflorum Thunb. was germinated for 12 h in growth medium containing 1·10^-4 M chlorotetracycline (CTC), or growing tubes were treated with 1·10^-4 M CTC for up to 2 h. These treatments have drastic effects: In the CTC-containing medium, out-growing tubes form only short tubes. Irregular wall thickenings are visible. Thirty minutes CTC-treatment cause growing tubes to bend and grow back toward the grain. Electron micrographs of CTC-treated tubes show that CTC affects the organelle distribution: The polar zonation of organelles is disturbed. Vesicle-and endoplasmic reticulum-accumulations are found in the wrong places, together with extensive wall thickenings and a very irregular plasma membrane. The structural details of most cell organelles look normal after CTC treatment, but the mitochondria possess unusual cristae, and microtubules are absent. The disoriented growth is interpreted as an effect of the ability of CTC to chelate intracellular calcium ions, to bind them to membranes, and thus to disturb the dynamics of the delicate Ca²⁺-equilibria thought to regulate oriented exocytosis.Abbreviations CTC chlorotetracycline - ER endoplasmic reticulum     

Growth inhibition and recovery in freeze-substitutedLilium longiflorum pollen tubes: structural effects of caffeine

Summary In an attempt to correlate structural effects with the known dissipation of the tip-focused Ca²⁺ gradient caused by caffeine, we have examined the ultrastructure of caffeine-treated lily pollen tubes prepared by rapid freeze fixation and freeze substitution. We show that treatment with caffeine results in a rapid rearrangement of secretory vesicles at the pollen tube tip; the normal cone-shaped array of vesicles is rapidly dispersed. In addition, microfilament bundles appear in the tip region, where they had previously been excluded. Delocalized vesicle fusion continues in the presence of caffeine but tube extension ceases. Removal of caffeine from the growth medium initially causes tip swelling, delocalized vesicle fusion and presence of microfilaments well into the tip before normal structure and growth resume, concurrent with the previously reported return to a normal Ca²⁺ gradient.Abbreviations ER endoplasmic reticulum - MES 2-[N-morpholino] ethanesulfonic acid - MFs microfilaments     

Histone H4 acetylation and DNA methylation dynamics during pollen development

Summary The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.     

Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.Li Wang and Yi-Min Liu are considered joint first authors     

A kinematic analysis of tepal growth in Lilium longiflorum

Time-lapse marking experiments indicate that the growth of tepals in Lilium longiforum Thunb. from 3.7 mm to maturity is triphasic. Phase I (tepal lengths 3.7–10 mm) is characterized by spatial and temporal variation in growth rate and, in the epidermis, a random distribution of mitoses with an acropetal increase in cell area. During phase II (10–90 mm) cell elongation and (later) cell division is restricted largely to basal regions. Cell division ceases when tepals are less than one-third of their mature length of 155 mm. Phase III (90–155 mm) is characterized by the gradual transition from basal to apical growth, and a modification of epidermal cell shape. A sharp peak in growth at the extreme tip of the tepal coincides with anthesis.Abbreviations LRGR local relative growth rate - RER relative elemental rate of growth     

Quantitative analysis of calcium gradients and activity in growing pollen tubes ofLilium longiflorum

Summary Pollen tubes ofLilium longiflorum were loaded with quin-2 to determine the cytoplasmic free calcium. Quin-2-fluorescence was detected at 500 nm with alternating excitation at 340 nm and 360 nm. The calcium²⁺-concentration was obtained using the intensity ratio R=I₃₄₀/I₃₆₀. The analysis exhibits a [Ca²⁺] of nearly 10^–7mol·l^–1 in the tip region and about 2·10^–8 mol·l^–1at the tube base, near the pollen grain. The data give evidence for the existence of a continuous gradient of free calcium within growing pollen tubes of various length.     

Enforced growth-rate fluctuation causes pectin ring formation in the cell wall of Lilium longiflorum pollen tubes

Normally growing lily (Lilium longiflorum Thunb.) pollen tubes cultured in standard sucrose medium display a relatively steady tip-growth pattern and a rather even pectin sheath in the cell wall. In an attempt to better understand pulsatory growth, observed in some species, e.g., Petunia, and its possible role in causing the formation of thickened cell wall rings, we have imposed marked fluctuations in the growth-rate of lily pollen tubes. The appropriate growth-perturbing conditions were achieved by modulating the medium osmolarity or by applying caffeine, a non-turgor inhibitor, in a specially designed incubation chamber with a controlled medium flow. The relatively non-esterified pectin deposition in the wall of the growth-interrupted pollen tubes was detected by immunofluorescence microscopy using a monoclonal antibody, JIM 5. The observations show that the periods of slow or inhibited growth correspond to the times when the thickened walls are deposited. Since the growth fluctuations were induced by both turgor- and non-turgor-related means, the proposed endogenous regulatory role of turgor pressure is questioned. Other factors, such as the tip-focused Ca²⁺ gradient which was demonstrated by ratiometric ion imaging, and the alteration in the extensibility of the cell wall, which correlated with pectin esterification/de-esterification, emerge as candidates for the regulation of growth fluctuations.     

Alkaline phytase from Lilium longiflorum: purification and structural characterization

Phytases catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytases are of great commercial importance because their use as food and animal feed supplement has been approved by many countries to alleviate environmental and nutritional problems. Although acid phytases have been extensively studied, information regarding alkaline phytases is limited. Alkaline phytases with unique catalytic properties have been identified in plants, however, there is no report on the purification or structural properties. In this paper, we describe the purification of alkaline phytase from plant tissue. The purification was challenging because of contamination from non-specific phosphatases and acid phytases and low endogenous concentration. The purification of alkaline phytase from pollen grains of Lilium longiflorum involved selective precipitation by heat and ammonium sulfate followed by anion exchange and chromatofocusing chromatography and, finally, gel electrophoresis. Alkaline phytase was purified approximately 3000-fold with an overall recovery of 4.2%. The native molecular mass was estimated to be in the range of 118+/-7 kDa by Ferguson plot analysis and Mr of denatured protein in the range of 52-55 kDa by SDS-PAGE suggesting that the enzyme is a homodimer. Separation by 2-D gel and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis of separated proteins indicates the presence of multiple mass and charge isoforms with pI values between 7.3 and 8.3. To our knowledge, this is the first alkaline phytase to be purified from plant sources. The unique properties suggest that the enzyme has the potential to be useful as a feed and food supplement.