The basic proteins of male gametic nuclei isolated from pollen grains of Lilium longiflorum

A method has been developed for the efficient isolation of generative and vegetative nuclei from the generative and vegetative cells, respectively, of pollen grains of Lilium longiflorum Thunb. First, large numbers of pollen protoplasts were isolated enzymatically from nearly mature pollen grains. After the protoplasts had been gently disrupted by a mechanical method, the generative cells could be separated from the other pollen contents, which included vegetative nuclei. The generative nuclei were isolated by suspending the purified generative cells in a buffer that contained a non-ionic deter gent. The isolated generative nuclei, like those within pollen grains, had highly condensed chromatin and the isolated material was without contamination by vegetative nuclei. When basic proteins, extracted from the preparation of generative nuclei by treatment with 0.4 N H₂SO₄, were compared with those from preparations of somatic and vegetative nuclei by two-dimensional gel electrophoresis, it was revealed that at least five proteins with apparent molecular masses of 35, 33, 22.5, 21 and 18.5 kDa (p35, p33, p22.5, p21 and p18.5), respectively, were specific for, or highly concentrated in, the generative nuclei. An examination of solubility in 5% perchloric acid and the mobility during electrophoresis indicated that two of these proteins (p35 and p33) resembled H1 histones while the three other proteins (p22.5, p21 and p18.5) resembled core histones. It is likely that these basic nuclear proteins are related to the condensation of chromatin or to the differentiation of male gametes in flowering plants, as is the case for analogous proteins present during spermatogenesis in animals.Abbreviations DAPI 4'6-diamidino-2-phenylindole - NIB nuclear isolation buffer This work was supported in part by Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

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.     

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     

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.     

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     

Demonstration of polar zinc distribution in pollen tubes ofLilium longiflorum with the Heidelberg proton microprobe

Summary The zinc distribution in pollen tubes ofLilium longiflorum was analysed by proton-induced X-ray emission (PIXE) with the Heidelberg proton microprobe. A very high zinc concentration was measured in the pollen tube tip region.On leave from Shanghai Institute of Nuclear Research, Academia Sinica, Shanghai, China     

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     

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     

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     

Changes in calcium and calmodulin levels during microsporogenesis,pollen development and germination in Gasteria verrucosa (Mill.) H. Duval

Summary The distribution of membrane calcium and calmodulin (CaM) has been fluorimetrically determined in the anther of Gasteria verrucosa with particular attention to sporogenous cells, meiocytes, microspores, pollen and stages of pollen germination and tube growth using chlortetracycline (CTC) and fluphenazine (FPZ). CTC and FPZ fluorescence in sporogenous cells is relatively higher than in the adjacent tapetal cells, indicating higher membrane calcium and CaM levels in the former cell type. However, during meiosis there is a significant increase in membrane calcium and CaM levels in the meiocytes compared to that found in the young microspores. CTC and FPZ fluorescence in the sporogenous cells, meiocytes and young microspores is punctate and slightly diffused throughout the cytoplasm. In the microspores of the tetrad and the young released microspores CTC fluorescence (CTCf) is polarized and mainly associated with the area opposite the future colporal region. FPZ fluorescence (FPZf) becomes polarized in the young microspore. Subsequently, there is a shift in the polarity, and most of the CTCf and FPZf in the old microspores and pollen is regionalized towards the colporal region, and the fluorescence is more diffused, indicating a change in the organellar-bound calcium and CaM. This final graded distribution of CTCf is maintained during pollen germination in that the growing pollen tubes invariably show a tip to base membrane-calcium gradient. In the tapetal cells a high level of Ca²⁺ is present during the microspore stage. During the preparation for anthesis the endothecium differentiation is marked by the presence of Ca²⁺. Post-treatment of labelled cells with a Ca²⁺ chelator such as EGTA resulted in a substantial decrease in diffuse and punctate CTCf. Alternatively, treatment of cells with non-ionic detergent Nonidet P-40 resulted in the total elimination of CTCf, suggesting that the observed CTC fluorescence was due to membrane-associated calcium. The cytological specification of CTC as a probe for calcium is discussed. From cytofluorometric measurements and atomic absorption, it became clear that the level of Ca²⁺ in the anther is high during the sporogenous and meiotic phases. An increase in CTCf and FPZf occurred after microspore mitosis. An interaction of Ca²⁺ transport from tapetum to the young pollen is postulated. These findings suggest that the level of Ca²⁺ in the anther during meiosis is generally relatively higher than at the sporogenous or young microspore stage. These findings are discussed in the light of available information on the role of Ca²⁺ and CaM-mediated processes such as cell division, callose synthesis and pollen-tube tip growth.     

A cytochemical study on the role of ATPases during pollen germination in Agapanthus umbelatus L'Her.

Summary Cytochemical detection of ATPase activity in the pollen grain (PG) and pollen tube (PT) of Agapanthus umbelatus showed that the enzymes concerned presented specific patterns of membrane distribution according to their ionic dependencies and to the timecourse of germination and tube growth. In the pollen tubes Ca²⁺-ATPases were mainly localized in mitochondria and ER membranes, while Mg²⁺-ATPases were found especially in the tonoplast and in the membrane of the P-particles. K⁺-ATPases showed a high activity at the plasma membrane. In the pollen grain similar patterns of ATPase activity were observed. The highest activity of all three types was observed at the plasma membrane of the grain and at the intine and inner exine layers of the cell wall. The activity observed in the pollen grain cell wall decreased with germination time. In vivo germination studies in the presence of specific inhibitors of the ATPases showed patterns of inhibition that could be correlated with the corresponding ATPase putative role.The results are discussed in terms of the ultrastructural organization of the PG and PT, especially those correlated with (1) formation and maintenance of ionic gradients throughout the PT, (2) polarized growth and (3) hydrodynamics of PT elongation.Abbreviations PT Pollen tube - PG pollen grain - PTW pollentube wall - PGW pollen-grain wall - ER endoplasmic reticulum - NEM N-ethylmaleimide     

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     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

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.     

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.     

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     

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca²⁺ precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca²⁺]_cyt, and in pollen and style maturation during the progamic phase.     

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