The Actin Cytoskeleton and Signaling Network during Pollen Tube Tip Growth

The organization and dynamics of the actin cytoskeleton play key roles in many aspects of plant cell development. The actin cytoskeleton responds to internal developmental cues and environmental signals and is involved in cell division, subcellular organelle movement, cell polarity and polar cell growth. The tipgrowing pollen tubes provide an ideal model system to investigate fundamental mechanisms of underlying polarized cell growth. In this system, most signaling cascades required for tip growth...     

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The dynamics of the actin cytoskeleton depends upon the unique constellation of ac- tin-binding proteins (ABPs), as well as their spatial distribution and local activation. However, the identification and characterization of actin-binding proteins in plant cells are still limited. At pre- sent, only a few plant ABPs have been identified in plant tissues, including profilin, ADF/cofilin, fimbrin, villin and several myosins. Compared with that in animals, there is still a long way for us …     

14-3-3 λ protein interacts with ADF1 to regulate actin cytoskeleton dynamics in Arabidopsis

Actin cytoskeleton dynamics is critical for variety of cellular events including cell elongation, division and morphogenesis, and is tightly regulated by numerous groups of actin binding proteins. However it is not well understood how these actin binding proteins are modulated in a physiological condition by their interaction proteins. In this study, we describe that Arabidopsis 14-3-3 λ protein interacted with actin depolymerizing factor 1(ADF1) in plant to regulate F-actin stability and dynamics. Loss of 14-3-3 λin Arabidopsis resulted in longer etiolated hypocotyls in dark and changed actin cytoskeleton architecture in hypocotyl cells. Overexpression of ADF1 repressed 14-3-3 λ mutant hypocotyl elongation and actin dynamic phenotype. In addition, the phosphorylation level of ADF1 was increased and the subcellular localization of ADF1 was altered in 14-3-3 λ mutant. Consistent with these observations, the actin filaments were more stable in 14-3-3 λ mutant. Our results indicate that 14-3-3 λ protein mediates F-actin dynamics possibly through inhibiting ADF1 phosphorylation in vivo.     

At FH16, an Arabidopsis Type II Formin, Binds and Bundles both Microfilaments and Microtubules, and Preferentially Binds to Microtubules

Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1 FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FHIFH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro, in addition, AtFH16 FHIFH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FHIFH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.     

nhibiting Self-Pollen： Self-Incompatibility in Papaver Involves Integration of Several Signaling Events

Cellular responses rely on signal perception and integration. A nice example of this is self incompatibility （SI）, which is an important mechanism to prevent inbreeding. It prevents self-fertilization by using a highly discriminatory cellular recognition and rejection mechanism. Most Sl systems are genetically specified by the S-locus, which has a pollen and a pistil S-component. A receptor-ligand interaction is used by Papaver rhoeas to control SI. S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to achieve rapid inhibition of tip growth. The incompatible Sl interaction triggers a Ca^2＋-dependent signaling cascade. A number of Sl-specific events are triggered in incompatible pollen, including rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases （SPPases）, Prp26.1; activation of a mitogen activated protein kinase, p56; programmed cell death （PCD） involving a caspase-3-1ike activity. These events contribute to prevent self-fertilizaUon. We are attempting to establish the functional significance of these events, and their possible involvement in integrating a coordinated signaling response. Here we describe the identification of these components shown to be involved in Sl, together with recent progress in identifying links between some of them. These data constitute the first steps in elucidating how SI signaling is integrated.     

Organelle trafficking,the cytoskeleton,and pollen tube growth

The pollen tube is fundamental for the reproduction of seed plants. Characteristically, it grows relatively quickly and uni‐directionally("polarized growth") to extend the male gametophyte to reach the female gametophyte. The pollen tube forms a channel through which the sperm cells move so that they can reach their targets in the ovule. To grow quickly and directionally, the pollen tube requires an intense movement of organelles and vesicles that allows the cell's contents to be distributed to sustain the growth rate. While the various organelles distribute more or less uniformly within the pollen tube, Golgi‐released secretory vesicles accumulate massively at the pollen tube apex, that is, the growing region. This intense movement of organelles and vesicles is dependent on the dynamics of the cytoskeleton,which reorganizes differentially in response to external signals and coordinates membrane trafficking with the growth rate of pollen tubes.     

Plant villins:Versatile actin regulatory proteins

Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology.Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Villins contain six gelsolin homology domains(G1–G6) and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a de fined role in modifying actin dynamics in the pollen Invitube; most of their in vivo activities remain to be ascertained.Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review,we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understanding of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.     

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 gn12-2 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.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Subcellular Localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible Iines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.     

Sexual Plant Reproduction: a Fertile and Flourishing Field

Since the very beginning of plant science, sexual plant reproduction (SPR) has proved an attractive and enduring topic for generations of botanists. With the rapid development of modern technology, a significant acceleration has occurred in our understanding on the developmental mechanisms of plant reproductive processes, particularly the evolution of double fertilisation, signalling in pollen tube orientation, molecular characterisation of plant gametes, maternal to zygotic transitions and parental gene involvement in early embryogenesis. This is reflected not only by several recent high-ranking research papers, but also by the frequent conferences and workshops on these topics. These include the 2008 XXth International Congress on SPR in Bras´齦ia and “Frontiers in SPR III” in Tucson, as well as “Cell-Cell Communication in Plant Reproduction” held in 2009 in Bath. The continuing efforts from around the world indicate that SPR is still a fertile and flourishing field, with great expectations for the coming decade. Investigations on SPR have been enhanced recently by their underlying relationship to agriculture. With the development of molecular crop breeding comes an increasing requirement for knowledge on molecular mechanisms of SPR, both general regulatory processes, and in particular, signalling for cell-cell communication in several critical steps. To reveal mechanisms behind some well-known phenomena in modern agriculture, such as compatibility of crop crosses and fertility determination of remote hybrids and heterosis, one must first precisely understand the basic developmental processes and molecular mechanisms involved in SPR. Thus, it is not surprising that the field has attracted much attention in recent years. In fact, advances in the study of SPR throw new light on investigations for solving many existing problems in agriculture. This special issue presents current research on multiple aspects of SPR, both pre- and post-fertilisation. Hence, this special issue of the Journal of Integrative Plant Biology features a diverse collection of papers. One paper, “The earliest normal flower in Liaoning Province, China” (see pages 800–811), presents evidence of component parts and morphology of early angiosperm flowers. Four papers, “A genome-wide functional characterisation of Arabidopsis regulatory calcium sensors in pollen tubes” (see pages 751–761), “GNOM-LIKE 2, encoding an ARF-GEF protein homologous to GNOM and GNL1, is essential for pollen germination in Arabidopsis” (see pages 762–773), “Regulation of actin dynamics in the pollen tubes:control of actin polymer level” (see pages 740–750) and “Pollen tube growth: a delicate equilibrium between secretory and endocytic pathways” (see pages 727–739), deal with molecular mechanisms of pollen tube growth and orientation, a hot field in SPR. Amongst these papers, Prof. Moscatelli offers a deep insight into dynamic control of vesicle trafficking in pollen tubes. In the signalling cascade of vesicle trafficking, calcium and actin dynamics play essential roles in the balance between secretory and endocytic pathways. GNOM-LIKE 2, as an ARF-GEF- and BFA-sensitive protein,may indirectly influence calcium and actin dynamics during pollen germination and pollen tube growth. “Premature tapetum degeneration: a major cause of abortive pollen development in photoperiod sensitive genic male sterility in rice” (see pages 774–781) presents new evidence for the interaction between tapetum and pollen development. “Comparative detection of calcium fluctuations in single female sexual cells of tobacco to distinguish calcium signals triggered by in vitro fertilisation” (see pages 782–791) reports observations on calcium dynamics during sperm-central cell fusion, and “Oil body biogenesis during Brassica napus embryogenesis” (see pages 792–799) focuses on post-fertilisation developmental events, mainly on oil body generation and accumulation during embryogenesis in both wild type and mutants. A review paper, “Analyses of sexual reproductive success in transgenic and/or mutant plants” (see pages 719–726), gives a critical evaluation of current techniques used for investigating genes that affect pistil development, and provides useful information and technical guidance to readers in this field. These papers coherently address the common theme: development and its mechanisms in plant reproductive processes. In this way, we hope to create an atmosphere in which extensive discussion can stimulate and lead to interactions between scientists with different perspectives in order to inspire future directions. We sincerely hope that this special issue of JIPB will provide a platform to highlight current advances and novel findings in the study of SPR. Finally, we express our appreciation to JIPB for supporting this special issue and to all authors for their great contributions. Mengxiang Sun, Professor The Editor for this Special Issue College of Life Sciences, Wuhan University, China     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Arabidopsis CNGC18 Is a Ca2＋-Permeable Channel

Dear Editor, Extracellular Ca2＋ influx focusing at the tips of pollen tubes is the main source of Ca2＋ for the pollen tube tip cytosolic Ca2＋ gradient, which is essential for both polar growth and orientation of pollen tubes in plants, and plasma membrane Ca2＋ channels were proposed to be present in the tips and function as key proteins by mediating and regulating extracellular Ca2＋ influx （for a review, see Guan et al., 2013）.     

Golgi 58K-like protein in pollens and pollen tubes ofLilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

Soluble expression and characterization of a GFP-fused pea actin isoform（PEAc1）

A pea actin isoform PEAcl with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its Nterminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating Sepharose^TM Fast Flow column. The purified fusion protein (PEAcl-GFP) efficiently inhibited DNase I activities before polymerization,and activated the myosin Mg-ATPase activities after polymerization. The PEAcl-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75μM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc 1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtainning soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAcl-GFP was also expressed in tobacco BY2 cells,which offers a new pathway for further studying its distribution and function in vivo.     

Role of plant myosins in motile organelles: Is a direct interaction required?

Plant organelles are highly motile, with speed values of 3–7 m m/s in cells of land plants and about20–60 m m/s in characean algal cells. This movement is believed to be important for rapid distribution of materials around the cell, for the plant's ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins.Most plants express multiple members of two main classes:myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in al kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate.     

Phosphorylation of Microtubule-associated Protein SB401 from Solanum berthaultii Regulates Its Effect on Microtubules

We reported previously that the protein SB401 from Solanum berthaultii binds to and bundles both microtubules and F-actin. In the current study, we investigated the regulation of SB401 activity by its phosphorylation. Our experimental results showed that the phosphorylation of SB401 by casein kinase II （CKII） downregulates the activities of SB401, namely the bundling of microtubules and enhancement of the polymerization of tubulin. However, phosphorylation of SB401 had no observable effect on its bundling of F-actin. Further investigation using extract of potato pollen indicated that a CKIl-like kinase may exist in potato pollen. Antibodies against CKII alpha recognized specifically a major band from the pollen extract and the pollen extract was able to phosphorylate the SB401 protein in vitro. The CKIl-like kinase showed a similar ability to downregulate the bundling of microtubules. Our experiments demonstrated that phosphorylation plays an important role in the regulation of SB401 activity. We propose that this phosphorylation may regulate the effects of SB401 on microtubules and the actin cytoskeleton.     

Roles of cortactin, an actin polymerization mediator, in cell endocytosis

Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene. Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents. Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 （SH3）-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation. Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.