Myosin XIK of Arabidopsis thaliana Accumulates at the Root Hair Tip and Is Required for Fast Root Hair Growth

Myosin motor proteins are thought to carry out important functions in the establishment and maintenance of cell polarity by moving cellular components such as organelles, vesicles, or protein complexes along the actin cytoskeleton. In Arabidopsis thaliana, disruption of the myosin XIK gene leads to reduced elongation of the highly polar root hairs, suggesting that the encoded motor protein is involved in this cell growth. Detailed live-cell observations in this study revealed that xik root hairs elongated more slowly and stopped growth sooner than those in wild type. Overall cellular organization including the actin cytoskeleton appeared normal, but actin filament dynamics were reduced in the mutant. Accumulation of RabA4b-containing vesicles, on the other hand, was not significantly different from wild type. A functional YFP-XIK fusion protein that could complement the mutant phenotype accumulated at the tip of growing root hairs in an actin-dependent manner. The distribution of YFP-XIK at the tip, however, did not match that of the ER or several tip-enriched markers including CFP-RabA4b. We conclude that the myosin XIK is required for normal actin dynamics and plays a role in the subapical region of growing root hairs to facilitate optimal growth. DOI: 10.1371/journal.pone.007674

Parteimedien in Krisenzeiten

Die vorliegende Arbeit beschäftigt sich mit der Konstruktion von Parteiwirklichkeiten in den Zentralorganen der SPÖ, ÖVP und KPÖ während der Ereignisse Ungarn-Aufstand 1956 und Prager Frühling 1968. Es sollte herausgefunden werden, wie die Parteien ihre Wirklichkeiten anhand von sechs Variablen aufbauen. Als Methode wurde die Diskursana-lyse nach Siegfried Jäger gewählt. Es wurden Aussagen aus der „Arbeiter-Zeitung“, „Das kleine Volksblatt“ und „Österreichische Volksstimme“ gesucht, die sich mit Russland, Österreich, der Sicherheit für Österreich, dem politischen Gegner, der Neutralität und der Reaktion der West-Staaten beschäftigen. Anschließend wurden die Diskursfragmente in Diskursstränge und schließlich in einen übergeordneten Diskurs eingebracht. Als Voraussetzung für die Interpretation wurde zunächst der kommunikationswissen-schaftliche Standort der Parteimedien bestimmt, anschließend der geschichtliche Rahmen erarbeitet, um die Ergebnisse interpretieren zu können. Als Grundlage der Interpretation diente der Konstruktivismus. Zwischen den Analysezeitpunkten sind deutliche Unterschiede zu beobachten. Die „Arbei-ter-Zeitung“ versucht 1956 Russland als gewalttätigen, illegitimen Herrscher darzustellen. Dies wird durch emotionale Gewaltdarstellungen und dem Absprechen der demokratischen Legitimation der Volksdemokratien erreicht. Ähnlich geht auch „Das kleine Volksblatt“ vor. 1968 wird in beiden Zeitungen auf Gewaltdarstellungen verzichtet, es kommt aber die Strategie der Darstellung Russlands als dumm, inkompetent und unverlässlich hinzu: Russ-land wird vom tschechoslowakischen Volk ganz einfach an falsche Orte geschickt, und die Russen merken dies nicht. Die „Österreichische Volksstimme“ macht aber die größte Ver-änderung durch: Von der Darstellung Russlands 1956 als Helfer und Befreier der Ungarn vom Faschismus wandelt sich die Einstellung zu einer differenzierten und kritischen Be-trachtungsweise, die den Einmarsch 1968 als unnötig verurteilt – dabei stützt sich das KPÖ-Organ größtenteils auf Zitate anderer europäischer Schwesterparteien. Die „Österrei-chische Volksstimme“ sieht 1956 die Gefahr eher in Österreichs Presse als in den Kämpfen im Nachbarland, da die Presse aktiv die Revolution unterstützt und Lügen verbreitet, um die Kämpfe weiter anzuheizen. Österreichs Rolle wird von der „Arbeiter-Zeitung“ und „Das kleine Volksblatt“ 1956 als Helfer und Verbündeter gesehen. Durch die ausführliche Beschreibung emotionaler Hilfs-aktionen wird Österreich kompromisslos als selbstloser Helfer dargestellt. Die „Österrei-chische Volksstimme“ sieht dies anders: Österreich wird als Unterstützer der Revolution in Ungarn gesehen – Hilfsaktionen werden ausgenutzt, um die unrechtmäßige, faschistische Erhebung mit Waffen zu unterstützen. 1968 werden Österreichs Bemühungen in allen drei Medien zwar positiv dargestellt, allerdings verliert der Österreichdiskurs an Wichtigkeit und Emotionalität. 1956 werden in der „Arbeiter-Zeitung“ und noch massiver in „Das kleine Volksblatt“ die Westmächte für ihr Nicht-Handeln verurteilt. Als Strategie wird der Vergleich mit der Su-ez-Krise benutzt, wo aus materiellem Interesse durch Großbritannien und Frankreich inter-veniert wurde, und so die Ungleichbehandlung Ungarns erörtert. 1968 gibt es in der „Ar-beiter-Zeitung“ einen ähnliche Taktik, hier wird als Vergleich der Krieg in Vietnam heran-gezogen. 1956 benutzt auch die „Österreichische Volksstimme“ die Suez-Krise als Mittel, die Westmächte als Aggressor darzustellen. 1968 wird in der „Volksstimme“ dann die Doppelmoral des Westens angeprangert: Die Krise in der ČSSR wird verurteilt, zum Viet-namkrieg aber geschwiegen

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis

Fine mapping in tomato using microsynteny with the Arabidopsis genome: the Diageotropica (Dgt) locus

BACKGROUND: The Arabidopsis thaliana genome sequence provides a catalog of reference genes applicable to comparative microsynteny analysis of other species, facilitating map-based cloning in economically important crops. We have applied such an analysis to the tomato expressed sequence tag (EST) database to expedite high-resolution mapping of the Diageotropica (Dgt) gene within the distal end of chromosome 1 in tomato (Lycopersicon esculentum). RESULTS: A BLAST search of the Arabidopsis database with nucleotide sequences of markers that flank the tomato dgt locus revealed regions of microsynteny between the distal end of chromosome 1 in tomato, two regions of Arabidopsis chromosome 4, and one on chromosome 2. Tomato ESTs homeologous to Arabidopsis gene sequences within those regions were converted into co-dominant molecular markers via cleaved amplified polymorphic sequence (CAPS) analysis and scored against an informative backcross mapping population. Six new microsyntenic EST (MEST) markers were rapidly identified in the dgt region, two of which further defined the placement of the Dgt gene and permitted the selection of a candidate tomato bacterial artificial chromosome clone for sequence analysis. CONCLUSIONS: Microsynteny-based comparative mapping combined with CAPS analysis of recombinant plants rapidly and economically narrowed the dgt mapping region from 0.8 to 0.15 cM. This approach should contribute to developing high-density maps of molecular markers to target-specific regions for positional cloning and marker-assisted selection in a variety of plants

Endoplasmic reticulum targeted GFP reveals ER organization in tobacco NT-1 cells during cell division

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Plant Physiology and Biochemistry, doi:10.1016/j.plaphy.2006.03.003.The endoplasmic reticulum (ER) of plant cells undergoes a drastic reorganization during cell division. In tobacco NT-1 cells that stably express a GFP construct targeted to the ER, we have mapped the reorganization of ER that occurs during mitosis and cytokinesis with confocal laser scanning microscopy. During division, the ER and nuclear envelope do not vesiculate. Instead, tubules of ER accumulate around the chromosomes after the nuclear envelope breaks down, with these tubules aligning parallel to the microtubules of the mitotic spindle. In cytokinesis, the phragmoplast is particularly rich in ER, and the transnuclear channels and invaginations present in many interphase cells appear to develop from ER tubules trapped in the developing phragmoplast. Drug studies, using oryzalin and latrunculin to disrupt the microtubules and actin microfilaments respectively, demonstrate that during division, the arrangement of ER is controlled by microtubules and not by actin, which is the reverse of the situation in interphase cells.Funding for this project included NASA grant # NAGW-4984 to the North Carolina NSCORT (NASA Specialized Center of Research and Training) (SLG, DAC, NSA), NSF REU Site Grant #0243930 (NSA), a Sigma Xi Grant-in-Aid Award (SLG), and Australian Research Council Discovery Grant no. DP0208806 (DAC)

Genome-Wide Identification, Functional Analysis and Expression Profiling of the Aux/IAA Gene Family in Tomato

Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components,among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk

Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array

Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 µm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly