Mechanism of inhibition of cytoplasmic streaming by a myosin inhibitor, 2,3-butanedione monoxime

Summary On the basis of the inhibition of myosin by 2,3-butanedione monoxime (BDM), the protein's involvement in various cell activities is discussed. However, it has not been established whether BDM inhibits plant myosin. In the present study, the effect of BDM on isolated plant myosin was analyzed in vitro. The sliding between myosin from lily (Lilium longiflorum) pollen tubes and actin filaments from skeletal muscle was inhibited to 25% at a concentration of 60 mM, indicating that BDM can be used as a myosin inhibitor for plant materials. Cytoplasmic streaming was completely inhibited by BDM at 30 mM in lily pollen tubes and at 70 mM in short root hair cells, and at 100 mM in long root hair cells ofHydrocharis dubia. However, BDM at high concentrations induced the disorganization of actin filament bundles in lily pollen tubes and short root hair cells. In addition, cortical microtubules were also fragmented in short root hair cells treated with BDM, suggesting a possible side effect of BDM.Abbreviations AF actin filament - BDM 2,3-butanedione monoxime - MT microtubule     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

21-kDa polypeptide,a low-molecular-weight cyclophilin,is released from pollen of higher plants into the extracellular medium in vitro

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca²⁺. This phenomenon was also dependent on pH and on the concentrations of MgCl₂ in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl₂ concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis-trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.Abbreviations CaM Calmodulin - CBB Coomassie-brilliant-blue - CsA Cyclosporin A - CyP Cyclophilin     

Biochemical, Immunochemical and Immunohistochemical Identification of Myosin Heavy Chains in Cultured Cells of Catharanthus roseus

In the pollen tubes of the lily Lilium longiflorum, myosin,composed of 170-kDa heavy chains is responsible for the intracellulartransport of organelles [Yokota and Shimmen (1994) Protoplasma177: 153]. Polypeptides of 170 kDa with similar antigenicityto this pollen-tube myosin have also been found in other angiospermcells [Yokota et al. (1995) Protoplasma 185: 178]. To clarifythe role of this type of myosin in cytoplasmic streaming, weprepared partially purified myosin fraction from cultured cellsof Catharanthus roseus by co-precipitation with F-actin. Ina motility assay in vitro with this fraction, rhodamine-phalloidin-labeledF-actin moved with an average velocity of 10.7 µm s^-1.This sliding velocity was similar to that of the cytoplasmicstreaming observed in intact cultured cells. Antibodies raisedagainst the 170-kDa heavy chain of pollen-tube myosin recognizedonly a single polypeptide of 170 kDa in this partially purifiedfraction. The same polypeptide was also identified by theseantibodies in a crude extract of proteins from cultured cells.The myosin-specific fluorescence was concentrated around thenuclei and was associated with particles of various sizes. Duallocalization using antibodies against myosin and against actinrevealed that these particles were preferentially co-localizedwith actin filaments. On the other hand, no component of thecrude extract or of the partially purified myosin fraction cross-reactedwith antibodies against heavy chains of myosin II from animalcells. These results suggest that the 170-kDa polypeptide is the myosinheavy chain and that this myosin generates the motive forcefor cytoplasmic streaming in cultured cells of Catharanthusroseus. (Received March 28, 1995; Accepted September 14, 1995)     

Isolation and characterization of plant myosin from pollen tubes of lily

Summary A plant myosin was isolated from pollen tubes of lily,Lilium longiflorum. Pollen tubes were homogenized in low ionic strength solution containing casein, and myosin from this crude extract was purified by co-precipitation with F-actin prepared from chicken breast muscle, followed by hydroxylapatite column and gel filtration column chromatography. Upon SDS-PAGE on 6% polyacrylamide gel, only 170 kDa polypeptide was detected in the purified myosin fraction. Furthermore, with immunoblotting using antiserum raised against 170 kDa polypeptide, only the 170 kDa component crossreacted in the crude sample of pollen tube proteins. This antiserum did not crossreact with the heavy chain of skeletal muscle myosin. The ATPase activity of pollen tube myosin was stimulated up to 60-fold by F-actin prepared from chicken breast muscle. The translocation velocity of rhodamine-phalloidin-labeled F-actin on a glass surface covered with pollen tube myosin ranged from 6.0 to 9.8 m/s with an average of 7.7 m/s. This velocity was similar to or a little faster than that of the cytoplasmic streaming that occurred in pollen tubes. These results suggested that myosin composed of a 170 kDa heavy chain produces the motive force for cytoplasmic streaming in pollen tube of lily.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazin-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate     

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is –98° ± 3° in lily and –124° ± 4° in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.     

Optimization of conditions for germination of cold-stored <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pollen

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical. F. Bou Daher and Y. Chebli contributed equally to this study.     

Cloning and sequence analysis of lily and tobacco guanylate kinases

Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phospharyl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74–84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.Abbreviation pI isoelectric point     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

Expression studies of SCA in lily and confirmation of its role in pollen tube adhesion

During pollination the pollen tube grows into the style and toward the ovary via the transmitting tract. In lily the growth of pollen tubes involves tube cell adhesion to transmitting tract cells. We reported two molecules involved in this adhesion event. One is a pectic polysaccharide and the other, a 9 kDa basic protein named SCA for stigma/stylar cysteine-rich adhesin. SCA, which shows some identity with LTP (lipid transfer protein), was localized to the transmitting tract epidermis of the style where pollen tubes adhere. The present studies on the expression of SCA indicate that the protein has a similar expression pattern with LTP1 in Arabidopsis and that the protein is abundant in both the stigma and the style. For further proof of its role in pollen tube adhesion the activity of Escherichia coli-expressed protein has been studied in an in vitro adhesion assay system.     

Immunolocalization of H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

Summary A heterogeneous distribution of H⁺-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H⁺-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H⁺-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H⁺-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.Abbreviations BSA bovine serum albumine - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - Tris 2-amino-2-hydroxymethyl-1,3-propandiol     

Molecular cloning,functional expression and characterization of two serine/threonine-specific protein kinases from<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> pollen

The existence of different kinds of kinases in pollen and pollen tubes suggests that kinase-mediated signaling pathways are likely involved in regulating pollen germination and pollen tube growth during the life cycle of higher plants. We have used RT-PCR and RACE to isolate full-length cDNAs for two pollen-expressed kinases, named NtPK1 and NtPK2, of Nicotiana tabacum. NtPK1 and NtPK2 encode proteins of 365 and 369 amino acids with calculated molecular masses of 39.2 kDa and 39.5 kDa, respectively, and both proteins possess the 12 sub-domains that are conserved among protein kinases. The nucleotide and deduced amino acid sequences of NtPK1 and NtPK2 share 88% and 91% identity, respectively, with the C-terminal region being the most conserved. RT-PCR analysis revealed that NtPK1 was specifically expressed in pollen and pollen tubes, and that NtPK2 was also expressed in pistil and petal. Immunoblot analysis using anti-NtPK1 and anti-NtPK2 antibodies confirmed that both NtPK1 and NtPK2 were produced in pollen and pollen tubes, and that NtPK2 was also produced in developing male gametophytes and other floral tissues. Biochemical fractionation experiments showed that, in all the tissues examined, NtPK1 and NtPK2 were present in the cytosolic fraction and not in the microsomal fraction. NtPK1 and NtPK2 were found to autophosphorylate on threonine and, for NtPK2, on serine as well. All the results taken together suggest that NtPK1 and NtPK2 are novel receptor-like cytosolic serine/threonine kinases, and could mediate signaling pathways required for pollen germination and/or pollen tube growth.The nucleotide sequence data of NtPK1 and NtPK2 reported in this paper will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases under the accession numbers AJ608156 and AJ608157, respectively     

Studies ofNajas pollen tubes. Fine structure and the dependence of chlorotetracycline fluorescence on external free ions

Summary The distribution of membrane-associated calcium was investigated in pollen grains and tubes of the underwater pollinated angiospermNajas marina L. using chlorotetracycline (CTC). Tubes grown in distilled water (pH 6) showed the highest fluorescence in a subapical region that tapered basally into a fluorescent strand centrally located in the tube and extending back towards the pollen grain. The apical cap had low fluorescence as did the cytoplasm surrounding the fluorescent strand, the tube base and the pollen grain. Tubes grown in different pond waters (pH 8) revealed no intracellular CTC fluorescence. Instead there was an external fluorescence forming a distinct layer around the whole tube, frequently enhanced in a subapical region to form an external collar.Modification of the patterns of fluorescence could be induced by manipulation pH of the growth media and content of specific ions. For example tubes grown in distilled water with 10^–3 M Mg²⁺ salts showed a similar CTC fluorescence as those grown in pond water. In contrast, Ca²⁺ enrichment had no visible influence on the patterns of fluorescence. The pattern of fluorescence displayed by tubes grown in distilled water, could be reproduced in pond water if the pH was artificially reduced to pH 6.Ultrastructurally, there was no detectable difference in the markedly polar distribution of organelles between pollen tubes grown in the various growth media. The secretory vesicles found in the pollen grain prior to germination become distributed throughout the pollen tube but are least concentrated in regions that show highest internal CTC fluorescence. These regions appear to have large amounts of endoplasmic reticulum and include mitochondria.These results are discussed in relation to the significance of calcium gradients for tip growth and limitations in the use of CTC.Abbreviations CTC chlorotetracycline - SV secretory vesicle - ER endoplasmic reticulum - PIXE proton induced X-ray emissions     

Unique and selective mitogenic effects of vanadate on SV40-transformed cells

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor -subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vanadate-induced mitogenesis is associated with the selective induction ofc-jun andjunB expression without significantly inducingc-fos orc-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation andc-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation andc-jun andjunB expression may serve the critical roles in mediating vanadate-induced mitogenesis in SV40-transformed cells.     

The kinesin-immunoreactive homologue from Nicotiana tabacum pollen tubes: Biochemical properties and subcellular localization

In plant cells, microtubule-based motor proteins have not been characterized to the same degree as in animal cells; therefore, it is not yet clear whether the movement of organelles and vesicles is also dependent on the microtubular cytoskeleton. In this work the kinesinimmunoreactive homologue from pollen tubes of Nicotiana tabacum L. has been purified and biochemically characterized. The protein preparation mainly contained a polypeptide with a relative molecular weight of approx. 100 kDa. This polypeptide bound to animal microtubules in an ATP-dependent manner and it further copurified with an ATPase activity fourfold-stimulated by the presence of microtubules. In addition, the sedimentation coefficient (approx. 9S) was similar to those previously shown for other kinesins. Immunofluorescence analyses revealed a partial co-distribution of the protein with microtubules in the pollen tube. These data clearly indicate that several properties of the kinesin-immunoreactive homologue are similar to those of kinesin proteins, and suggest that molecular mechanisms analogous to those of animal cells may drive the microtubule-based motility of organelles and vesicles in plants.Abbreviations AE-LPLC anion-exchange low-pressure liquid chromatography - AMPPNP 5-adenylylimidodiphosphate - PKH pollen kinesin homologue - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Assembly of an antibody and its derived antibody fragment inNicotiana andArabidopsis

The yield and assembly of an IgG1 antibody and its derived F_ab fragment were compared inNicotiana andArabidopsis. The results obtained showed a lot of interclonal variability. For 45% of the primary transgenic calluses, antigen-binding entities represented less than 0.1% of the total soluble protein (TSP). Only two of the 103 analysed transformants contained more than 1% of antigen-binding protein, with 1.26% being the highest yield. Analogous amounts of complete antibody and F_ab accumulated in primary callus tissue. Moreover, yields were in the same range for both species as far as primary callus tissue is concerned. However, the accumulation of the F_ab fragment in leaf tissue of regenerated plants differed significantly betweenNicotiana andArabidopsis. The F_ab fragment accumulated to only 0.044% of TSP inNicotiana leaves but up to 1.3% inArabidopsis leaves. Furthermore, both species showed differences in the assembly pattern of the complete antibody. WhereasArabidopsis contained primarily fully assembled antibodies of 150 kDa,Nicotiana showed an abundance of fragments in the 50 kDa range.     

Characterization and function of wall-bound exo-β-glucanases of Lilium longiflorum pollen tubes

Two exo-β-glucanases (LP-ExoI, 83 kDa and LP-ExoII, 71 kDa) were extracted and partially purified from the cell wall of Lilium longiflorum pollen tubes. Both LP-ExoI and LP-ExoII hydrolyzed laminarin (1,3-β-glucan). These enzymes also exhibited some activity toward 1,3:1,4-β-glucans of Hordeum vulgare and Cetraria islandica and the 1,6-β-glucan of Umbilicaria papullosa. The pH for optimum activity for both exo-β-glucanases was 5.5. Methylation analysis of the reaction products revealed that purified LP-ExoI decreased both 1,3- and 1,4-glucosyl linkages in hemicellulosic polysaccharides isolated from the cell wall of lily pollen tubes. D-gluconolactone and nojirimycin, inhibitors of glucosidase, inhibited activities of both exo-β-glucanases, as well as growth of the lily pollen tubes. These results disclosed that the wall-bound exo-β-glucanases play an important role in the regulation of lily pollen tube growth. Received: 3 January 2000 / Revision accepted: 8 March 2000     

Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope

Summary Myosins providing the motors for the actin-based motility that occurs in diverse plants have proved difficult to study. To facilitate those studies, we describe polymerase chain reaction primers that reliably amplify part of the myosin head from diverse plants, consensus sequences that characterise the amplified product as encoding a class V or class VIII myosin, and a monoclonal antibody that recognises an epitope conserved in the head of most plant, fungal, and animal myosins. A pair of stringent oligonucleotide primers was designed that, when used in the polymerase chain reaction, amplified at least eleven different myosins from five species of angiosperms and one sequence from each of the fernAzolla and the algaeNitella andPhaeodactylum. The amplified products, comprising 126 to 135 nucleotides encoding part of the myosin head domain, can be used as myosin-specific probes to screen genomic and cDNA libraries. To identify the products of plant myosin genes, we raised a monoclonal antibody (anti-CHE) to a nine amino acid peptide matching a conserved head epitope showing not more than single amino acid substitutions in most published myosin genes. This antibody recognises rabbit skeletal myosin and multiple polypeptides of >100 kDa in four angiosperms and in the algaNitella. Relating the M_r values of immunoreactive bands inArabidopsis extracts to the predicted M_r values of the products of five myosin genes supports the view that the antibody recognises both myosins V and VIII together with the products of some as yet unsequenced genes. The previously described MB170 antibodies may, in contrast, be specific for one or more type V myosins. Together, the polymerase chain reaction primers and the antibody represent versatile tools for identifying and categorising myosins in diverse plants.