Chasmogamous and Cleistogamous Pollination in Salpiglossis sinuata

Pollen from chasmogamous flowers of Salpiglossis sinuata L. could not be induced to germinate in vitro unless stigmatal extract was applied to the culture medium. The substance that induces pollen germination in the stigmatal extract is water-soluble and heat-stable. Crosses could not be achieved between chasmogamous and cleistogamous flowers because of structural incompatibility. Pollinated pistils of chasmogamous flowers release a large amount of ethylene. The burst of ethylene release is due to an interaction between pollen tubes and stylar tissue and is directly proportional to the quantity of pollen placed on the stigma. Cleistogamous flower buds also produce a burst of ethylene at the time of pollination within the closed flower. The ethylene release may be a cause of reduced corolla development associated with cleistogamous flowers.     

PLC—IP3信号途径参与花粉管伸长调控的显微注射实验

以百合（Ｌｉｌｉｕｍ ｄａｖｉｄｉｉ Ｄｕｃｈ．）花粉为材料,通过显微注射肌醇磷脂信使系统中重要组成成分或其抗体,研究该信使系统牟花粉管伸长的影响。发现显微注射动物来源磷脂酶Ｃ（ＰＬＣ）β１－３抗体显著抑制花粉管的伸长生长,而注射ＰＬＣβ４抗体对花粉管伸长无影响;显微注射三磷酸肌醇（ＩＰ３）,可显著促进花粉管伸长生长;显微注射动物来源的ＩＰ３Ｒ２、ＩＰ３Ｒ３抗体可显著抑制花粉管伸长生长,而注射ＩＰ     

花粉管引导机制的研究进展

花粉管引导是指显花植物在受精过程中,雌蕊组织与花粉管相互作用使花粉管定向生长并最终到达胚囊的过程,其机制颇为复杂。该文基于调控花粉管生长的孢子体引导和配子体细胞引导两个主要过程,阐述雌蕊中不同蛋白分子和其它小分子物质的浓度梯度在花粉管的孢子体组织引导中的作用,以及胚囊中不同类型的细胞及其相关基因与蛋白在花粉管的配子体细胞引导中的作用。同时,该文也对精细胞在花粉管引导中的作用进行了阐述。     

A Gain-of-Function Mutation of Arabidopsis Lipid Transfer Protein 5 Disturbs Pollen Tube Tip Growth and Fertilization

During compatible pollination of the angiosperms, pollen tubes grow in the pistil transmitting tract (TT) and are guided to the ovule for fertilization. Lily (Lilium longiflorum) stigma/style Cys-rich adhesin (SCA), a plant lipid transfer protein (LTP), is a small, secreted peptide involved in pollen tube adhesion-mediated guidance. Here, we used a reverse genetic approach to study biological roles of Arabidopsis thaliana LTP5, a SCA-like LTP. The T-DNA insertional gain-of-function mutant plant for LTP5 (ltp5-1) exhibited ballooned pollen tubes, delayed pollen tube growth, and decreased numbers of fertilized eggs. Our reciprocal cross-pollination study revealed that ltp5-1 results in both male and female partial sterility. RT-PCR and β-glucuronidase analyses showed that LTP5 is present in pollen and the pistil TT in low levels. Pollen-targeted overexpression of either ltp5-1 or wild-type LTP5 resulted in defects in polar tip growth of pollen tubes and thereby decreased seed set, suggesting that mutant ltp5-1 acts as a dominant-active form of wild-type LTP5 in pollen tube growth. The ltp5-1 protein has additional hydrophobic C-terminal sequences, compared with LTP5. In our structural homology/molecular dynamics modeling, Tyr-91 in ltp5-1, replacing Val-91 in LTP5, was predicted to interact with Arg-45 and Tyr-81, which are known to interact with a lipid ligand in maize (Zea mays) LTP. Thus, Arabidopsis LTP5 plays a significant role in reproduction.     

花粉萌发和花粉管生长发育的信号转导

显花植物授粉是一个复杂的发育过程。从花粉落在柱头上开始,经过粘附、识别、水合、萌发,花粉管在花柱内生长,直至到达子房发生双受精作用,整个过程发生在雌、雄两性细胞和组织之间,受到严格的遗传控制和细胞控制。一方面雌雄配子的基因型决定两者是否亲和,另一方面雌雄两性细胞间发生复杂的相互作用,细胞外信号分子是这些过程的主要调控因子。当花粉或花粉管细胞感知外部信号后,必然通过信号转导级联反应,达到控制萌发、调整花粉管生长方向等目的。这一系列动力学的细胞事件,关系到受精的成败。因此研究此过程中的信号及其转换机…     

肌醇磷脂信号途径参与胞外钙调素启动花粉萌发和花粉管伸长

高等植物有性生殖是植物发育生物学研究的重要内容之一,而作为雄配子体的花粉在雌蕊柱头上萌发及花粉管在花柱内的持续生长是有性生殖实现的关键。已有许多研究表明Ca2 在花粉萌发和花粉管生长过程中起重要作用。最近,我室在多年细胞外钙调素（calinodulin,CaM）存在。性质及生物学功能研究（孙大业等1995;Sun等1994,1995;Tang等1996）的基础上,通过不过膜的大分子CaM拈抗剂或抗体并结合恢复实验证实细胞外CaM对花粉的萌发和花粉管的伸长具有启动作用（马力耕和孙大业1996）,并发现G蛋白、质膜Caz”通道及胞内依赖Caz”的蛋白…     

Genetic Variation in Sorghum for the Inhibition of Maize Pollen Tube Growth

Aniline blue fluorescence was used to study the growth of maizepollen tubes in the stigmas of 13 diverse sorghum accessions.In 12, only short maize pollen tubes were formed, but in thesingle exception (Sorghum nervosum Nr481) maize pollen tubesgrew at least as far as the base of the style. The S. bicolorgenotypes S9B and CMS (a cytoplasmic male sterile line) werehybridized with Nr481, and analysis of maize pollen tube growthin F₁ plants, and BC₁ plants using Nr481 as the recurrent parent,suggested that differences in inhibition of pollen tube growthwere due to variation at a single locus, which we propose todesignate lap (Inhibition of alien pollen tubes). AccessionNr481 appears to be homozygous for a recessive allele permittingmaize pollen tube growth. Attempts were made to produce sorghumx maize hybrids using Nr481 and CMS derivatives which were knownto allow maize pollen tube growth to the base of the style.A putative hybrid endosperm was obtained in one Nr481 x Seneca60 maize cross, but this was not repeatable and no hybrid plantswere produced. A fundamental problem may be the large size ofthe maize pollen tube, which could have difficulty growing throughthe sorghum ovary and in entering the micropyle. Sorghum bicolor spp. bicolor (L.) Moench, Zea mays L, sorghum, maize, pollen tube growth, hybridization barriers     

花粉管钙信号特性及其调控研究进展

花粉管在花柱中生长受多个信号分子的协同调控,钙离子在其中发挥着重要作用.钙是一种重要的第二信使,它将外界的多种生物或非生物信息转化为对细胞内基因表达以及细胞生理反应的调控.钙信号表达方式是胞内自由钙浓度的特异性变化.该文对国内外近年来有关花粉管生长中钙信号特性及其调控的研究进展,如花粉管尖端自由钙离子浓度梯度与胞内钙振荡、花粉管质膜钙转运体的鉴定及其调控特性、花粉管钙信号与微丝和ROP蛋白的关系以及花粉管钙信号与植物自交不亲和性反应的关系等进行综述,为深入开展相关研究提供参考.     

Effect of Growth Regulators and Light on Pollen Germination and Pollen Tube Growth in Pinus roxburghii Sarg

Pine (Pinus roxburghii) pollen grown in suspension cultureswas used to study the effects of growth regulators and lightconditions on germination and pollen tube growth. Indol-3-ylacetic acid, gibberellic acid, ethylene, abscisic acid and cyclicAMP (cAMP) at low concentrations (1–10 mg 1^–1) promotedgermination and tube growth. Addition of 1 and 10 mg 1^–1cAMP to any of the growth regulators had a promotory effect.Pollen tube growth decreased in white light as compared to thedark, and was increased in red light. Far-red light counteractedthe effect of red light. The effect of growth regulators incausing the enhanced tube growth appears to be manifested throughsubstances such as cAMP, and phytochrome seems to be involved. Pinus roxburghii, pine, pollen germination, pollen tube growth, growth regulators, cyclic AMP, phytochrome     

花粉管细胞结构与生长机制研究进展

花粉管的极性顶端生长是一个复杂的动力学过程,在高等植物有性生殖过程中起着重要的作用。花粉管的生长过程包括许多方面,其中最为重要的是花粉管细胞骨架动态和胞质运动。本文较全面地综述了花粉管的结构、细胞骨架、胞质运动、囊泡转运及循环、线粒体运动以及内质网和高尔基体之间囊泡运动等。     

花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用EGTA及钙调素抗血清处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,而注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调素及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调素对花粉萌发和花粉管伸长均有促进作用。     

Interference of the Histone Deacetylase Inhibits Pollen Germination and Pollen Tube Growth in Picea wilsonii Mast

Histone deacetylase (HDAC) is a crucial component in the regulation of gene expression in various cellular processes in animal and plant cells. HDAC has been reported to play a role in embryogenesis. However, the effect of HDAC on androgamete development remains unclear, especially in gymnosperms. In this study, we used the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB) to examine the role of HDAC in Picea wilsonii pollen germination and pollen tube elongation. Measurements of the tip-focused Ca²⁺ gradient revealed that TSA and NaB influenced this gradient. Immunofluorescence showed that actin filaments were disrupted into disorganized fragments. As a result, the vesicle trafficking was disturbed, as determined by FM4-64 labeling. Moreover, the distribution of pectins and callose in cell walls was significantly altered in response to TSA and NaB. Our results suggest that HDAC affects pollen germination and polarized pollen tube growth in Picea wilsonii by affecting the intracellular Ca²⁺ concentration gradient, actin organization patterns, vesicle trafficking, as well as the deposition and configuration of cell wall components.     

GA_3对含笑花粉萌发及花粉管生长的影响

以含笑（Michelia figo）花粉为试材,采用花粉离体培养法,研究GA3对含笑花粉萌发和花粉管生长的影响。结果表明,GA3可以促进含笑花粉提早萌发,20～200 mg/L GA3对含笑花粉萌发和花粉管生长起促进作用,浓度超过200 mg/L花粉萌发和花粉管生长均受到抑制。以GA3200 mg/L的促进作用最好。     

雌蕊胞外基质在花粉管生长中的作用

雌蕊胞外基质对雌蕊与花粉的识别以及花粉管的定向生长有着重要的作用,是近年来植物生殖生物学的研究热点之一。与花粉萌发和花粉管生长相关的雌蕊胞外基质种类主要包括阿拉伯半乳糖蛋白、类伸展素糖蛋白、富含脯氨酸糖蛋白、钙调素、S—糖蛋白、果胶以及子房的特异性物质等。本文着重介绍这些雌蕊胞外基质的生理功能及其研究进展。     

Inhibition of Camellia japonica Pollen Tube Growth by Maltose

Various oligosaccharides were studied with regard to their effecton the in vitro growth of Camellia japonica pollen tube. Sucrose,raffinose, melezitose, cellobiose, turanose and isomaltose,especially the first four, promoted pollen tube growth, whilemaltotriose, trehalose, gentiobiose, palatinose, melibiose,lactose and lactulose had little effect. Maltose strongly inhibitednot only the tube growth on sugar-free medium but also sugar-stimulatedgrowth, except in the case of sucrose stimulation. Glycosidaseactivities toward the growth-stimulating oligosaccharides weredetected in the extract of sucrose-grown pollen, but the activitiesof -glucosidase and -galactosidase were much lower than thoseof ß-fructosidase and ß-glucosidase. Maltosesuppressed the increase in UDP-glucose level of the glucose-grownpollen but not that of the sucrose-grown one. These resultssuggest that maltose acts, directly or indirectly, somewherein the pathway from glucose to UDP-glucose via glucose-1-phosphate,but does not interfere with the direct conversion of sucroseto UDP-glucose. (Received December 1, 1984; Accepted May 24, 1985)     

Pollen Tube Growth Inhibitors Involved in the Ovary of Self-Incompatible Japanese Pear

The growth inhibitors of pollen tubes in the pistils of Japanesepear ‘Chojuro’ were studied in vitro to elucidatethe physiological mechanism of self-incompatibility. Addition of water extracts from ovaries into a well dug in anagar medium containing sucrose and boric acid inhibited thegrowth of incompatible pollen tubes more strongly than thatof compatible ones. The substance, tentatively called Sinhibitor(self-inhibitor), was detectable in a relatively wide rangeof concentrations of the extract, from 10- to 60-fold dilution.However, it was absent in the stylar extract. S-inhibitor was stable, even though the extract was heated to100?C for 10 min. The chromatogram of the S-inhibitor followinga Sephadex G-10 gel filtration showed 2 peaks of inhibition:one peak corresponded to the peaks of protein and phenol (phenolsmay be conjugated with proteins), and the other to that of reducingsugar. The water extract from mature ovaries when diluted 10-fold wasa stronger growth inhibitor of incompatible pollen tubes comparedwith that from immature ones. This substance, tentatively calledA-inhibitor (adult-inhibitor), appeared to be different fromS-inhibitor. (Received May 20, 1986; Accepted December 22, 1986)     

Elevation of Pollen Mitochondrial DNA Copy Number by WHIRLY2: Altered Respiration and Pollen Tube Growth in Arabidopsis

In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information.Reflecting their endosymbiotic origin, mitochondria contain DNA genomes (mtDNA) encoding several key proteins for oxidative phosphorylation. As most genes identified in the mitochondrial genome are indispensable for mitochondrial function, it is generally believed that each mitochondrion must possess at least one full copy of the genome. Indeed, this seems to be the case in animals. For example, although the number of mitochondria per cell varies in human, mouse, rabbit, and rat cell lines, the mtDNA copy number per mitochondrion remains constant at 2.6 ± 0.3 (Robin and Wong, 1988). Also, in mouse egg cells, each mitochondrion contains an estimated one to two copies of the mtDNA (Pikó and Matsumoto, 1976).Plant cells, however, have very few copies of the mtDNA compared with the number of mitochondria. For example, in the Cucurbitaceae, cells containing 110 to 140 copies of the mtDNA have 360 to 1,100 mitochondria (Bendich and Gauriloff, 1984). In Arabidopsis (Arabidopsis thaliana), leaf cells each contain approximately 670 mitochondria (Sheahan et al., 2005) and approximately 50 copies of the mtDNA (Draper and Hays, 2000). Thus, in plant cells, each mitochondrion does not possess one complete copy of the mtDNA, a phenomenon that occurs commonly in somatic cells of plants (Preuten et al., 2010). In addition, work in Arabidopsis, barley (Hordeum vulgare), and tobacco (Nicotiana tabacum) showed that cells in leaves, stems, and roots contain few copies of the mtDNA (40–160), whereas cells in root tips contain more copies (300–450; Preuten et al., 2010). This is consistent with the mitochondrial nucleoid diminishment previously observed in developing root and shoot tips (Fujie et al., 1993, 1994), which suggests that the low copy numbers in plant cells result from a decrease in the mtDNA copy number in nondividing cells during development.One question raised by these findings is whether some mitochondria have complete mtDNAs while others have no mtDNA or whether mitochondria have partial mtDNAs. Using techniques for the direct visualization of small amounts of DNA, our group revealed that up to two-thirds of mitochondria in Arabidopsis mesophyll cells totally lack mtDNA and the remaining one-third of mitochondria possess mtDNA of about 100 kb on average (Wang et al., 2010). This agrees well with a previously reported value for mtDNA copy number (about 50 copies per cell; Draper and Hays, 2000) and is consistent with the idea that plant mitochondrial genomes exist as submolecules smaller than the total genomic sizes (Satoh et al., 1993; Kubo and Newton, 2008). Among plant cells possessing low mtDNA copy numbers, the vegetative cell in the pollen grains is an extreme case; a mature pollen grain of Antirrhinum majus, containing many more mitochondria than a somatic cell, possesses only 16 copies of the mtDNA (Wang et al., 2010). Similar to the changes observed in somatic cells, this extremely low level of mtDNA in pollen vegetative cells results from a rapid decrease in mtDNA copy number during pollen development (Sodmergen et al., 1991; Nagata et al., 1999). In A. majus, the vegetative cell in its initial developmental stage has 482.7 copies of the mtDNA per cell, indicating a 30-fold decrease (482.7/16) during development (Wang et al., 2010). These results from both somatic and reproductive cells led to the intriguing idea that the mtDNA copy number in plants decreases in parallel with cell differentiation, to a very low value, and thus that several mitochondria must share the genetic information carried on a single copy of the mtDNA. Plant cell mitochondria undergo frequent and coupled fusions and divisions, which may explain how mitochondria share this information (Arimura et al., 2004). However, the biological significance of why plant cells lose their mtDNA, and how this benefits these cells, remains unknown. Given that pollen germination, pollen tube elongation, and sperm cell delivery all require energy conversion, the extremely low mtDNA copy numbers, such as in pollen vegetative cells, must not compromise mitochondrial function.The mtDNA copy numbers remain constant in various tissues, however, indicating that cellular mechanisms accurately regulate the levels of mtDNA in relation to cell type (Robin and Wong, 1988; Preuten et al., 2010). In yeast and animals, this regulation involves the core enzymes of mtDNA replication, such as DNA polymerase-γ (Sharief et al., 1999), RNA polymerase (Wanrooij et al., 2008), and mitochondrial helicase (Liu et al., 2009), as well as a group of DNA-binding proteins such as ARS-binding factor2 protein in yeast (Saccharomyces cerevisiae; Newman et al., 1996), MITOCHONDRIAL TRANSCRIPTION FACTOR A (TFAM) in human (Alam et al., 2003), and mitochondrial single-stranded DNA binding protein in Drosophila spp. (Maier et al., 2001). Overexpression of TFAM causes an increase in the mtDNA copy number, and RNA interference of TFAM decreases the mtDNA copy number (Ekstrand et al., 2004; Kanki et al., 2004). Also, the homozygous knockout of TFAM in mouse results in embryos that lack mtDNA and thus fail to survive (Larsson et al., 1998). Clearly, protein factors within mitochondrial nucleoids play a crucial role in the regulation of mtDNA copy number.Recent investigation in Arabidopsis revealed that, similar to the case in yeast and animal cells, DNA polymerase, the core enzyme of mtDNA replication, functions to maintain mtDNA levels in plants. Mutation of Arabidopsis PolIA or PolIB (homologs of bacterial DNA polymerase I) causes a reduction in mtDNA copy number, and double mutation of these proteins is lethal (Parent et al., 2011). Also, an Mg²⁺-dependent exonuclease, DEFECTIVE IN POLLEN ORGANELLE DNA DEGRADATION1 (DPD1), degrades organelle DNA, helping to produce the proper amounts of mtDNA in pollen cells (Matsushima et al., 2011; Tang et al., 2012). These results provide insights into the molecular control of mtDNA levels in plants, via both mtDNA replication and mtDNA degradation. Except for these enzymes, however, other protein factors (such as TFAM in animals) have not been identified in plants. The DNA-binding proteins, such as MutS Homolog1 (MSH1), Organellar Single-Strand DNA Binding Protein1 (OSB1), Recombinase A1 (RecA1), RecA3, and WHIRLY2 (WHY2), identified so far in plant mitochondria likely participate in genomic maintenance by affecting substoichiometric shifting (Abdelnoor et al., 2003), stoichiometric transmission (Zaegel et al., 2006), genomic stability (Shedge et al., 2007; Odahara et al., 2009), and DNA repair (Cappadocia et al., 2010). None of these plant nucleoid factors (DNA-binding proteins) has been implicated in the control of mtDNA copy number; thus, the mechanisms by which nonenzyme protein factors regulate mtDNA copy number in plants remain obscure.To test whether nucleoid DNA-binding proteins can affect mtDNA copy number, we examined the effect of producing Arabidopsis WHY2, a single-stranded DNA-binding protein (Cappadocia et al., 2010), in the pollen vegetative cell, which generally does not express WHY2 (Honys and Twell, 2004). We found that expression of WHY2 resulted in a 10-fold increase in mtDNA copy number in the pollen vegetative cell. This increase affected mitochondrial respiration, mitochondrial size, and pollen tube growth. Thus, our results uncover a novel function for WHY2, a member of the plant Whirly protein family, in regulating mtDNA amounts and indicate that, in plants, low mtDNA copy number does not compromise mitochondrial function but rather promotes proper mitochondrial function.     

Analysis of nuclear and mitochondrial genomes of transplastomic Salpiglossis sinuata plants obtained by transfer of transformed plastids from N. tabacum (+S. sinuata) cybrid

New model system of plastid transformation has been proposed using a wild representative of Solanaceae family--S. sinuata. Earlier obtained cybrid plants N. tabacum (+ S. sinuata) were used for transformation experiments by PEG treatment of protoplasts with aadA gene that confers resistance to spectinomycin. Transformed S. sinuata plastome was transferred from N. tabacum (+ S. sinuata) cybrid to S. sinuata wild type plants by somatic hybridization. Molecular analysis of nuclear and mitochondrial DNA has been performed.     

Fine-Tuning of the Cytoplasmic Ca2+ Concentration Is Essential for Pollen Tube Growth

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca²⁺ concentration ([Ca²⁺]) gradient and regular oscillations of the cytosolic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region. Whether this [Ca²⁺] gradient and/or [Ca²⁺]_cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca²⁺]_cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca²⁺]_cyt gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca²⁺]_cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca²⁺]_cyt in the tip region is essential for pollen tube growth, whereas a regular [Ca²⁺] oscillation is not. Next, we monitored [Ca²⁺] dynamics in the endoplasmic reticulum ([Ca²⁺]_ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca²⁺ compared with yellow cameleon 3.60. The [Ca²⁺]_ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 μm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca²⁺-ATPases, inhibited growth and decreased the [Ca²⁺]_ER. Our observations suggest that the ER serves as one of the Ca²⁺ stores in the pollen tube and cyclopiazonic acid-sensitive Ca²⁺-ATPases in the ER are required for pollen tube growth.In many flowering plants, a pollen grain that lands on the top surface of a stigma will hydrate and germinate a pollen tube. Following germination, the pollen tube enters the style and grows through the wall of transmitting tract cells on the way to the ovary, where the tube emerges to release the sperm for double fertilization. Therefore, pollen tube growth is essential for reproduction in flowering plants.Since Brewbaker and Kwack (1963) revealed that Ca²⁺ is essential for in vitro pollen tube cultures, the relationship between the Ca²⁺ concentration ([Ca²⁺]) and pollen tube growth has been further examined under in vitro germination culture conditions. Ratiometric ion imaging using fluorescent dye has revealed that the apical domain of a pollen tube grown in vitro contains a tip-focused [Ca²⁺] gradient (Pierson et al., 1994, 1996; Cheung and Wu, 2008) and that the cytoplasmic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region and the growth rate oscillate with the same periodicity (Pierson et al., 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997). Therefore, oscillation of the [Ca²⁺]_cyt has been thought to correlate with pollen tube growth. It is not clear, however, whether regular [Ca²⁺]_cyt oscillations in the tip region occur in pollen tubes growing through stigmas and styles.The [Ca²⁺]_cyt is controlled temporally and spatially by transporters in the membranes of intracellular compartments and in the plasma membrane (Sze et al., 2000). Studies using a Ca²⁺-sensitive vibrating electrode revealed Ca²⁺ influx in the tip region of the pollen tube (Pierson et al., 1994; Holdaway-Clarke et al., 1997; Franklin-Tong et al., 2002). Stretch-activated Ca²⁺ channels have been found in the plasma membrane using patch-clamp electrophysiology (Kuhtreiber and Jaffe, 1990; Dutta and Robinson, 2004). Recently, CNGC18 was identified as a Ca²⁺-permeable channel in the plasma membrane that is essential for pollen tube growth (Frietsch et al., 2007). The intracellular compartments that store Ca²⁺ in the pollen tube and the relevant Ca²⁺ transporters, however, have yet to be identified.Yellow cameleons are genetically encoded Ca²⁺ indicators that were developed to monitor the [Ca²⁺] in living cells (Miyawaki et al., 1997). These indicators are chimeric proteins consisting of enhanced cyan fluorescent protein (ECFP), calmodulin (CaM), a glycylglycine linker, the CaM-binding domain of myosin light chain kinase (M13), and enhanced yellow fluorescent protein (EYFP). When the CaM domain binds Ca²⁺, the domain associates with the M13 peptide and induces fluorescence resonance energy transfer (FRET) between ECFP and EYFP. Several types of cameleons have been developed by tuning the CaM domain binding affinity for Ca²⁺. Yellow cameleon 2.1 (YC2.1) is a high-affinity indicator that has been used to monitor the [Ca²⁺]_cyt in Arabidopsis (Arabidopsis thaliana) guard cells (Allen et al., 1999, 2000, 2001), Lilium longiflorum and Nicotiana tabacum pollen tubes (Watahiki et al., 2004), and the root hair of Medicago truncatula (Miwa et al., 2006). YC3.1 is a low-affinity indicator that has been used to monitor the [Ca²⁺]_cyt during pollen germination and in papilla cells of Arabidopsis (Iwano et al., 2004).Recently, YC3.60 was developed as a new YC variant (Nagai et al., 2004), in which the acceptor fluorophore is a circularly permuted version of Venus rather than EYFP (Nagai et al., 2002). YC3.60 has a monophasic Ca²⁺ dependency with a dissociation constant (K_d) of 0.25 μm. Compared with YC3.1, YC3.60 is equally bright with a 5- to 6-fold larger dynamic range. Thus, YC3.60 results in a markedly enhanced signal-to-noise ratio, thereby enabling Ca²⁺ imaging experiments that were not possible with conventional YCs. On the other hand, YC4.60 was developed by mutating the Ca²⁺-binding loop of CaM in YC3.60. Because YC4.60 has a significantly lower Ca²⁺ affinity with a biphasic Ca²⁺ dependency (K_d: 58 nm and 14.4 μm), it allows changes in [Ca²⁺] dynamics to be detected against a high background [Ca²⁺] (Nagai et al., 2004).To examine whether the [Ca²⁺]_cyt oscillates in pollen tubes growing through a stigma after pollination (in vivo condition), in those placed in germination medium immediately after passing through a style (semi-in vivo condition), or in those grown in germination medium (in vitro condition), we generated transgenic Arabidopsis and N. tabacum lines expressing the YC3.60 gene in their pollen grains and monitored Ca²⁺ dynamics in the pollen tube tip. We also examined how inhibitors of pollen tube growth affect Ca²⁺ dynamics in pollen tubes growing under the semi-in vivo condition. To examine Ca²⁺ dynamics in the endoplasmic reticulum (ER), we generated transgenic Arabidopsis plants expressing YC4.60 in the pollen tube ER. The results are discussed in relation to the physiological relevance of [Ca²⁺] oscillations for pollen tube growth.