cytochalasin-d and alpha-naphthylphthalamic-acid

Enhancement of endoreduplication in dark-grown hypocotyl is a common feature in dicotyledonous polysomatic plants, and TIBA-mediated inhibition of the endoreduplication is partially due to abnormal actin organization. Many higher plant species use endoreduplication during cell differentiation. However, the mechanisms underlying this process have remained elusive. In this study, we examined endoreduplication in hypocotyls and cotyledons in response to light in some dicotyledonous plant species. Enhancement of endoreduplication was found in the dark-grown hypocotyls of all the polysomatic species analyzed across five different families, indicating that this process is a common feature in dicotyledonous plants having polysomatic tissues. Conversely, endoreduplication was enhanced in the light-grown cotyledons in four of the five species analyzed. We also analyzed the effect of a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA) on endoreduplication in hypocotyl and cotyledon tissues of radish (Raphanus sativus L. var. longipinnatus Bailey). TIBA was found to inhibit and promote endoreduplication in hypocotyls and cotyledons, respectively, suggesting that the endoreduplication mechanism differs in these organs. To gain insight into the effect of TIBA, radish and spinach (Spinacia oleracea L.) seedlings were treated with a vesicle-trafficking inhibitor, brefeldin A, and an actin polymerization inhibitor, cytochalasin D. Both of the inhibitors partially inhibited endoreduplication of the dark-grown hypocotyl tissues, suggesting that the prominent inhibition of endoreduplication by TIBA might be attributed to its multifaceted role.

Topics: Biological Transport; Brefeldin A; Cotyledon; Cytochalasin D; Endoreduplication; Fluorenes; Hypocotyl; Indoleacetic Acids; Isobutyrates; Light; Phthalimides; Ploidies; Raphanus; Spinacia oleracea; Triiodobenzoic Acids

The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

Topics: Actins; Biological Transport; Carrier Proteins; Cytochalasin D; Cytoskeleton; Gravitropism; Hypocotyl; Indoleacetic Acids; Nucleic Acid Synthesis Inhibitors; Phthalimides; Plant Growth Regulators; Plant Proteins; Vegetables

The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

Topics: Actins; Biological Transport; Carrier Proteins; Cucurbitaceae; Cytochalasin D; Cytoskeleton; Herbicides; Hypocotyl; Indoleacetic Acids; Phalloidine; Phthalimides; Plant Growth Regulators; Plant Proteins; Tromethamine

To determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays cv Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer-controlled video digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to that of vertical roots. Application of cytochalasin B or cytochalasin D resulted in extensive disruption of MFs in the cortex and epidermis, but only partially affected MFs in the stele. Despite the cytochalasin B-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots.

Topics: Actin Cytoskeleton; Actins; Cytochalasin B; Cytochalasin D; Cytoskeleton; Gravitation; Gravitropism; Herbicides; Microscopy, Confocal; Microtubules; Nucleic Acid Synthesis Inhibitors; Phthalimides; Plant Epidermis; Plant Roots; Zea mays