Cytoplasmic Orientation of the Naphthylphthalamic Acid-Binding Protein in Zucchini Plasma Membrane Vesicles

Abstract

Polar transport of the plant hormone auxin is blocked by substances such as N-1-naphthylphthalamic acid (NPA), which inhibit auxin efflux and block polar auxin transport. To understand how auxin transport is regulated in vivo, it is necessary to discern whether auxin transport inhibitors act at the intra-or extracellular side of the plasma membrane. Populations of predominantly inside-in plasma membrane vesicles were subjected to treatments that reverse the orientation. These treatments, which included osmotic shock, cycles of freezing and thawing, and incubation with 0.05% Brii-58, all increased NPA-binding activity and the accessibility of the binding protein to protease digestion. Marker activities for inside-out vesicles also increased, indicating that these treatments act by altering the membrane orientation. Finally, binding data were analyzed by multiple analyses and indicated that neither the affinity nor abundance of binding sites changed. Kinetic analyses indicated that the change in NPA-binding activity by Brij-58 treatment was due to an increase in the initial rates of both association and dissociation of this ligand. These experiments indicated that the NPA-binding site is on the cytoplasmic face of the plasma membrane in zucchini (Cucurbita pepo 1. cv Burpee Fordhook).The auxins are a class of phytohormones that control plant growth, development, and response to the environment through regulation of elongation, cell division, differentiation, and tropic responses to light and gravity. Auxins, of which IAA is the predominant naturally occurring type, are delivered to plant cells by a unique polar transport mechanism (for review, see Goldsmith, 1977;Lomax et al., 1995). Polar transport is critica1 for the growth and development of plants and their ability to respond to environmental stimuli. Auxins are transported from their source in the shoot apex at the growing tip toward the base of stems. Polar auxin transport depends on the proton gradient across the plasma membrane of plant cells and has been described as chemiosmotic (Rubery and Sheldrake, 1974). The ionization states of IAA in the extracellular space and in the cytoplasm affect the membrane permeability of this phytohormone and lead to an accumulationThe work performed at Wake Forest University was supported 421of the anionic form of the molecule within the cytoplasm. Polar transport is believed to be mediated by an uptake 2H+/IAA-symport carrier (Lomax et al., 1985;Sabater and Rubery, 1987) and an IAA-efflux carrier (Rubery and Sheldrake, 1974). Basal localization of this IAA-efflux carrier has been proposed to drive the polarity of IAA transport (Rubery and Sheldrake, 1974;Jacobs and Gilbert, 1983).Severa1 synthetic auxin transport inhibitors, or phytotropins, have been identified, but NPA is the best characterized (Rubery, 1990). These phytotropins specifically block auxin transport at the site of the efflux carrier (Rubery, 1990). Natural inhibitors, or flavonoids, have also been shown to block transport at the site of the efflux carrie...

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

“…Moreover, our live-cell imaging analyses established that TWD1 influences both actin bundling and actin filament dynamics at the cortex of hypocotyl epidermal cells ( Figure 2, Table 2). This role for TWD1 and physical interaction with ACT7 is in line with previous in vitro and in vivo data supporting the concept that a peripheral NPA binding protein is associated with the cytoskeleton (Cox and Muday, 1994;Dixon et al, 1996;Butler et al, 1998). This holds especially true in light of evidence indicating that TWD1 is attached to the PM via an inplane membrane anchor (Scheidt et al, 2007;Bailly et al, 2014).…”

“…Based on biochemical in vitro assays, NPA binding proteins were reported to be PM associated, and NPA binding activity was localized to the cytoplasmic face of the membrane (Cox and Muday, 1994). Moreover, a peripheral NPA binding protein was suggested to be associated with the cytoskeleton (Cox and Muday, 1994;Dixon et al, 1996;Butler et al, 1998). ABCB-type auxin transporters and TWD1 were originally identified by NPA chromatography and subsequently verified as NPA targets Geisler et al, 2003;Rojas-Pierce et al, 2007;Nagashima et al, 2008;Kim et al, 2010).…”

The transparent testa4 Mutation Prevents Flavonoid Synthesis and Alters Auxin Transport and the Response of Arabidopsis Roots to Gravity and Light[W]

“…The rest of the cytoplasm of these cells is filled with flavonoids with slightly greater fluorescence intensity in naringenin-fed tt4(2YY6) roots compared with Col. The cytoplasmic localization of flavonoid accumulation is consistent with cytoplasmic orientation of high affinity membrane associated naphthylphthalamic acid (NPA) binding activity from zucchini (Cucurbita pepo), which may be the site of flavonoid action (Dixon et al, 1996). DPBA fluorescence was also detectable in the nuclei of Col and naringenin-fed tt4(2YY6), although some naringenin-fed tt4(2YY6) samples lacked nuclear staining.…”

Mutation of the Rice Narrow leaf1 Gene, Which Encodes a Novel Protein, Affects Vein Patterning and Polar Auxin Transport

“…4, B and C). It was proposed that the action of NPA to inhibit auxin transport was achieved through interacting with the so-called NPA-binding protein, which has long been considered part of the auxin efflux carrier complex (Dixon et al, 1996;Ruegger et al, 1997;Blakeslee et al, 2005). On the other hand, a characteristic feature of NPA action is that auxin accumulates in treated cells, and the effect of NPA on PAT was generally interpreted as an inhibition of auxin efflux (Morris et al, 2004).…”