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     

Purification and characterization of histone H1 from meiotic cells of a monocotyledonous plant, Lilium longiflorum.

To identify molecules involved in regulating meiotic chromatin structure, nuclear proteins from meiocytes of Lilium longiflorum were chromatographed on hydroxylapatite and the bound and unbound proteins were examined. An abundant nuclear protein was purified from the unbound fraction and by the following criteria was identified as a histone H1 molecule. The protein is soluble in acidic (perchloric and sulfuric acid) solutions, and its amino acid composition and the sequence of its amino terminus are similar to that of known histone H1s. An antiserum was produced against the protein to facilitate subsequent studies. Immunoblotting experiments demonstrated that histone H1 immunostaining declines in the developmental interval spanning the diplotene to tetrads stages. Concommitant with this decline is the appearance of several lower molecular mass, cross-reacting proteins. Neither the identity nor roles of these proteins is known. Immunoblotting experiments also demonstrate that, while the level of the protein is relatively constant in nuclei prepared from meiotic and vegetative cells, histone H1 is apparently enriched in total cellular extracts of meiotic cells compared with vegetative cells. This difference was found to be at least 16-fold. I conclude that in meiotic cells, histone H1 accounts for more of the total cellular protein than it does in vegetative cells. The difference in its relative abundance as a percent of the total cellular protein is probably in part due to differences in the ratio of nuclear to cytoplasmic volume in the different cell types, or the purging of sporophytic proteins from the cytoplasm of the meiocytes, or both.     

Immunocytochemical visualization of the centromeres during male and female meiosis in Lilium longiflorum

Immunofluorescence staining with an antiserum raised against a presumptive meiotic histone, which has been shown to appear prior to male meiosis in liliaceous plants, preferentially stained the centromere (kinetochore) region of meiotic chromosomes in microsporocytes and megasporocytes. Using this antiserum, we were able clearly to visualize the centromeres at all important meiotic stages in microsporocytes, namely, the association and fusion of centromeres of homologous chromosomes at zygotene-pachytene in prophase I, the disjunction of the homologous centromeres at diplotene, the doubling of each centromere at metaphase I and nonseparation of the sister centromeres at anaphase I, by confocal laser scanning microscopy. Thus, this report provides a complete picture of the behavior of centromeres during meiosis in a eukaryote for the first time. This antiserum also decorated centromeres during female meiosis in cryo-sectioned megasporocytes, but did not stain the centromeres of mitotic chromosomes in root-tip meristem. From these observations, it is suggested that a meiosis-specific centromere protein is required for the meiosis-specific behavior of the centromere. Received: 12 May 1997; in revised form: 20 August 1997 / Accepted: 25 August 1997     

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     

Synthesis of beta-mannosides using the transglycosylation activity of endo-beta-mannosidase from Lilium longiflorum

Endo-beta-mannosidase is an endoglycosidase that hydrolyzes only the Man beta 1-4GlcNAc linkage of the core region of N-linked sugar chains. Recently, endo-beta-mannosidase was purified to homogeneity from Lilium longiflorum (Lily) flowers, its corresponding gene was cloned and important catalytic amino acid residues were identified [Ishimizu T., Sasaki A., Okutani S., Maeda M., Yamagishi M. & Hase S. (2004) J. Biol. Chem.279, 38555-38562]. In the presence of Man beta 1-4GlcNAc beta 1-4GlcNAc-peptides as a donor substrate and p-nitrophenyl beta-N-acetylglucosaminide as an acceptor substrate, the enzyme transferred mannose to the acceptor substrate by a beta1-4-linkage regio-specifically and stereo-specifically to give Man beta 1-4GlcNAc beta 1-pNP as a transfer product. Further studies indicated that not only p-nitrophenyl beta-N-acetylglucosaminide but also p-nitrophenyl beta-glucoside and p-nitrophenyl beta-mannoside worked as acceptor substrates, however, p-nitrophenyl beta-N-acetylgalactosaminide did not work, indicating that the configuration of the hydroxyl group at the C4 position of an acceptor is important. Besides mannose, oligomannoses were also transferred. In the presence of (Man)(n)Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc-peptides (n = 0-2) and pyridylamino GlcNAc beta 1-4GlcNAc, the enzyme transferred (Man)(n)Man alpha 1-6Man en bloc to the acceptor substrate to produce pyridylamino (Man)(n)Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc (n =0-2). Thus, the lily endo-beta-mannosidase is useful for the enzymatic preparation of oligosaccharides containing the mannosyl beta 1,4-structure, chemical preparations of which have been frequently reported to be difficult.     

Characterization of an anther-specific glycoprotein in Lilium longiflorum

Antiserum was raised in rabbits against a lily (Lilium longiflorum) anther-specific glycoprotein (LLA-75). LLA-75 protein bound to concanavalin A, suggesting that it was a glycoprotein. Monospecific anti-LLA-75 antibodies were prepared to investigate its distribution during anther development. Immunoblot analyses of total protein from floral and vegetative organs show that LLA-75 glycoproteins accumulated to detectable levels primarily in one discrete stage of anther development, during the microspore and early pollen phase when the tapetum is actively secreting. A cross-reactive protein with molecular mass of 43.0 kD was also observed. Immunoblots of two-dimensional polyacrylamide gels of lily anther proteins indicated that the four isoforms of LLA-75 glycoprotein ranged from isoelectric point 5.6 to 6.1. These affinity purified antibodies to LLA-75 cross-reacted with anther proteins in two dicot species of the Bignoniaceae. In situ localization data using antirabbit immunoglobulin G (IgG) conjugated with gold particles was not definitive but demonstrated that LLA-75 glycoprotein in lily anthers occurred prominently in tapetal tissue and all other tissues to a lesser degree.     

Temperature effects on self incompatibility in Lilium longiflorum

Summary Detached pistils of the clonal variety, Lilium longiflorum Aral No. 5, were submerged before pollination in 50°C water for 0, 1, 2, 3, 4, 5, 6 or 7 min and then immediately compatibly and incompatibly pollinated. Incompatibility, as indicated by pollen tube length after 48 h at 23.5°C, was eliminated by a 1–2 min submersion while compatibility was removed by a 4–5 min one. The window of incubation temperatures at which incompatible and compatible pollen tubes are clearly differentiated occurred between 15 and 30°C.     

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.     

Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb

Friable callus was obtained from styles and flower pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige and Skoog (MS) media containing either 2 μm dicamba or 2 μm picloram. Cell suspension cultures were established by suspending the callus of L. longiflorum Snow Queen in liquid medium containing 2 μm dicamba. Through a purification process, a fine fast-growing cell suspension was obtained. This suspension was composed of a homogenous population of small dense cells, which tended to organise into embryo like structures (ELS). In liquid culture with the auxin dicamba, the ELS underwent continuous callus formation. When transferred to solidified hormone-free MS medium, the ELS germinated, forming complete plantlets. Histological investigation showed that in the ELS both shoot and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact somatic embryos. Received: 4 April 1997 / Revision received: 13 May 1997 / Accepted: 15 June 1997     

Regulation of self-incompatibility by acetylcholine and cAMP in Lilium longiflorum

Elongation of pollen tubes in pistils of Lilium longiflorum cv. Hinomoto after self-incompatible pollination was here found to be promoted by acetylcholine (ACh) and other choline derivatives, such as acetylthiocholine, l-alpha-phosphatidylcholine and chlorocholinechloride [CCC; (2-chloroethyl) trimethyl ammonium chloride]. Moreover, the elongation was promoted by neostigmine, a potent inhibitor of acetylcholinesterase (AChE; acetylcholine-decomposing enzyme) (EC 3.1.1.7.) and activities of this and choline acetyltransferase (ChAT; acetylcholine-forming enzyme) (EC 2.3.1.6.) in pistils were associated with self-incompatibility. The activity of ChAT was lower after self-incompatible as compared with cross-compatible pollination. Application of cAMP promoted ChAT activities in both cases, whereas activity of AChE in pistils after self-pollination was higher than that after cross-compatible pollination and was suppressed by cAMP in both cases. Furthermore, AChE activity was inhibited by treatment with neostigmine or heating. Our results indicate that the self-incompatibility with self-pollination is due to decrease of ACh and cAMP, causing reduction of ChAT and AC (adenylate cyclase) and concise elevation of AChE and PDE (cAMP phosphodiesterase), and therefore suppressed growth of pollen tubes.     

Nucleic acids in relation to cell division in Lilium longiflorum

Methods are described for preparing cell suspensions of Lilium microsporocytes, microspores and pollen grains; for obtaining cell counts of these suspensions; and for their analysis for pentose nucleic acid (PNA) and desoxypentose nucleic acid (DNA).The results of these analyses have been calculated to nucleic acid content in μμg per microsporocyte, microspore or pollen grain, and the results related to logarithm of flower bud length, an index of the developmental status of the cells, and of their temporal relationship to meiosis, microspore mitosis and opening of the flower.DNA content per cell drops sharply at the end of meiosis, with the formation of four microspores from each microsporocyte. It then increases gradually during the microspore interphase between meiosis and the microspore mitosis. At microspore mitosis DNA content doubles rapidly. In the development of the resulting binucleate pollen grain, from microspore mitosis until the opening of the flower, there is a further gradual increase of DNA content. PNA content of these cells follows the same pattern up to microspore mitosis at a level about twice that of DNA, increases sharply at mitosis, and continues to increase rapidly at a rate nine times that for DNA in the maturing pollen grain.The absolute amounts of DNA and PNA are great. At the time of anthesis the two-celled pollen grain contains about 375 μμg of DNA and 1705 μμg of PNA.     

Thigmotropism in the growth of pollen tubes of Lilium longiflorum

The pollen tubes of Lilium longiflorum were clearly seen togrow regularly along minute network filaments. By analyzingthe nature of this growth regularity, we found that the pollentubes showed a kind of thigmotropism in their growth. (Received October 14, 1974; )     

Cytochemical Analysis of Callose Localization in Lilium longiflorum Pollen Tubes

Callose, a ß, 1–3 glucan as a component of plantcells has received sporadic attention. Here, we report an attemptto determine whether aniline blue and lacmoid are indeed specificfor visualizing callose. We also re-evaluate, based on a checkfor stain specificity, the localization of callose in elongatingLilium longiflorum, cv. ‘Ace’ pollen tubes. Specificityof these stains was checked by chemical and enzymatic extractionprocedures which solubilize proteins and polysaccharides. Resultsherein question the generally accepted validity of the fluorescent-anilineblue method for detecting callose. Lacmoid either possessesan affinity for both callose and protein or for callose as aglycoprotein. As for callose localization, the walls of thenon-growing region of the lily pollen tube contain callose,probably as a glycoprotein. Presence of the callosicglycoproteinin the wall of the growing tube-tip is dependent on tube length.Callose plugs exhibiting an affinity for aniline blue or lacmoidwere never seen. Phase-contrast microscopy revealed non-stainablewall ingrowths in fixed-tubes and free-moving cytoplasmic masseswithin living tubes.     

The identification of a pollen-specific LEA-like protein in Lilium longiflorum

A novel pollen‐specific LEA‐like protein, LP28, was detected in Lilium longiflorum using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24·2 kDa and a pI of 4·7. The amino acid sequence is highly hydrophilic except for the N‐terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollen‐specific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.     

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.     

Two classes of pollen-specific, heat-stable proteins in Lilium longiflorum

Several floral organ-specific proteins in lily anthers do not accumulate to detectable levels until just before anthesis. Antisera were raised against three of these proteins. designated LLA, in pollen of Lilium longiflorum Thunb. cv. Snow Queen. Monospecific antibodies were further prepared from antisera to investigate the specificity and distribution of these proteins during development. In an effort to study the function of these gene products, pollen protein was heat-treated at 90°C for 10 min. Monospecific anti-LLA-32 and -23 antibodies recognized two of these heat-stable proteins with molecular masses of 32 and 23 kDa. Accumulation of the two proteins in anther development was correlated with desiccation that occurred naturally in the pollen. Immunob-lot analyses of total protein from floral and vegetative organs confirmed that both LLA-32 and -23 proteins were pollen-specific. The proteins showed consistent patterns of expression during development and their levels decreased when pollen germinated. The properties of the two proteins differed in responsiveness to both polyethylene glycol 8000 and abscisic acid, and in solubility characteristics. Analysis of amino acid composition indicates that both LLA-32 and -23 proteins are rich in glutamic acid/glutamine and glycine, a characteristic of heat-stable proteins. However, LLA-23 has more polar amino acid residues with a polarity of 57%, two-fold higher than that of the LLA-32. Immunoblot analyses showed that LLA-32 and -23 proteins were immunologically unrelated to the dehydrin-like protein in lily seeds. It concluded that the two classes of pollen-specific proteins have some features in common with each other and with dehydrins. but that each is distinct.