Extensin-like Glycoproteins in the Maize Pollen Tube Wall

We recently described the cloning and characterization of Pex1, a maize pollen-specific gene with an extensin-like domain. Here, we report that antibodies raised against a Pex fusion protein and a Pex synthetic peptide recognize a protein doublet with an apparent molecular mass of ~300 kD as well as larger proteins in pollen extracts. These proteins were not detected in extracts of seedling, endosperm, ear, silk, root, leaf, wounded leaf, meiotic tassel, or young microspore. After deglycosylation, only the protein doublet was detected by the anti-Pex antiserum, suggesting that the higher molecular mass proteins represent a glycosylated form of the Pex proteins. The anti-Pex antiserum was also used in immunolocalization experiments with in vitro-germinated pollen. With the aid of a confocal light microscope, the Pex proteins were localized to the pollen tube wall. The Pex proteins could not be removed with high salt, SDS, or chaotropic or reducing agents, suggesting a very tight association with the pollen tube wall. Immunocytochemical analysis at the ultrastructural level localized the Pex proteins to the intine in mature pollen and to the callosic sheath of the pollen tube wall in germinated pollen. Localization to the pollen tube wall strongly suggests that the Pex proteins play a role in pollen tube growth during pollination.     

The Apical Actin Fringe Contributes to Localized Cell Wall Deposition and Polarized Growth in the Lily Pollen Tube

In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube.Pollen tubes provide an excellent model for studying the molecular and physiological processes that lead to polarized cell growth. Because all plant cell growth results from the regulated yielding of the cell wall in response to uniform turgor pressure (Winship et al., 2010; Rojas et al., 2011), the cell wall of the pollen tube must yield only at a particular spot: the cell apex, or tip. To accomplish the extraordinary growth rates seen in many species, and to balance the thinning of the apical wall due to rapid expansion, the pollen tube delivers prodigious amounts of wall material, largely methoxylated pectins, to the tip in a coordinated manner. Recent studies suggest that the targeted exocytosis increases the extensibility of the cell wall matrix at the tip, which then yields to the existing turgor pressure, permitting the tip to extend or grow (McKenna et al., 2009; Hepler et al., 2013). There are many factors that influence exocytosis in growing pollen tubes; in this study, we investigate the role of the apical actin fringe.For many years, it has been known that an actin structure exists near the pollen tube tip, yet its exact form has been a matter of some contention (Kost et al., 1998; Lovy-Wheeler et al., 2005; Wilsen et al., 2006; Cheung et al., 2008; Vidali et al., 2009; Qu et al., 2013). The apical actin structure has been variously described as a fringe, a basket, a collar, or a mesh. Using rapid freeze fixation of lily (Lilium formosanum) pollen tubes followed by staining with anti-actin antibodies, the structure appears as a dense fringe of longitudinally oriented microfilaments, beginning 1 to 5 µm behind the apex and extending 5 to 10 µm basally. The actin filaments are positioned in the cortical cytoplasm close to the plasma membrane (Lovy-Wheeler et al., 2005). More recently, we used Lifeact-mEGFP, a probe that consistently labels this palisade of longitudinally oriented microfilaments in living cells (Vidali et al., 2009; Fig. 1A, left column). For the purposes of this study, we will refer to this apical organization of actin as a fringe.Open in a separate windowFigure 1.The actin fringe and the thickened pollen tube tip wall are stable, although dynamic, structures during pollen tube growth. A, The left column shows a pollen tube transformed with Lifeact-mEGFP imaged with a spinning-disc confocal microscope. Maximal projections from every 15 s are shown. The right column shows epifluorescence images of a pollen tube stained with PI. Again, images captured every 15 s are shown. Bars = 10 μm. B, The data from the pollen tube in A expressing Lifeact-mEGFP were subjected to kymograph analysis using an 11-pixel strip along the image’s midline. C, The first three frames from the pollen tube in A and B were assigned the colors red, blue, and green, respectively, and then overlaid. Areas with white show the overlap of all three. The fringe is stable, but most of its constituent actin is not shared between frames.Many lines of evidence demonstrate that actin is required for pollen tube growth. Latrunculin B (LatB), which blocks actin polymerization, inhibits pollen tube growth and disrupts the cortical fringe at concentrations as low as 2 nm. Higher concentrations are needed to block pollen grain germination and cytoplasmic streaming (Gibbon et al., 1999; Vidali et al., 2001). Actin-binding proteins, including actin depolymerizing factor-cofilin, formin, profilin, and villin, and signaling proteins, such as Rho-of-Plants (ROP) GTPases and their effectors (ROP interacting crib-containing proteins [RICs]), also have been shown to play critical roles in growth and actin dynamics (Fu et al., 2001; Vidali et al., 2001; Allwood et al., 2002; Chen et al., 2002; Cheung and Wu, 2004; McKenna et al., 2004; Gu et al., 2005; Ye et al., 2009; Cheung et al., 2010; Staiger et al., 2010; Zhang et al., 2010a; Qu et al., 2013; van Gisbergen and Bezanilla, 2013).Our understanding of the process of exocytosis and pollen tube elongation has been influenced by ultrastructural images of pollen tube tips, which reveal an apical zone dense with vesicles (Cresti et al., 1987; Heslop-Harrison, 1987; Lancelle et al., 1987; Steer and Steer, 1989; Lancelle and Hepler, 1992; Derksen et al., 1995). It has long been assumed that these represent exocytotic vesicles destined to deliver new cell wall material. This model of polarized secretion has been challenged in recent years in studies using FM dyes. Two groups have suggested that exocytosis occurs in a circumpolar annular zone (Bove et al., 2008; Zonia and Munnik, 2008). However, other studies, using fluorescent beads attached to the cell surface, indicate that the maximal rate of expansion, and of necessity the greatest deposition of cell wall material, occurs at the apex along the polar axis of the tube (Dumais et al., 2006; Rojas et al., 2011). Similarly, our experiments with propidium iodide (PI; McKenna et al., 2009; Rounds et al., 2011a) and pectin methyl esterase fused to GFP (McKenna et al., 2009) show that the wall is thickest at the very tip and suggest that wall materials are deposited at the polar axis, consistent with the initial model of exocytosis (Lancelle and Hepler, 1992). Experiments using tobacco (Nicotiana tabacum) pollen and a receptor-like kinase fused to GFP also indicate that exocytosis occurs largely at the apical polar axis (Lee et al., 2008).Many researchers argue that apical actin is critical for exocytosis (Lee et al., 2008; Cheung et al., 2010; Qin and Yang, 2011; Yan and Yang, 2012). More specifically, recent work suggests that the fringe participates in targeting vesicles and thereby contributes to changes in growth direction (Kroeger et al., 2009; Bou Daher and Geitmann, 2011; Dong et al., 2012). In this article, using three different inhibitors, namely brefeldin A (BFA), LatB, and potassium cyanide (KCN), we test the hypothesis that polarized pectin deposition in pollen tubes requires the actin fringe. Our data show that during normal growth, pectin deposition is focused to the apex along the polar axis of the tube. However, when growth is modulated, different end points arise, depending on the inhibitor. With BFA, exocytosis stops completely, and the fringe disappears, with the appearance of an actin aggregate at the base of the clear zone. LatB, as shown previously (Vidali et al., 2009), incompletely degrades the actin fringe and leaves a rim of F-actin around the apical dome. Here, we show that, in the presence of LatB, pectin deposition continues, with the focus of this activity shifting in position frequently as the slowly elongating pollen tube changes direction. With KCN, the actin fringe degrades completely, but exocytosis continues and becomes depolarized, with pectin deposits now occurring across a wide arc of the apical dome. This dome often swells as deposition continues, only stopping once normal growth resumes. Taken together, these results support a role for the actin fringe in controlling the polarity of growth in the lily pollen tube.     

ANXUR Receptor-Like Kinases Coordinate Cell Wall Integrity with Growth at the Pollen Tube Tip Via NADPH Oxidases

It has become increasingly apparent that the extracellular matrix (ECM), which in plants corresponds to the cell wall, can influence intracellular activities in ways that go far beyond their supposedly passive mechanical support. In plants, growing cells use mechanisms sensing cell wall integrity to coordinate cell wall performance with the internal growth machinery to avoid growth cessation or loss of integrity. How this coordination precisely works is unknown. Previously, we reported that in the tip-growing pollen tube the ANXUR receptor-like kinases (RLKs) of the CrRLK1L subfamily are essential to sustain growth without loss of cell wall integrity in Arabidopsis. Here, we show that over-expression of the ANXUR RLKs inhibits growth by over-activating exocytosis and the over-accumulation of secreted cell wall material. Moreover, the characterization of mutations in two partially redundant pollen-expressed NADPH oxidases coupled with genetic interaction studies demonstrate that the ANXUR RLKs function upstream of these NADPH oxidases. Using the H₂O₂-sensitive HyPer and the Ca²⁺-sensitive YC3.60 sensors in NADPH oxidase-deficient mutants, we reveal that NADPH oxidases generate tip-localized, pulsating H₂O₂ production that functions, possibly through Ca²⁺ channel activation, to maintain a steady tip-focused Ca²⁺ gradient during growth. Our findings support a model where ECM-sensing receptors regulate reactive oxygen species production, Ca²⁺ homeostasis, and exocytosis to coordinate ECM-performance with the internal growth machinery.     

Pollen Cell Wall Patterns Form from Modulated Phases

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拟南芥授粉前后花粉与乳突细胞的超微结构

利用透射电子显微镜技术,对自交亲和植物拟南芥授粉前后花粉和乳突细胞的超微结构进行了观察。发现花粉和柱头乳突细胞一些未经报道的超微结构特征,可能与拟南芥花粉和乳突细胞的识别及花粉管生长相关:(1)成熟花粉中,电子透明的、体积较大的小液泡(直径200～1000nm)呈均匀分布。部分小液泡内含有多层膜状结构物质,推测可能是膜的一种储存形式,与花粉萌发时大量出现的小囊泡有关。(2)花粉萌发时,小液泡由均匀分布变为不均匀分布。(3)授粉前后的乳突细胞顶端和侧端的内壁上有明显的壁内突结构,粘附的花粉开始萌发时的乳突细胞壁内突处可观察到直径50～100nm的小泡存在,表明拟南芥乳突细胞具有一定的分泌功能。     

花粉管细胞壁结构及胞质运动

花粉管的极性顶端生长是将雄配子体运输到子房的过程,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壁的合成和胞质运动。本文就细胞壁的结构及组成,生殖细胞和营养核的移位,细胞器以及分泌小泡的运动等方面作了较全面论述。     

ABCG Transporters Are Required for Suberin and Pollen Wall Extracellular Barriers in Arabidopsis

Effective regulation of water balance in plants requires localized extracellular barriers that control water and solute movement. We describe a clade of five Arabidopsis thaliana ABCG half-transporters that are required for synthesis of an effective suberin barrier in roots and seed coats (ABCG2, ABCG6, and ABCG20) and for synthesis of an intact pollen wall (ABCG1 and ABCG16). Seed coats of abcg2 abcg6 abcg20 triple mutant plants had increased permeability to tetrazolium red and decreased suberin content. The root system of triple mutant plants was more permeable to water and salts in a zone complementary to that affected by the Casparian strip. Suberin of mutant roots and seed coats had distorted lamellar structure and reduced proportions of aliphatic components. Root wax from the mutant was deficient in alkylhydroxycinnamate esters. These mutant plants also had few lateral roots and precocious secondary growth in primary roots. abcg1 abcg16 double mutants defective in the other two members of the clade had pollen with defects in the nexine layer of the tapetum-derived exine pollen wall and in the pollen-derived intine layer. Mutant pollen collapsed at the time of anther desiccation. These mutants reveal transport requirements for barrier synthesis as well as physiological and developmental consequences of barrier deficiency.     

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.     

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40–50%), hemicellulose (25–30%), and lignin (20–30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas Mäule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.     

ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.     

Genetic Evidence for a Long-Range Activity That Directs Pollen Tube Guidance in Arabidopsis

The fertilization process of plants is governed by different kinds of cell-cell interactions. In higher plants, these interactions are required both for recognition of the pollen grain by the female reproductive system and to direct the growth of the pollen tube inside the ovary. Despite many years of study, the signaling mechanisms that guide the pollen tube toward its target, the ovule, are largely unknown. Two distinct types of principles, mechanical and chemotropic, have been suggested to account for the directed growth of the pollen tube. The first of these two types of models implies that the guidance of the pollen tube depends on the architecture and chemical properties of the female reproductive tissues, whereas the latter suggests that the ovule provides a signal for the target-directed growth of the pollen tube. To examine such a role for the ovules, we analyzed the growth path of pollen tubes in mutants defective in ovule development in Arabidopsis. The results presented here provide unique in vivo evidence for an ovule-derived, long-range activity controlling pollen tube guidance. A morphological comparison of the ovule mutants used in this study indicates that within the ovule, the haploid embryo sac plays an important role in this long-range signaling process.     

Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments

The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca²⁺, in vitro. Analysis of a mutant that bears a point mutation at the Ca²⁺ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca²⁺ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.     

硼缺乏导致花粉管细胞壁多糖分布的改变

集应用免疫细胞化学及显微红外光谱分析技术,深入研究了硼元素对花粉管生长的调节作用。用识别甲酯化果胶的单克隆抗体JIM7和识别酸性果胶的JIM5对离体培养的百合（LiliumlongiflorumThunb．）及烟草（NicotianatabacumL.cv．“PetitHavana”）花粉管进行免疫荧光标记,发现无硼培养导致细胞壁果胶成分呈异常分布,酸性果胶在花粉管顶端大量富集;苯胺蓝诱导荧光法揭示,无硼培养引起胼胝质在顶端细胞壁积累。通过显微红外光谱（FTIR）分析,进一步验证了无硼培养导致细胞壁酸性果胶质含量的增加,并发现酚酯含量比正常情况减少,而游离酚类化合物含量明显增加。上述结果表明,硼可能作为一种相关因子影响关键酶活性,改变细胞壁多糖网状结构以至细胞壁的延展性,从而调节花粉管生长;酚类积累对质膜完整性的影响也会对调节花粉管生长有间接作用。     

Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]_cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]_cyt of the pollen tubes in vivo, we show that [pH]_cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]_cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]_cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]_cyt and its effect on several components in the context of SI-induced events and PCD.Programmed cell death (PCD) in plants is relatively well documented and characterized (Jones and Dangl, 1996; van Doorn, 2011; van Doorn et al., 2011). There is considerable biochemical evidence for the involvement of caspase-like activities in plant PCD (van Doorn and Woltering, 2005). For example, the vacuolar processing enzyme has YVADase (caspase-1-like) activity (Hatsugai et al., 2004; Rojo et al., 2004; Hara-Nishimura et al., 2005), DEVDase (caspase-3-like) and YVADases are associated with PCD in several plant systems (del Pozo and Lam, 1998; Korthout et al., 2000; Danon et al., 2004), and VEIDase (caspase-6-like) is the main caspase-like activity involved in embryonic pattern formation (Bozhkov et al., 2004). However, because plants have no caspase gene homologs (Sanmartín et al., 2005), the nature of their caspase-like enzymes is the subject of considerable debate. Vacuolar cell death is one of two major classes of PCD in plants (van Doorn et al., 2011). It is thought that collapse of the vacuole is a key irreversible step in several plant PCD systems, including during tissue and organ formation, such as the classic differentiation of tracheary elements (Hara-Nishimura and Hatsugai, 2011). Exactly how this is achieved and what processes are involved remain unknown. Until very recently, it was generally thought that the rupturing vacuole releases proteases, hydrolases, and nucleases, allowing cellular disassembly by an autophagy-like process. Some PCD systems cannot be assigned to either class; these include PCD triggered by the hypersensitive response to biotrophic pathogens, PCD in cereal endosperm, and self-incompatibility (SI)-induced PCD (van Doorn et al., 2011).SI is a genetically controlled pollen-pistil cell-cell recognition system. Self-pollen is recognized by the stigma as being genetically identical, resulting in inhibition of pollen tube growth. Most SI systems use tightly linked polymorphic genes: the pollen (male) and pistil (female) S-determinants. In field poppy (Papaver rhoeas), the S-determinants are a 14-kD signaling ligand field poppy stigma S (PrsS) and a unique transmembrane protein field poppy pollen S (PrpS; Foote et al., 1994; Wheeler et al., 2010). These interact in an S-specific manner, and increases in cytosolic free calcium ([Ca2+]_cyt) are triggered in incompatible pollen tubes (Franklin-Tong et al., 1993), resulting in phosphorylation of soluble inorganic pyrophosphatases (sPPases; Rudd et al., 1996; de Graaf et al., 2006), activation of a Mitogen-Activated Protein Kinase (MAPK; Rudd et al., 2003), and increases in reactive oxygen species (ROS) and nitric oxide (Wilkins et al., 2011, 2014). Most of these components are integrated into a signaling network leading to PCD (Bosch et al., 2008; Wilkins et al., 2014). The actin cytoskeleton is a key target in the field poppy SI response, undergoing depolymerization (Snowman et al., 2002) followed by polymerization into highly stable F-actin foci decorated with the actin binding proteins (ABPs) Actin-Depolymerizing Factor (ADF) and Cyclase-Associated Protein (CAP; Poulter et al., 2010, 2011), with both processes being involved in mediating PCD (Thomas et al., 2006). A major player in SI-mediated PCD is a caspase-3-like/DEVDase-like activity (Thomas and Franklin-Tong, 2004; Bosch and Franklin-Tong, 2007). The SI-induced caspase-3-like/DEVDase exhibits maximum substrate cleavage in vitro at pH 5, with peak activity 5 h after SI induction in vivo (Bosch and Franklin-Tong, 2007). The low pH optimum for this caspase-3-like/DEVDase activity is unusual, because most of the cytosolic plant caspase-like activities identified to date have optimal activity close to normal physiological pH (approximate pH, 6.5–7.0; Korthout et al., 2000; Bozhkov et al., 2004; Coffeen and Wolpert, 2004). Because the SI-induced cytosolic-located DEVDase requires a low pH for activity, this suggested that, during SI, the pollen tube cytosol undergoes dramatic acidification. In vivo pH measurements of the cytosol at 1 to 4 h after SI induction confirmed this, when cytosolic pH ([pH]_cyt) had dropped from pH 6.9 to pH 5.5 (Bosch and Franklin-Tong, 2007). This fits the in vitro pH optimum of the caspase-3-like/DEVDase almost exactly, implicating pollen cytosolic acidification as playing a vital role in creating optimal conditions for the activation of the caspase-3-like/DEVDase-like activity and progression of PCD.Under normal cellular conditions, [pH]_cyt is between approximately 6.9 and 7.5 (Kurkdjian and Guern, 1989; Felle, 2001). Pollen tubes, like other tip-growing cells, have [pH]_cyt gradients (Gibbon and Kropf, 1994; Feijó et al., 1999). The [pH]_cyt of the pollen tube shank is an approximate pH of 6.9 to 7.11 (Fricker et al., 1997; Messerli and Robinson, 1998). There has been much debate about the [pH]_cyt gradient, comprising an apical domain with an approximate pH of 6.8 and a subapical alkaline band with an approximate pH of 7.2 to 7.8 in Lilium longiflorum and Lilium formosanum pollen tubes (Fricker et al., 1997; Messerli and Robinson, 1998; Feijó et al., 2001; Lovy-Wheeler et al., 2006). Oscillations of [pH]_cyt between approximate pH values of 6.9 and 7.3 have been linked to tip growth in L. formosanum pollen tubes (Lovy-Wheeler et al., 2006). The vacuole and the apoplast have a highly acidic pH between pH 5 and pH 6 (Katsuhara et al., 1989; Feijó et al., 1999). The majority of studies of pH changes in plant cells reports modest, transient changes in [pH]_cyt of approximately 0.4 and 0.7 pH units during development, gravitropic responses, decreases in light intensity, and addition of elicitors, hormones, and other treatments. For example, during root hair development in Arabidopsis (Arabidopsis thaliana), root [pH]_cyt was elevated from an approximate pH of 7.3 to 7.7 (Bibikova et al., 1998). Root gravitropic responses stimulate small transient [pH]_cyt alterations (Scott and Allen, 1999; Fasano et al., 2001; Johannes et al., 2001). More recently, it has been shown that the [pH]_cyt drops during PCD controlling root cap development; however, exactly how many units the [pH]_cyt decreased was not measured (Fendrych et al., 2014). Other studies investigating [pH]_cyt in response to physiologically relevant signals also report small transient alterations. Light-adapted cells respond to a decrease in light intensity with a rapid transient cytosolic acidification by approximately 0.3 pH units (Felle et al., 1986). Addition of nodulation factors resulted in an increase of 0.2 pH units in root hairs (Felle et al., 1998), and abscisic acid increased the [pH]_cyt of guard cells by 0.3 pH units (Blatt and Armstrong, 1993). Changes in [pH]_cyt are thought to activate stress responses (Felle, 2001). Elicitor treatments resulted in a [pH]_cyt drop of between 0.4 and 0.7 pH units in suspension cells (Mathieu et al., 1996; Kuchitsu et al., 1997), a drop of 0.2 pH units in Nitellopsis obtusa cells treated with salt (Katsuhara et al., 1989), and a drop of 0.3 to 0.7 pH units in Eschscholzia californica (Roos et al., 1998).Here, we investigate SI-induced acidification of the cytosol, providing measurements of physiologically relevant temporal alterations in [pH]_cyt, and identify key targets of this, providing mechanistic insights into these events. The SI-induced acidification plays a pivotal role in the activation of a caspase-3-like/DEVDase activity, the formation of punctate F-actin foci, and ABP localization during SI PCD. We investigate the vacuole as a potential contributor to SI-induced [pH]_cyt acidification.     

AUXIN RESPONSE FACTOR17 Is Essential for Pollen Wall Pattern Formation in Arabidopsis

In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression.In angiosperms, the pollen wall is the most complex plant cell wall. It consists of the inner wall, the intine, and the outer wall, the exine. The exine is further divided into sexine and nexine layers. The sculptured sexine includes three major parts: baculum, tectum, and tryphine (Heslop-Harrison, 1971; Piffanelli et al., 1998; Ariizumi and Toriyama, 2011; Fig. 1A). Production of a functional pollen wall requires the precise spatial and temporal cooperation of gametophytic and sporophytic tissues and metabolic events (Blackmore et al., 2007). The intine layer is controlled gametophytically, while the exine is regulated sporophytically. The sporophytic tapetum cells provide material for pollen wall formation, while primexine determines pollen wall patterning (Heslop-Harrison, 1968).Open in a separate windowFigure 1.Schematic representation of the pollen wall and primexine development. A, The innermost layer adjacent to the plasma membrane is the intine. The bacula (Ba), tectum (Te), and tryphine (T) make up the sexine layer. The nexine is located between the intine and the sexine layers. The exine includes the nexine and sexine layers. B, Primexine (Pr) appears between callose (Cl) and plasma membrane (Pm) at the early tetrad stage (left panel). Subsequently, the plasma membrane becomes undulated (middle panel) and sporopollenin deposits on the peak of the undulated plasma membrane to form bacula and tectum (right panel).After meiosis, four microspores were encased in callose to form a tetrad. Subsequently, the primexine develops between the callose layer and the microspore membrane (Fig. 1B), and the microspore plasma membrane becomes undulated (Fig. 1B; Fitzgerald and Knox, 1995; Southworth and Jernstedt, 1995). Sporopollenin precursors then accumulate on the peak of the undulated microspore membrane to form the bacula and tectum (Fig. 1B; Fitzgerald and Knox, 1995). After callose degradation, individual microspores are released from the tetrad, and the bacula and tectum continue to grow into exine with further sporopollenin deposition (Fitzgerald and Knox, 1995; Blackmore et al., 2007).The callose has been reported to affect primexine deposition and pollen wall pattern formation. The peripheral callose layer, secreted by the microsporocyte, acts as the mold for primexine (Waterkeyn and Bienfait, 1970; Heslop-Harrison, 1971). CALLOSE SYNTHASE5 (CalS5) is the major enzyme responsible for the biosynthesis of the callose peripheral of the tetrad (Dong et al., 2005; Nishikawa et al., 2005). Mutation of Cals5 and abnormal CalS5 pre-mRNA splicing resulted in defective peripheral callose deposition and primexine formation (Dong et al., 2005; Nishikawa et al., 2005; Huang et al., 2013). Besides CalS5, four membrane-associated proteins have also been reported to be involved in primexine formation: DEFECTIVE EXINE FORMATION1 (DEX1; Paxson-Sowders et al., 1997, 2001), NO EXINE FORMATION1 (NEF1; Ariizumi et al., 2004), RUPTURED POLLEN GRAIN1 (RPG1; Guan et al., 2008; Sun et al., 2013), and NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU; Chang et al., 2012). Mutation of DEX1 results in delayed primexine formation (Paxson-Sowders et al., 2001). The primexine in nef1 is coarse compared with the wild type (Ariizumi et al., 2004). The loss-of-function rpg1 shows reduced primexine deposition (Guan et al., 2008; Sun et al., 2013), while the npu mutant does not deposit any primexine (Chang et al., 2012). Recently, it was reported that Arabidopsis (Arabidopsis thaliana) CYCLIN-DEPENDENT KINASE G1 (CDKG1) associates with the spliceosome to regulate the CalS5 pre-mRNA splicing for pollen wall formation (Huang et al., 2013). Clearly, disrupted primexine deposition leads to aberrant pollen wall patterning and ruptured pollen grains in these mutants.The plant hormone auxin has multiple roles in plant reproductive development (Aloni et al., 2006; Sundberg and Østergaard, 2009). Knocking out the two auxin biosynthesis genes, YUC2 and YUC6, caused an essentially sterile phenotype in Arabidopsis (Cheng et al., 2006). Auxin transport is essential for anther development; defects in auxin flow in anther filaments resulted in abnormal pollen mitosis and pollen development (Feng et al., 2006). Ding et al. (2012) showed that the endoplasmic reticulum-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis. Evidence for the localization, biosynthesis, and transport of auxin indicates that auxin regulates anther dehiscence, pollen maturation, and filament elongation during late anther development (Cecchetti et al., 2004, 2008). The role of auxin in pollen wall development has not been reported.The auxin signaling pathway requires the auxin response factor (ARF) family proteins (Quint and Gray, 2006; Guilfoyle and Hagen, 2007; Mockaitis and Estelle, 2008; Vanneste and Friml, 2009). ARF proteins can either activate or repress the expression of target genes by directly binding to auxin response elements (AuxRE; TGTCTC/GAGACA) in the promoters (Ulmasov et al., 1999; Tiwari et al., 2003). The Arabidopsis ARF family contains 23 members. A subgroup in the ARF family, ARF10, ARF16, and ARF17, are targets of miRNA160 (Okushima et al., 2005b; Wang et al., 2005). Plants expressing miR160-resistant ARF17 exhibited pleiotropic developmental defects, including abnormal stamen structure and reduced fertility (Mallory et al., 2005). This indicates a potential role for ARF17 in plant fertility, although the detailed function remains unknown. In addition, ARF17 was also proposed to negatively regulate adventitious root formation (Sorin et al., 2005; Gutierrez et al., 2009), although an ARF17 knockout mutant was not reported and its phenotype is unknown.In this work, we isolated and characterized a loss-of-function mutant of ARF17. Results from cytological observations suggest that ARF17 controls callose biosynthesis and primexine deposition. Consistent with this, the ARF17 protein is highly abundant in microsporocytes and tetrads. Furthermore, we demonstrate that the ARF17 protein is able to bind the promoter region of CalS5. Our results suggest that ARF17 regulates pollen wall pattern formation in Arabidopsis.     

Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes.Pollen tubes play a crucial role in flowering plant reproduction. A pollen tube is the vegetative cell of the male gametophyte. It undergoes rapid polarized growth in order to transport the two nonmotile sperm cells to an ovule. This rapid growth is supported by the constant delivery of secretory vesicles to the pollen tube tip, where they fuse with the plasma membrane to enlarge the cell (Bove et al., 2008; Bou Daher and Geitmann, 2011; Chebli et al., 2013). This vesicle delivery is assumed to be driven by the rapid movement of organelles and cytosol throughout the cell, a process that is commonly referred to as cytoplasmic streaming (Shimmen, 2007). Cytoplasmic streaming in angiosperm pollen tubes forms a reverse fountain: organelles moving toward the tip travel along the cell membrane, while organelles moving away from the tip travel through the center of the tube (Heslop-Harrison and Heslop-Harrison, 1990; Derksen et al., 2002). Drug treatments revealed that pollen tube cytoplasmic streaming and tip growth depend on actin filaments (Franke et al., 1972; Mascarenhas and Lafountain, 1972; Heslop-Harrison and Heslop-Harrison, 1989; Parton et al., 2001; Vidali et al., 2001). Curiously, very low concentrations of actin polymerization inhibitors can prevent growth without completely stopping cytoplasmic streaming, indicating that cytoplasmic streaming is not sufficient for pollen tube growth (Vidali et al., 2001). At the same time, however, drug treatments have not been able to specifically inhibit cytoplasmic streaming; thus, it is unknown whether cytoplasmic streaming is necessary for pollen tube growth.Myosins are actin-based motor proteins that actively transport organelles throughout the cell and are responsible for cytoplasmic streaming in plants (Shimmen, 2007; Sparkes, 2011; Madison and Nebenführ, 2013). Myosins can be grouped into at least 30 different classes based on amino acid sequence similarity of the motor domain, of which only class VIII and class XI myosins are found in plants (Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Class VIII and class XI myosins have similar domain architecture. The N-terminal motor domain binds actin and hydrolyzes ATP (Tominaga et al., 2003) and is often preceded by an SH3-like (for sarcoma homology3) domain of unknown function. The neck domain, containing IQ (Ile-Gln) motifs, acts as a lever arm and is bound by calmodulin-like proteins that mediate calcium regulation of motor activity (Kinkema and Schiefelbein, 1994; Yokota et al., 1999; Tominaga et al., 2012). The coiled-coil domain facilitates dimerization (Li and Nebenführ, 2008), and the globular tail functions as the cargo-binding domain (Li and Nebenführ, 2007). Class VIII myosins also contain an N-terminal extension, MyTH8 (for myosin tail homology8; Mühlhausen and Kollmar, 2013), and class XI myosins contain a dilute domain in the C-terminal globular tail (Kinkema and Schiefelbein, 1994; Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Recently, Mühlhausen and Kollmar (2013) proposed a new nomenclature for plant myosins based on a comprehensive phylogenetic analysis of all known plant myosins that clearly identifies paralogs and makes interspecies comparisons easier (Madison and Nebenführ, 2013).The localization of class VIII myosins, as determined by immunolocalization and the expression of fluorescently labeled full-length or tail constructs, has implicated these myosins in cell-to-cell communication, cell division, and endocytosis in angiosperms and moss (Reichelt et al., 1999; Van Damme et al., 2004; Avisar et al., 2008; Golomb et al., 2008; Sattarzadeh et al., 2008; Yuan et al., 2011; Haraguchi et al., 2014; Wu and Bezanilla, 2014). On the other hand, class XI myosin mutants have been studied extensively in Arabidopsis (Arabidopsis thaliana), which revealed roles for class XI myosins in cell expansion and organelle motility (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Very few studies have examined the reproductive tissues of class XI myosin mutants. In rice (Oryza sativa), one myosin XI was shown to be required for normal pollen development under short-day conditions (Jiang et al., 2007). In Arabidopsis, class XI myosins are required for stigmatic papillae elongation, which is necessary for normal fertility (Ojangu et al., 2012). Even though pollen tubes of myosin XI mutants have not been examined, the tip growth of another tip-growing plant cell has been thoroughly examined in myosin mutants. Root hairs are tubular outgrowths of root epidermal cells that function to increase the surface area of the root for water and nutrient uptake. Two myosin XI mutants have shorter root hairs, of which the myo11e1 (xik; myosin XI K) mutation has been shown to be associated with a slower root hair growth rate and reduced actin dynamics compared with the wild type (Ojangu et al., 2007; Peremyslov et al., 2008; Park and Nebenführ, 2013). Higher order mutants have a further reduction in root hair growth and have altered actin organization (Prokhnevsky et al., 2008; Peremyslov et al., 2010). Disruption of actin organization was also observed in myosin XI mutants of the moss Physcomitrella patens (Vidali et al., 2010), where these motors appear to coordinate the formation of actin filaments in the apical dome of the tip-growing protonemal cells (Furt et al., 2013). Interestingly, organelle movements in P. patens are much slower than in angiosperms and do not seem to depend on myosin motors (Furt et al., 2012).The function of myosins in pollen tubes is currently not known, although it is generally assumed that they are responsible for the prominent cytoplasmic streaming observed in these cells by associating with organelle surfaces (Kohno and Shimmen, 1988; Shimmen, 2007). Myosin from lily (Lilium longiflorum) pollen tubes was isolated biochemically and shown to move actin filaments with a speed of about 8 µm s⁻¹ (Yokota and Shimmen, 1994) in a calcium-dependent manner (Yokota et al., 1999). Antibodies against this myosin labeled small structures in both the tip region and along the shank (Yokota et al., 1995), consistent with the proposed role of this motor in moving secretory vesicles to the apex.In Arabidopsis, six of 13 myosin XI genes are highly expressed in pollen: Myo11A1 (XIA), Myo11A2 (XID), Myo11B1 (XIB), Myo11C1 (XIC), Myo11C2 (XIE), and Myo11D (XIJ; Peremyslov et al., 2011; Sparkes, 2011). The original gene names (Reddy and Day, 2001) are given in parentheses. Myo11D is the only short-tailed myosin XI in Arabidopsis (Mühlhausen and Kollmar, 2013) and lacks the typical myosin XI globular tail involved in cargo binding (Li and Nebenführ, 2007). The remaining genes have the same domain architecture as the conventional class XI myosins that have been shown to be involved in the elongation of trichomes, stigmatic papillae, and root hairs (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Therefore, we predicted that these five pollen-expressed, conventional class XI myosins are required for the rapid elongation of pollen tubes. In this study, we examined transfer DNA (T-DNA) insertion mutants of Myo11A1, Myo11A2, Myo11B1, Myo11C1, and Myo11C2 for defects in fertility and pollen tube growth. Organelle motility and actin organization were also examined in myo11c1 myo11c2 pollen tubes.     

Biochemical Characteristics of Pollen Wall Glycoproteins Separated on Ricin-Sepharose 4B in Cucurbita pepo

By affinity chromatography on ricin-Septarose 4B. pollen wall glycoproteins of Cucurbita pepo were isolated from pollen wall proteins which were prepared by extracting the pollen with isotonic Tris-HC1 buffer. Gas chromatographic analyses showed that the carbohydrate fraction of the pollen wall glycoproteins contained 0.7% rhamnose, 8.1% fucose, 5.1% arabinose, 5.1% xylose, 36.5% galactose, 36.2% glucose, and two unknown components. Isoelectric focusing PAGE showed that the pollen wall glycoproteins contained 6 components, with their respective pI at 3.8, 4.8, 5.4, 6.2, 6.7 and 7.6, and that the relative content of each of them was 40.6%, 15.6% 8.3%, 14.4%, 7,9%, and 13.2%, respectively. The amino acid composition of the 6 glycoproteins was determined with amino acid analyzer. Serine, glutamic acid and glycine were some of the predominent amino acids. These three, amino acids constitute more than 49% of the total amino acids.     

Development of the Generative Cell Wall in Monotropa uniflora L. Pollen

During an ultrastructural survey of an achlorophyllus dicotyledonous plant, Monotropa uniflora L., a stage of pollen development was encountered which suggests a relationship between the activity of the rough endoplasmic reticulum and the development of the generative cell wall.