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To reach the female gametophyte, growing pollen tubes must penetrate different tissues within the pistil, the female reproductive organ of a flower. Past research has identified various chemotropic cues that guide pollen tubes through the transmitting tract of the pistil, which represents the longest segment of its growth path. In addition, physical mechanisms also play a role in pollen tube guidance; however, these processes remain poorly understood. Here we show that pollen tubes from plants with solid transmitting tracts actively respond to the stiffness of the environment. We found that pollen tubes from Nicotiana tabacum and other plant species with a solid or semisolid transmitting tract increase their growth rate in response to an increasing matrix stiffness. By contrast, pollen tubes from Lilium longiflorum and other plant species with a hollow transmitting tract decrease their growth rate with increasing matrix stiffness, even though the forces needed to maintain a constant growth rate remain far below the maximum penetration force these pollen tubes are able to generate. Moreover, when confronted with a transition from a softer to a stiffer matrix, pollen tubes from N. tabacum display a greater ability to penetrate into a stiffer matrix compared with pollen tubes from L. longiflorum, even though the maximum force generated by pollen tubes from N. tabacum (11 µN) is smaller than the maximum force generated by pollen tubes from L. longiflorum (36 µN). These findings demonstrate a mechano-sensitive growth behavior, termed here durotropic growth, that is only expressed in pollen tubes from plants with a solid or semisolid transmitting tract and thus may contribute to an effective pollen tube guidance within the pistil.

The general role of cellular membranes is to provide a barrier and to generate separate reaction spaces. However, additional functions of membrane domains enriched in certain classes of lipids have been discovered, which represent an important area of ongoing research. Such membrane domains can be found in cells at different size scales (e.g., nanodomains, microdomains), represent membrane regions with special physical properties and play important roles in the direct or indirect propagation of signaling processes. Domain formation within the plasma membrane (PM) does not only involve the accumulation of specific lipids, but also the recruitment of specific transmembrane or PM-associated peripheral proteins. Phosphatidic acid (PA) is increasingly recognized as an important signaling lipid and component of PM domains. This lipid is involved in the regulation not only of biotic or abiotic stress responses, but also of pollen tube tip growth and of other forms of polar cell expansion. Although many PA-binding proteins have been characterized, a conserved PA interaction motif could not be identified in these proteins. Consequently, protein binding to PA cannot be predicted based on sequence analysis, but has to be biochemically tested using lipid strip or liposome assays. Although these assays are often informative, they are generally based on the use of artificial model membranes, which compared to natural membranes contain fewer lipid types often at non-physiological concentrations. In this chapter, we describe an alternative in vivo assay that can be employed to analyze protein binding to PA at the PM of normally elongating tobacco pollen tubes. This assay is based on the use of n-butanol (n-ButOH), which inhibits phospholipase D (PLD) and thereby blocks a major biosynthetic pathway that generates PA within the PM from substrates like phosphatidylcholine (PC) or phosphatidylethanolamine (PE). PLD inhibition reduces the PA content of the PM and consequently the level of PM association of PA-binding proteins, which can be analyzed using fluorescence microscopy. Methods enabling n-ButOH treatment of cultured tobacco pollen tubes expressing YFP-tagged PA-binding proteins as well as the quantitative determination of the PM association of these proteins are described.

The general role of cellular membranes is to provide a barrier and to generate separate reaction spaces. However, additional functions of membrane domains enriched in certain classes of lipids have been discovered, which represent an important area of ongoing research. Such membrane domains can be found in cells at different size scales (e.g., nanodomains, microdomains), represent membrane regions with special physical properties and play important roles in the direct or indirect propagation of signaling processes. Domain formation within the plasma membrane (PM) does not only involve the accumulation of specific lipids, but also the recruitment of specific transmembrane or PM-associated peripheral proteins. Phosphatidic acid (PA) is increasingly recognized as an important signaling lipid and component of PM domains. This lipid is involved in the regulation not only of biotic or abiotic stress responses, but also of pollen tube tip growth and of other forms of polar cell expansion. Although many PA-binding proteins have been characterized, a conserved PA interaction motif could not be identified in these proteins. Consequently, protein binding to PA cannot be predicted based on sequence analysis, but has to be biochemically tested using lipid strip or liposome assays. Although these assays are often informative, they are generally based on the use of artificial model membranes, which compared to natural membranes contain fewer lipid types often at non-physiological concentrations. In this chapter, we describe an alternative in vivo assay that can be employed to analyze protein binding to PA at the PM of normally elongating tobacco pollen tubes. This assay is based on the use of n-butanol (n-ButOH), which inhibits phospholipase D (PLD) and thereby blocks a major biosynthetic pathway that generates PA within the PM from substrates like phosphatidylcholine (PC) or phosphatidylethanolamine (PE). PLD inhibition reduces the PA content of the PM and consequently the level of PM association of PA-binding proteins, which can be analyzed using fluorescence microscopy. Methods enabling n-ButOH treatment of cultured tobacco pollen tubes expressing YFP-tagged PA-binding proteins as well as the quantitative determination of the PM association of these proteins are described.

Pollen tube tip growth depends on balancing secretion of cell wall material with endocytic recycling of excess material incorporated into the plasma membrane (PM). The classical model of tip growth, which predicts bulk secretion, occurs apically, and is compensated by subapical endocytosis, has been challenged in recent years. Many signaling proteins and lipids with important functions in the regulation of membrane traffic underlying tip growth associate with distinct regions of the pollen tube PM, and understanding the mechanisms responsible for the targeting of these regulatory factors to specific PM domains requires quantitative information concerning the sites of bulk secretion and endocytosis. Here, we quantitatively characterized the spatial organization of membrane traffic during tip growth by analyzing steady-state distributions and dynamics of FM4-64-labeled lipids and YFP-tagged transmembrane (TM) proteins in tobacco (Nicotiana tabacum) pollen tubes growing normally or treated with Brefeldin A to block secretion. We established that (1) secretion delivers TM proteins and recycled membrane lipids to the same apical PM domain, and (2) FM4-64-labeled lipids, but not the analyzed TM proteins, undergo endocytic recycling within a clearly defined subapical region. We mathematically modeled the steady-state PM distributions of all analyzed markers to better understand differences between them and to support the experimental data. Finally, we mapped subapical F-actin fringe and trans-Golgi network positioning relative to sites of bulk secretion and endocytosis to further characterize functions of these structures in apical membrane traffic. Our results support and further define the classical model of apical membrane traffic at the tip of elongating pollen tubes.

RAC/ROP GTPases coordinate actin dynamics and membrane traffic during polar plant cell expansion. In tobacco (Nicotiana tabacum), pollen tube tip growth is controlled by the RAC/ROP GTPase RAC5, which specifically accumulates at the apical plasma membrane. Here, we describe the functional characterization of RISAP, a RAC5 effector identified by yeast (Saccharomyces cerevisiae) two-hybrid screening. RISAP belongs to a family of putative myosin receptors containing a domain of unknown function 593 (DUF593) and binds via its DUF593 to the globular tail domain of a tobacco pollen tube myosin XI. It also interacts with F-actin and is associated with a subapical trans-Golgi network (TGN) compartment, whose cytoplasmic position at the pollen tube tip is maintained by the actin cytoskeleton. In this TGN compartment, apical secretion and endocytic membrane recycling pathways required for tip growth appear to converge. RISAP overexpression interferes with apical membrane traffic and blocks tip growth. RAC5 constitutively binds to the N terminus of RISAP and interacts in an activation-dependent manner with the C-terminal half of this protein. In pollen tubes, interaction between RAC5 and RISAP is detectable at the subapical TGN compartment. We present a model of RISAP regulation and function that integrates all these findings.

Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells. The biosensor consists of a PA-binding domain of yeast SNARE Spo20p fused to fluorescent proteins. Live-cell imaging of PA dynamics in transiently transformed tobacco (Nicotiana tabacum) pollen tubes was performed using confocal laser scanning microscopy. In growing pollen tubes, PA shows distinct annulus-like fluorescence pattern in the plasma membrane behind the extreme tip. Coexpression studies with markers for other plasmalemma signaling lipids phosphatidylinositol 4,5-bisphosphate and diacylglycerol revealed limited colocalization at the shoulders of the apex. PA distribution and concentrations show distinct responses to various lipid signaling inhibitors. Fluorescence recovery after photobleaching (FRAP) analysis suggests high PA turnover in the plasma membrane. Our data show that a biosensor based on the Spo20p-PA binding domain is suitable for live-cell imaging of PA also in plant cells. In tobacco pollen tubes, distinct subapical PA maximum corroborates its involvement in the regulation of endocytosis and actin dynamics.

Tetraspanins are small transmembrane proteins that laterally associate with each other and cluster with numerous partner proteins as well as lipids. These interactions result in the formation of a distinct class of membrane domains, the tetraspanin-enriched microdomains (TEMs), which influence numerous cellular processes such as cell adhesion and fusion, intracellular membrane trafficking, signaling, morphogenesis, motility as well as interaction with pathogens and cancer development. The majority of information available about tetraspanins is based on studies using animal models or cell lines, but tetraspanins are also present in fungi and plants. Recent studies indicate that tetraspanins have important functions in plant development, reproduction and stress responses. Here we provide a brief summary of the current state of tetraspanin research in plants.

Reactive oxygen species (ROS) generated by NADPH oxidase (NOX) are crucial for tip growth of pollen tubes. However, the regulation of NOX activity in pollen tubes remains unknown. Using purified plasma membrane fractions from tobacco and olive pollen and tobacco BY-2 cells, we demonstrate that pollen NOX is activated by calcium ions and low abundant signaling phospholipids, such as phosphatidic acid and phosphatidylinositol 4,5-bisphosphate in vitro and in vivo. Our data also suggest possible synergism between Ca(2+) and phospholipid-mediated NOX activation in pollen. Rac/Rop small GTPases are also necessary for normal pollen tube growth and have been proposed to regulate ROS production in root hairs. We show here elevated ROS formation in pollen tubes overexpressing wild-type NtRac5 and constitutively active NtRac5, while overexpression of dominant-negative NtRac5 led to a decrease of ROS in pollen tubes. We also show that PA formed by distinct phospholipases D (PLD) is involved in pathways both upstream and downstream of NOX-mediated ROS generation and identify NtPLDδ as a PLD isoform acting in the ROS response pathway.

Polarized tip growth is a fundamental cellular process in many eukaryotic organisms, mediating growth of neuronal axons and dendrites or fungal hyphae. In plants, pollen and root hairs are cellular model systems for analysing tip growth. Cell growth depends on membrane traffic. The regulation of this membrane traffic is largely unknown for tip-growing cells, in contrast to cells exhibiting intercalary growth. Here we show that in Arabidopsis, GBF1-related exchange factors for the ARF GTPases (ARF GEFs) GNOM and GNL2 play essential roles in polar tip growth of root hairs and pollen, respectively. When expressed from the same promoter, GNL2 (in contrast to the early-secretory ARF GEF GNL1) is able to replace GNOM in polar recycling of the auxin efflux regulator PIN1 from endosomes to the basal plasma membrane in non-tip growing cells. Thus, polar recycling facilitates polar tip growth, and GNL2 seems to have evolved to meet the specific requirement of fast-growing pollen in higher plants.

Phosphatidylinositol-4-monophosphate 5-kinases produce phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] and have been implicated in vesicle trafficking and cytoskeletal rearrangements. Here, we adopted a reverse genetics approach to investigate the function of the Arabidopsis thaliana pollen-expressed gene encoding phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4). Pollen germination, tube growth, and polarity were significantly impaired in homozygous mutant plants lacking PIP5K4 transcript. In vitro, supplementation with PtdIns(4,5)P2 rescued these phenotypes. In vivo, mutant pollen fertilized ovules, leading to normal seed set and silique length. However, fertilization took longer than in wild-type plants, and the pip5k4 null mutant allele was transmitted through the pollen at a reduced frequency. Analysis of endocytic events using FM1-43 (or FM4-64) suggested a reduction in endocytosis and membrane recycling in pip5k4 null mutant pollen tubes. Imaging of elongating tobacco (Nicotiana tabacum) pollen tubes transiently transformed with a PIP5K4-green fluorescent protein fusion construct revealed that the protein localized to the plasma membrane, particularly in the subapical region. Overexpression of PIP5K4-GFP delocalized the protein to the apical region of the plasma membrane, perturbed pollen tube growth, and caused apical cell wall thickening. Thus, PIP5K4 plays a crucial role in regulating the polarity of pollen tubes. This study supports a model for membrane secretion and recycling where the apical and subapical regions appear to contain the components required to promote and sustain growth.

Rho of plants (ROP) proteins, also known as RAC proteins, are Rho-related GTPases that function as molecular switches in a multitude of signaling cascades involved in the regulation of the actin and microtubule cytoskeleton, of vesicle trafficking, and of plant responses to hormones, stresses, or

Summary In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca2+ ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.

Polarized Rac/Rop GTPase signaling plays a key role in polar cell growth, which is essential for plant morphogenesis. The molecular and cellular mechanisms responsible for the polarization of Rac/Rop signaling during polar cell growth are only partially understood. Mutant variants of Rac/Rop GTPases lacking specific functions are important tools to investigate these mechanisms, and have been employed to develop a model suggesting that RhoGAP (GTPase activating protein) and RhoGDI (Guanine Nucleotide Dissociation Inhibitor) mediated recycling of Rac/Rop GTPases maintains apical polarization of Rac/Rop activity in pollen tubes, which elongate by 'tip growth' (an extreme form of polar cell growth). Despite the importance of these mutant variants for Rac/Rop functional characterization, their distinct intracellular distributions have not been thoroughly comparatively and quantitatively analyzed. Furthermore, support for the proposed RhoGAP and RhoGDI functions in apical polarization of Rac/Rop activity based on the analysis of in vivo interactions between these proteins and Rac/Rop GTPases has been missing. Here, extensive fluorescent protein tagging and bimolecular fluorescence complementation (BiFC) analyses are described of the intracellular distributions of wild type and mutant variants of the tobacco pollen tube Rac/Rop GTPase Nt-Rac5, as well as of interactions of these Nt-Rac5 variants with RhoGAP and RhoGDI proteins, in normally growing transiently transformed pollen tubes. Presented results substantially enhance our understanding of apical dynamics of pollen tube Rac/Rop signaling proteins, confirm previously proposed RhoGAP and RhoGDI functions in Rac/Rop polarization and provide important technical insights facilitating future in vivo protein localization and BiFC experiments in pollen tubes.

Phosphatidyl inositol 4,5-bisphosphate (PI 4,5-P2) accumulates in a Rac/Rop-dependent manner in the pollen tube tip plasma membrane, where it may control actin organization and membrane traffic. PI 4,5-P2 is hydrolyzed by phospholipase C (PLC) activity to the signaling molecules inositol 1,4,5-trisphosphate and diacyl glycerol (DAG). To investigate PLC activity during tip growth, we cloned Nt PLC3, specifically expressed in tobacco (Nicotiana tabacum) pollen tubes. Recombinant Nt PLC3 displayed Ca2+-dependent PI 4,5-P2–hydrolyzing activity sensitive to U-73122 and to mutations in the active site. Nt PLC3 overexpression, but not that of inactive mutants, inhibited pollen tube growth. Yellow fluorescent protein (YFP) fused to Nt PLC3, or to its EF and C2 domains, accumulated laterally at the pollen tube tip plasma membrane in a pattern complementary to the distribution of PI 4,5-P2. The DAG marker Cys1:YFP displayed a similar intracellular localization as PI 4,5-P2. Blocking endocytic membrane recycling affected the intracellular distribution of DAG but not of PI 4,5-P2. U-73122 at low micromolar concentrations inhibited and partially depolarized pollen tube growth, caused PI 4,5-P2 spreading at the apex, and abolished DAG membrane accumulation. We show that Nt PLC3 is targeted by its EF and C2 domains to the plasma membrane laterally at the pollen tube tip and that it maintains, together with endocytic membrane recycling, an apical domain enriched in PI 4,5-P2 and DAG required for polar cell growth.

Rac-type small GTPases are known to function in some cellular processes in plants. To further understand the involvement of Rac type GTPases in plant development, we isolated from cultured Zinnia cells a gene (ZeRAC2) encoding a new Rac-type small GTPase. ZeRAC2 mRNA accumulates preferentially in xylogenic culture and transiently at the time when visible tracheary elements appear. Experiments with ZeRAC2 recombinant proteins demonstrated that ZeRAC2 binds to and hydrolyzes GTP. A GFP-ZeRAC2 fusion protein was localized to the plasma membrane. Together with the fact that ZeRAC2 possesses a putative geranylgeranylation site at the C-terminus, this suggests that ZeRAC2 acts on the plasma membrane. In situ hybridization indicated that ZeRAC2 mRNA accumulates preferentially in xylem parenchyma and tracheary element precursor cells, and surprisingly the accumulation is restricted to the site facing developing tracheary elements.

The eukaryotic cytoskeleton is a dynamic filamentous network with various cellular and developmental functions. Plant cells display a singular architecture, necessitating a structurally and functionally unique cytoskeleton and plant specific control mechanisms.

Different alpha-tubulin cDNA sequences fused in an antisense orientation to a CaMV 35S promoter were introduced into Arabidopsis thaliana plants. Several independent transgenic lines that showed a moderate but clear reduction of alpha-tubulin gene expression (TUA6/AS lines) were obtained and phenotypically characterized. Although no apparent abnormalities were detected in the aerial parts of TUA6/AS plants, root development was severely affected. Cells in TUA6/AS root tips were found to contain aberrant microtubular structures, to expand abnormally and to be unable to undergo regular cell division. These cellular defects caused a dramatic radial expansion of the root tip and inhibited root elongation. In addition, TUA6/AS roots displayed ectopic formation of root hairs, root hair branching and a reduced ability to respond to gravitropic challenges. Our results contribute to an improved understanding of the different roles microtubules play during root development and demonstrate that reverse genetics is a powerful tool to analyze cytoskeletal functions during plant organogenesis.

Actin depolymerizing factor (ADF) is a key regulator of the organization of the actin cytoskeleton during various cellular activities. We found that ADF genes in Arabidopsis form a large family consisting of at least nine members, four of which were cloned and sequenced in this study. Comparison of genomic and cDNA sequences showed that the AtADF1, AtADF5, and AtADF6 genes all contain two introns at conserved positions. Analysis of transgenic Arabidopsis plants carrying promoter-GUS fusion constructs revealed that AtADF1 and AtADF6 are expressed in the vascular tissues of all organs, whereas expression of AtADF5 is restricted to the root tip meristem. GFP-AtADF1, GFP-AtADF5, and GFP-AtADF6 fusion proteins were found to bind to actin filaments in vivo, and to reorganize the actin cytoskeleton when transiently expressed in plant cells.

The formation of the cell plate, a unique structure in dividing plant cells, is pivotal for cytokinesis. A mutation in the Arabidopsis KORRIGAN (KOR) gene causes the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to severely abnormal seedling morphology. The mutant, designed kor1-2, was identified as a stronger allele than the previously identified kor1-1, which appears to be defective only in cell elongation. KOR1 encodes an endo-1,4-beta-d-glucanase with a transmembrane domain and two putative polarized targeting signals in the cytosolic tail. When expressed in tobacco BY2 cells, a KOR1-GFP (green fluorescence protein) fusion protein was localized to growing cell plates. Substitution mutations in the polarized targeting motifs of KOR1 caused the fusion proteins to localize to the plasma membrane as well. Expression of these mutant genes in kor1-2 plants complemented only the cell elongation defect but not the cytokinesis defect, indicating that polarized targeting of KOR1 to forming cell plates is essential for cytokinesis. Our results suggest that KOR1 plays a critical role during cytokinesis.

In an attempt to elucidate the biological function of villin-like actin-binding proteins in plants we have cloned several genes encoding Arabidopsis proteins with high homology to animal villin. We found that Arabidopsis contains at least four villin-like genes (AtVLNs) encoding four different VLN isoforms. Two AtVLN isoforms are more closely related to mammalian villin in their primary structure and are also antigenically related, whereas the other two contain significant changes in the C-terminal headpiece domain. RNA and promoter/β-glucuronidase expression studies demonstrated that AtVLN genes are expressed in all organs, with elevated expression levels in certain types of cells. These results suggest that AtVLNs have less-specialized functions than mammalian villin, which is found only in the microvilli of brush border cells. Immunoblot experiments using a monoclonal antibody against pig villin showed that AtVLNs are widely distributed in a variety of plant tissues. Green fluorescent protein fused to full-length AtVLN and individual AtVLN headpiece domains can bind to both animal and plant actin filaments in vivo.

Ein Verband gleichartiger Zellen wird als Gewebe bezeichnet. Die Gleichartigkeit bezieht sich in erster Linie auf das Aussehen der Zellen, aber auch auf ihre physiologischen Leistungen. Gewebe sind also primar morphologische Einheiten. Mehrzellige Funktionseinheiten werden als Organe bezeichnet; diese sind haufig aus mehreren Geweben aufgebaut. Das Studium der Gewebe ist Gegenstand der Histologie (griech. histos, Gewebe).

Viele Entwicklungsprozesse werden durch Phytohormone reguliert. Phytohormone sind niedermolekulare Signalstoffe, die in allen oder fast allen Pflanzen vorkommen, in niedrigen Konzentrationen (≤10–6M) charakteristische physiologische Reaktionen auslosen und deren Bildungs- und Wirkorte in der Regel nicht identisch sind. Phytohormone dienen also der interzellularen Kommunikation und Regulation in vielzelligen Organismen und haben damit im Prinzip die gleiche Funktion wie tierische und menschliche Hormone. Im Gegensatz zu diesen regulieren Phytohormone allerdings nur in seltenen Fallen (z.B. Schlieszellkontrolle durch Abscisinsaure, s. Abschn. 17.4; Regulation der Keimung durch Gibberelline, s. Abschn. 17.3) physiologische Vorgange in bereits ausdifferenzierten Organismen. Eine viel wichtigere Rolle spielen sie bei der Kontrolle von Wachstum und Differenzierung wahrend der Pflanzenentwicklung, wobei sie haufig auf komplexe und weitgehend unverstandene Weise miteinander zu interagieren scheinen.

Wachstum und Differenzierung, also die Morphogenese, werden nicht allein durch endogene Prozesse gesteuert. Im Rahmen der genetisch festgelegten Grenzen wird die Pflanzenentwicklung masgeblich durch Ausenfaktoren beeinflusst. Die standortgebundenen Pflanzen sind im Unterschied zu frei beweglichen Organismen den oft erheblich schwankenden auseren Bedingungen an ihrem Standort ausgesetzt und mussen in angemessener Weise darauf reagieren konnen.

Unter dem Begriff der Korrelationen werden Wechselwirkungen zusammengefasst, die der Koordinierung von Entwicklungsprozessen uber die Grenzen eines Phytomers (s. Einleitung Kap. 14) hinweg dienen. Obwohl solche systemische Prozesse bereits bei relativ einfach aufgebauten Pflanzen beobachtet werden konnen, sind sie doch in weiter entwickelten, komplexen Pflanzen besonders auffallig. Korrelative Wechselwirkungen, die uber Nahrstoffkonkurrenz bzw. die gegenseitige Belieferung mit Nahrstoffen hinausgehen, werden in der Regel durch Phytohormone (s. Kap. 1) vermittelt, die durch Leitbahnen des Xylems und des Phloems transportiert werden. In einigen Fallen scheinen aber auch Makromolekule als Signalmolekule uber weite Distanzen in Pflanzen transportiert zu werden und an korrelativer Regulation beteiligt zu sein. Es gibt Hinweise darauf, dass regulatorische Proteine (z.B. Transkriptionsfaktoren, s. Abschn. 8.3) oder mRNA-Molekule, die solche Proteine codieren, nicht nur durch Plasmodesmen von Zelle zu Zelle wandern (s. Abschn. 15.4.1), sondern in einigen Fallen sogar in Siebzellen des Phloems transportiert und damit in der ganzen Pflanze verteilt werden konnen.

Gestalt und Funktionen von Zellen sind Gegenstand der Zellbiologie. In ihr vereinigen sich Feinstrukturforschung, Molekularbiologie und Biochemie sowie viele Aspekte der Physiologie. Vor 1950, noch bevor die modernen Methoden der Zellforschung etabliert waren, wurde die Zellbiologie als Cytologie bezeichnet (griech. kýtos, Blase, Zelle). Sie war weitgehend auf die Lichtmikroskopie von Zellen beschrankt.

Unter Entwicklung versteht man die Gesamtheit aller Prozesse des Form- und Funktionswechsels im Lebenszyklus eines ein- oder vielzelligen Organismus. Entwicklungsprozesse konnen auf der Ebene von Molekulen, Kompartimenten, Zellen, Geweben und Organen ablaufen. Die Entwicklungsbiologie befasst sich mit der Kausalanalyse der Entwicklungsvorgange. Ziel ist es, die molekularen Ablaufe zu verstehen, durch welche die genetische Information (der Genotyp) unter Einwirkung der Umwelt realisiert wird (Phanotyp). Genotyp- und umweltabhangige Entwicklung fuhrt bei den Nachkommen sowohl zu einer getreuen Reproduktion der charakteristischen Arteigenschaften der Elterngeneration, als auch zu individueller Variabilitat in der Auspragung des Phanotyps im Rahmen der durch den Genotyp festgelegten Grenzen. Diese Plastizitat der Entwicklung dient bei Pflanzen vor allem der Anpassung des Organismus an unterschiedliche Standortgegebenheiten. Die Entwicklungszyklen der Pflanzen unterschiedlicher systematischer Stellung werden in Kap. 24 dargestellt.

Wie bereits bei arbeitsteiligen prokaryotischen Zellverbanden (z.B. Heterocystenbildung N2-fixierender Cyanobakterien), wird auch bei vielzelligen Pflanzen die Determination und die dadurch festgelegte Differenzierung einer Zelle durch ihre Lage im Zellverband bzw. im Organismus bestimmt. So entwickeln sich z.B. die Leitbundel im Spross einer Pflanze immer in einem bestimmten Abstand von der Oberflache, wahrend die Epidermis normalerweise direkt an der Oberflache entsteht. In Spross- und Wurzelachsen von Kormophyten weist die Schichtung der Gewebe von ausen nach innen stets eine charakteristische Abfolge auf, die das Vorhandensein radialer Positionsinformation in diesen Achsen belegt (Abb. 15.1). Dagegen geht beispielsweise die Differenzierung von regelmasig verteilten Schlieszellen und Trichomen in Epidermen bzw. von Wurzelhaaren in Rhizodermen auf charakteristische Musterbildungsprozesse zuruck, die Ausdruck einer in der Flache wirkenden tangentialen Positionsinformation ist. Mikrochirurgische Experimente an Wurzeln von Arabidopsis thaliana belegen auserdem das Vorhandensein einer entlang der Organlangsachse ausgebildeten longitudinalen Positionsinformation, die die Zelldifferenzierung der von den Meristeminitialen abgegebenen Tochterzellen determiniert (Abb. 15.1). Das korrekte positionsabhangige Differenzieren jeder einzelnen Pflanzenzelle hangt also von ihrer Fahigkeit ab, die unterschiedlichen sich uberlagernden Positionsinformationen wahrnehmen und interpretieren zu konnen.

A flexible, comparatively inexpensive system based on a liquid nitrogen-cooled slow-scan CCD (charge coupled device) camera is presented, which can be employed for quantitative low-light (bioluminescence, chemiluminescence or fluorescence) imaging. Using this camera system and the firefly luciferase (LUC) as a screenable marker, transgenic tobacco lines have been produced by direct gene transfer. Bioluminescence emitted from single tobacco cells transiently expressing the firefly luciferase gene (Luc) as well as from stably transformed calli, regenerated shoots, plantlets and T-1 seedlings could be monitored in vivo with no effect on the viability of the material analysed. The patterns of light emission from sections through Luc-expressing leaves and bioluminescent single protoplasts isolated from such leaves were also imaged microscopically. The assay used to detect in vivo LUC activity was optimized by quantifying bioluminescence emitted from Luc-expressing tobacco protoplasts and leaves incubated in different substrate solutions and determining the kinetics of light emission during incubation in the substrate solution.

An efficient system has been established that allows well controlled DNA microinjection into tobacco (Nicotiana tabacum) mesophyll protoplasts with partially regenerated cell walls and subsequent analysis of transient as well as stable expression of injected reporter genes in particular targeted cells or derived clones. The system represents an effective tool to study parameters important for the successful transformation of plant cells by microinjection and other techniques. Protoplasts were immobilized in a very thin layer of medium solidified with agarose or alginate. DNA microinjection was routinely monitored by coinjecting FITC-dextran and aimed at the cytoplasm of target cells. The injection procedure was optimized for efficient delivery of injection solution into this compartment. Cells were found to be at the optimal stage for microinjection about 24 h after immobilization in solid medium. Embedded cells could be kept at this stage for up to 4 d by incubating them at 4°C in the dark. Within 1 h successful delivery of injection solution was routinely possible into 20-40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913, a plasmid containing the neo (neomycin phosphotransferase II) gene, stably transformed, paromomycin-resistant clones could be recovered through selection. Transgenic tobacco lines have been established from such clones. Injection solutions containing pSHI9l3 at a concentration of either 50 μg ml -1 or 1 mg ml -1 have been tested. With 1 mg ml -1 plasmid DNA the percentage of resistant clones per successfully injected cell was determined to be about 3.5 times higher. Incubation of embedded protoplasts at 4°C before microinjection was found to reduce the percentage of resistant clones obtained per injected cell. Protoplasts were immobilized above a grid pattern and the location of injected cells was recorded by Polaroid photography. The fate of particular targeted cells could be observed. Isolation and individual culture of clones derived from injected cells was possible. Following cytoplasmic coinjection of FITC-dextran and 1 mg ml -1 plasmid DNA on average about 20% of the targeted cells developed into microcalli and roughly 50% of these calli were stably transformed. Transient expression of the firefly luciferase gene (Luc) was non-destructively analysed 24 h after injection of pAMLuc. Approximately 50% of the injected cells that were alive at this time point expressed the Luc gene transiently. Apparently, stable integration of the injected genes occurred in essentially all transiently expressing cells that developed into clones.

Polarized cell expansion plays an important role in plant morphogenesis. Tip growth is a dramatic form of this process, which is widely used as a model to study its regulation by RAC/ROP GTPase signalling. During the dominant haploid phase of its life cycle, the moss Physcomitrella patens contains different types of cells that expand by tip growth. Physcomitrella is a highly attractive experimental system because its genome has been sequenced, and transgene integration by homologous recombination occurs in this plant at frequencies allowing effective gene targeting. Furthermore, together with the vascular spikemoss Selaginella moellendorffii, whose genome has also been sequenced, the non-vascular moss Physcomitrella provides an evolutionary link between green algae and angiosperms. BLAST searches established that the Physcomitrella and Selaginella genomes encode not only putative RAC/ROP GTPases, but also homologues of all known regulators of polarized RAC/ROP signalling, as well as of key effectors acting in signalling cascades downstream of RAC/ROP activity. Nucleotide sequence relationships within seven different families of Physcomitrella, Selaginella, Arabidopsis thaliana and Nicotiana tabacum (tobacco) genes with distinct functions in RAC/ROP signalling were characterized based on extensive maximum likelihood and Neighbor-Joining analyses. The results of these analyses are interpreted in the light of current knowledge concerning expression patterns and molecular functions of RAC/ROP signalling proteins in angiosperms. A key aim of this study is to facilitate the use of Physcomitrella as a model to investigate the molecular control of tip growth in plants.

Pollen tubes grow rapidly in a strictly polarized manner as they transport male reproductive cells through female flower tissues to bring about fertilization. Vegetative pollen tube cells are an excellent model system to investigate processes underlying directional cell expansion. In this chapter, we describe materials and methods required for (1) the identification of novel factors essential for polarized cell growth through the isolation and analysis of Arabidopsis mutants with defects in pollen tube growth and (2) the detailed functional characterization of pollen tube proteins based on transient transformation and microscopic analysis of cultured tobacco pollen tubes.

Rho GTPases play central roles in the regulation of essential cellular processes, such as directional expansion, motility, and division. RhoGEFs (Guanine Nucleotide Exchange Factors) have key functions in the stimulus-induced spatio-temporal control of Rho GTPase activity. RhoGAPs (GTPase activating proteins) and RhoGDIs (Guanine nucleotide dissociation inhibitors) have long been seen as less important regulators of Rho GTPase activity, with functions largely restricted to the constitutive attenuation of Rho signaling. Extended families of diverse RhoGAPs, as well as small families of structurally similar RhoGDIs, have been identified in yeast, animals, and plants. Recent research has established that members of these protein families play much more important and complex roles than previously anticipated in the regulation of Rho GTPase activity and cellular processes. Non-plant RhoGAPs and RhoGDIs were shown to be tightly regulated by upstream signaling, and the same is likely to be true for their plant homologs as well. The recent functional characterization of plant RhoGAPs and RhoGDIs has allowed exciting and universally important insights into the molecular mechanisms underlying the control of Rho GTPase activity by these proteins.

Spatially restricted signaling by Rho GTPases is essential for the polarization of eukaryotic cells, which is required for the morphogenesis, mobility and division of single cells, and for the development of multicellular organisms. Rac-Rop GTPases, which constitute a plant-specific Rho GTPase subfamily, accumulate at the apical plasma membrane of pollen tubes and root hairs, where they control rapid polar cell expansion by a process known as tip growth. Here, recent insights into the spatial control of Rac-Rop-dependent signaling in tip-growing plant cells by regulatory proteins (i.e. Rho GTPase-activating proteins, Rho guanine nucleotide dissociation inhibitors, Rho guanine nucleotide-exchange factors and phosphoinositide-specific phospholipase C) and lipids [phosphatidylinositol (4,5)-bisphosphate and diacyl glycerol] are summarized. A model is presented, which integrates the current knowledge concerning the molecular mechanisms that maintain the polarization of Rho signaling in plant cells.

Rac/Rop-type Rho-family small GTPases accumulate at the plasma membrane in the tip of pollen tubes and control the polar growth of these cells. Nt-RhoGDI2, a homolog of guanine nucleotide dissociation inhibitors (GDIs) regulating Rho signaling in animals and yeast, is co-expressed with the Rac/Rop GTPase Nt-Rac5 specifically in tobacco (Nicotiana tabacum) pollen tubes. The two proteins interact with each other in yeast two-hybrid assays, preferentially when Nt-Rac5 is prenylated. Transient over-expression of Nt-Rac5 and Nt-RhoGDI2 depolarized or inhibited tobacco pollen tube growth, respectively. Interestingly, pollen tubes over-expressing both proteins grew normally, demonstrating that the two proteins functionally interact in vivo. Nt-RhoGDI2 was localized to the pollen tube cytoplasm and effectively transferred co-over-expressed YFP-Nt-Rac5 fusion proteins from the plasma membrane to this compartment. A single amino acid exchange (R69A), which abolished binding to Nt-RhoGDI2, caused Nt-Rac5 to be mis-localized to the flanks of pollen tubes and strongly compromised its ability to depolarize pollen tube growth upon over-expression. Based on these observations, we propose that Nt-RhoGDI2-mediated recycling of Nt-Rac5 from the flanks of the tip to the apex has an essential function in the maintenance of polarized Rac/Rop signaling and cell expansion in pollen tubes. Similar mechanisms may generally play a role in the polarized accumulation of Rho GTPases in specific membrane domains, an important process whose regulation has not been well characterized in any cell type to date.

The functions of microtubules and actin filaments during various processes that are essential for the growth, reproduction and survival of single plant cells have been well characterized. A large number of plant structural cytoskeletal or cytoskeleton–associated proteins, as well as genes encoding such proteins, have been identified. Although many of these genes and proteins have been partially characterized with respect to their functions, a coherent picture of how they interact to execute cytoskeletal functions in plant cells has yet to emerge. Cytoskeleton–controlled cellular processes are expected to play crucial roles during plant cell differentiation and organogenesis, but what exactly these roles are has only been investigated in a limited number of studies in the whole plant context. The intent of this review is to discuss the results of these studies in the light of what is known about the cellular functions of the plant cytoskeleton, and about the proteins and genes that are required for them. Directions are outlined for future work to advance our understanding of how the cytoskeleton contributes to plant organogenesis and development.

The eukaryotic cytoskeleton is a dynamic filamentous network with various cellular and developmental functions. Plant cells display a singular architecture, necessitating a structurally and functionally unique cytoskeleton and plant specific control mechanisms.

Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis.

The actin cytoskeleton has essential functions in the survival, the division, the growth and the morphogenesis of plant cells, and in the development of plant organs. A key approach to investigating these functions is the observation of the organization and the dynamic behavior of filamentous actin structures in plants cells. Visualizing plant actin filaments has been notoriously difficult, because they are highly sensitive to standard fixation and cell permeabilization procedures. This chapter demonstrates that the expression in plant cells of an optimized mutant version of the green fluorescent protein (GFP) from Aequorea victoria fused to the well characterized F-actin binding domain of mouse talin labels the actin cytoskeleton and allows non-invasive observation of its dynamic organization by fluorescence microscopy.

The plant cytoskeleton has crucial functions in a number of cellular processes that are essential for cell morphogenesis, organogenesis and development. These functions have been intensively investigated using single cell model systems. With the recent characterization of plant mutants that show aberrant organogenesis resulting from primary defects in cytoskeletal organization, an integrated understanding of the importance of the cytoskeleton for plant development has begun to emerge. Newly established techniques that allow the non-destructive visualization of microtubules or actin filaments in living plant cells and organs will further advance this understanding.

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the ‘distorted’ class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.