Inhibition of RNA and protein synthesis in pollen tube development of Pinus bungeana by actinomycin D and cycloheximide

* The effects of actinomycin D and cycloheximide on RNA and protein synthesis were investigated during pollen tube development of Pinus bungeana. * RNA and protein contents, protein expression patterns, cell wall components and ultrastructural changes of pollen tubes were studied using spectrophotometry, SDS-PAGE electrophoresis, Fourier transformed infrared (FTIR) microspectroscopy and transmission electron microscopy (TEM). * Pollen grains germinated in the presence of actinomycin D, but tube elongation and RNA synthesis were inhibited. By contrast, cycloheximide inhibited pollen germination and protein synthesis, induced abnormal tube morphology, and retarded the tube growth rate. SDS-PAGE analysis showed that protein expression patterns changed distinctly, with some proteins being specific for each phase. FTIR microspectroscopy established significant changes in the chemical composition of pollen tube walls. TEM analysis revealed the inhibitors caused disintegration of organelles involved in the secretory system. * These results suggested RNA necessary for pollen germination and early tube growth were present already in the pollen grains before germination, while the initiation of germination and the maintenance of pollen tube elongation depended on continuous protein synthesis.     

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.     

GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 ( gnl2-1 ) and gnl2-2 in Arabidopsis thaliana , in which the pollen grains failed to germinate in vitro and in vivo . GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.     

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.     

Latrunculin B has different effects on pollen germination and tube growth

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.     

The effect of cherry leaf roll virus infection on the performance of birch pollen and studies on virus replication in germinating pollen

The rates of germ tube elongation in vitro of pollen from cherry leaf roll virus (CLRV)-infected birch (Betula pendula) did not differ significantly from those of pollen from virus-free trees. Differences in percentage germination of pollen collected from trees at different sites were significant but percentages of germination of pollen from virus-infected and virus-free trees did not differ greatly from one another. In vivo, pollen from infected trees germinated on healthy and CLRV-infected stigmas and callose plugs formed in both types of tissues. However, the extent of callose plug formation was greater in the pollen tubes of virus-free grains germinating in infected stigmas than in reciprocal crosses. CLRV coat antigen was detected by ELISA in stigmatic tissue, from healthy trees, on which virus-carrying pollen grains had germinated. Using fluorescein isothiocyanate conjugated to CLRV-specific γ-globulin, viral coat antigen was detected throughout germ tubes from virus-carrying but not virus-free pollen germinating in vitro. Protoplasts released following Driselase digestion of pollen germ tubes from virus-infected trees contained CLRV antigen detectable by ELISA. During germination of virus-infected pollen there was little synthesis of viral coat protein or nucleic acid but, following inoculation with purified virus particles, protoplasts made from healthy germinating pollen produced increasing amounts of CLRV-specific antigen implying that CLRV replicated in this system.     

Characterization of pollen tube development in Pinus strobus (Eastern white pine) through proteomic analysis of differentially expressed proteins

The differentially expressed proteins in pollen tubes indicate their specific roles in this stage of male gametophyte development. To isolate these proteins, 2-DE was done using ungerminated pollen and 2-day-old pollen tubes of Pinus strobus. Results show that 645 and 647 protein spots were clearly resolved from pollen grains and pollen tubes, respectively. Thirty-eight protein spots were expressed only in pollen tubes, while 19 increased in intensity. MALDI-TOF MS was used to generate tryptic peptide masses that were submitted to Mascot for identification. Of the differentially expressed proteins, 12% matched with hypothetical proteins, 33% did not hit any protein, and for the 55%, a putative function was assigned based on similarity of sequences with previously characterized proteins. Therefore, pollen tube development can be characterized by the cellular activities that involve metabolism, stress/defense response, gene regulation, signal transduction, and cell wall formation. This study expands our understanding of the changes in protein expression associated with pollen tube development and provides insights into the molecular programs that separate the development of the pollen tubes from pollen grains. This is the first report that describes a global analysis of differentially expressed proteins from the pollen tube of any seed plant.     

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

 We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. Received: 28 October 1996 / Revision accepted: 24 January 1997     

AtAMT1;4, a Pollen-Specific High-Affinity Ammonium Transporter of the Plasma Membrane in Arabidopsis

Pollen represents an important nitrogen sink in flowers to ensurepollen viability. Since pollen cells are symplasmically isolatedduring maturation and germination, membrane transporters arerequired for nitrogen import across the pollen plasma membrane.This study describes the characterization of the ammonium transporterAtAMT1;4, a so far uncharacterized member of the ArabidopsisAMT1 family, which is suggested to be involved in transportingammonium into pollen. The AtAMT1;4 gene encodes a functionalammonium transporter when heterologously expressed in yeastor when overexpressed in Arabidopsis roots. Concentration-dependentanalysis of ¹⁵N-labeled ammonium influx into roots of AtAMT1;4-transformedplants allowed characterization of AtAMT1;4 as a high-affinitytransporter with a K_m of 17 µM. RNA and protein gel blotanalysis showed expression of AtAMT1;4 in flowers, and promoter–genefusions to the green fluorescent protein (GFP) further definedits exclusive expression in pollen grains and pollen tubes.The AtAMT1;4 protein appeared to be localized to the plasmamembrane as indicated by protein gel blot analysis of plasmamembrane-enriched membrane fractions and by visualization ofGFP-tagged AtAMT1;4 protein in pollen grains and pollen tubes.However, no phenotype related to pollen function could be observedin a transposon-tagged line, in which AtAMT1;4 expression isdisrupted. These results suggest that AtAMT1;4 mediates ammoniumuptake across the plasma membrane of pollen to contribute tonitrogen nutrition of pollen via ammonium uptake or retrieval.     

烟草脱外壁花粉人工萌发与离体授粉实验系统的建立

通过花蕾低温处理、花药漂浮培养与花粉短时酶解程序可脱去花粉外壁,分离出烟草(Nicotianatabacum L.)的脱外壁花粉。研究了分离过程中的酶液渗透压、培养基中聚乙二醇(PEG)与蔗糖以及添加水解乳蛋白等因素对脱外壁花粉人工萌发的影响。在含30％PEG-6000与0.1％水解乳蛋白的D_2培养基中,萌发率最高达57.8％;花粉管生长正常,培养24h后一半以上的花粉管中生殖细胞分裂成精子。用微滴和贴滤纸小片的方法将30～40粒脱外壁花粉授予柱头上,近一半能萌发花粉管并在花柱中生长。采取授粉后子房培养方法,获得了种子与幼苗。从而建立了脱外壁花粉离体授粉实验系统。讨论了脱外壁花粉人工萌发与离体授粉实验系统的建立对于研究外壁在花粉萌发中的生物学功能以及开拓新的转基因技术等方面的意义。     

MALE GAMETOPHYTE DEFECTIVE 2, Encoding a Sialyltransferase-like Protein, is Required for Normal Pollen Germination and Pollen Tube Growth in Arabidopsis

Sialyltransferases (SiaTs) exist widely in vertebrates and play important roles in a variety of biological processes. In plants, several genes have also been identified to encode the proteins that share homology with the vertebrate SiaTs. However, very little is known about their functions in plants. Here we report the identification and characterization of a novel Arabidopsis gene, MALE GAMETOPHYTE DEFECTIVE 2 [MGP2) that encodes a sialyltransferase-like protein. MGP2 was expressed in all tissues including pollen grains and pollen tubes. The MGP2 protein was targeted to Golgi apparatus. Knockout of MGP2 significantly inhibited the pollen germination and retarded pollen tube growth in vitro and in vivo, but did not affect female gametophytic functions. These results suggest that the sialyltransferase-like protein MGP2 is important for normal pollen germination and pollen tube growth, giving a novel insight into the biological roles of the sialyltransferase-like proteins in plants.     

Germination and <Emphasis Type="Italic">In Vitro</Emphasis> Growth of Petunia Male Gametophyte Are Affected by Exogenous Hormones and Involve the Changes in the Endogenous Hormone Level

The data obtained characterize the changes in the contents of endogenous phytohormones (IAA, cytokinins, GA, and ABA) in germinating pollen grains and growing pollen tubes of a self-compatible clone of petunia (sPetunia hybrida L.) within an 8-h period under in vitro conditions. The hydration and initiation of germination of pollen grains brought the ABA content down to a zero level, while the levels of GA, IAA, and cytokinins increased 1.5–2-fold. Later, in the growing pollen tubes, the GA content increased twofold, while the levels of IAA and cytokinins decreased. The exogenous ABA and GA₃ considerably promoted pollen germination and pollen tube growth; however, only the treatment with GA₃ produced the maximum length of pollen tubes. The exogenous IAA promoted and the exogenous cytokinins hindered the growth of pollen tubes. The membrane potential, as assessed with a potential-sensitive dye diS-C₃-(5), considerably increased in the pollen grains treated with ABA and benzyladenine, whereas IAA and GA₃ did not practically affect it. The authors conclude that the mature pollen grains contain the complete set of hormones essential for pollen germination and pollen tube growth. ABA, GA, and IAA together with cytokinins control the processes of pollen grain hydration, germination, and pollen tube growth, respectively.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 584–590.Original Russian Text Copyright © 2005 by Kovaleva, Zakharova, Minkina, Timofeeva, Andreev.     

Expression of Bra r 1 gene in transgenic tobacco and Bra r 1 promoter activity in pollen of various plant species

Bra r 1 encodes a Ca2+-binding protein specifically expressed in anthers of Brassica rapa. In this study, we isolated a genomic clone of Bra r 1 and found sequences similar to Pollen Box core motifs and LAT56/59 box, pollen-specific cis-acting element, in the 5' upstream region of Bra r 1. Reporter gene fusion revealed that the Bra r 1 promoter directs male gametophytic expression in Nicotiana tabacum, Arabidopsis thaliana and B. napus, showing strong expression in mature pollen grains similar to that of endogenous Bra r 1. Genomic DNA of Bra r 1 was introduced into tobacco plants and the highest accumulation of Bra r 1 protein was observed in mature pollen in the same manner as reporter gene expression. Using in vitro-germinated pollen tubes of transgenic tobacco, we firstly demonstrated the subcellular localization of Bra r 1 in pollen tubes. Bra r 1 protein was distributed throughout the pollen tube of transgenic tobacco and slightly intense signals of Bra r 1 were observed in the tip region. In long-germinated pollen tubes, Bra r 1 was detected only in the cytoplasmic compartments while no signals were observed in the empty part of the pollen tube, indicating that cytoplasmic movement toward the tube tip is accompanied by Bra r 1. Hence, we suggest that Bra r 1 is involved in pollen germination and pollen tube growth.     

栽培稻与其野生近缘种的可交配性研究

通过人工授粉方法研究栽培稻与二倍体和四倍体野生稻之间的可交配性.以栽培稻为对照,用光学显微镜观察不同野生稻花粉在同一栽培种柱头上的萌发生长情况.结果表明,在栽培稻柱头上普通野生稻(AA)花粉萌发最好,与对照萌发情况相近.药用野生稻(CC)萌发差,表现为柱头上花粉附着量少,开始萌发时间迟,萌发量少,花粉管扭曲、缠绕、伸长慢等.四倍体野生稻未观察到有萌发现象.说明普通野生稻与栽培稻亲缘关系近,可交配性好;药用野生稻与栽培稻可交配性差;四倍体野生稻与栽培稻可交配性极差.由此推断,转基因水稻与普通野生稻通过花粉途径发生基因漂移的可能性很大,而与药用野生稻和其他基因组野生稻发生基因漂移的可能性很小.     

紫萼花粉粒和花粉管中微丝的超微结构

用常规化学固定和化学固定前用鬼笔环肽处理两种电镜样品制作技术,分别研究了紫萼[Hosta venteicosa (=H.coerulea]成熟花粉粒和幼花粉管中的微丝的超微结构。结果表明,在常规电镜固定中花粉粒中的微丝能保存,但在花粉管中的则遭受破坏。用鬼笔环肽处理后化学固定的方法,微丝在花粉管中能良好地保存。在花粉粒中平行的微丝形成束,表现为具分布的特点,即限于分布在它们功能的区域,并且微丝束经常紧密地与营养核贴近。在幼花粉管中微丝束表现为在线粒体、质体、内质网、小泡和小液泡的表面通过,并常常与脂体紧密联结。这些现象表明在花粉萌发和花粉管生长时,微丝与营养核及与其它细胞器的运动之间存在某些联系的迹象。