Biomedical subjects

Aaron P Smith

Publications and source records attributed to Aaron P Smith.

4 recordsLinked to original sources

PubMed2005

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1.

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport.

ATP-Binding Cassette Transporters↗

PubMed2004

Proteomic analysis of Arabidopsis glutathione S-transferases from benoxacor- and copper-treated seedlings.

Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions.

Arabidopsis↗

PubMed2004

NMR difference spectroscopy with a dual saddle-coil difference probe.

A new difference probe for nuclear magnetic resonance (NMR) spectroscopy is presented. The difference probe uses two saddle-shaped coils to excite and detect two samples simultaneously. The samples are held in a specially modified 3-mm NMR tube with an Ultem plastic disk to separate the samples. The probe's resonant circuit contains two crossed diodes that passively switch the relative phase of each coil during the NMR experiment. The result is a difference spectrum from the two samples. The degree of cancellation of common signals was determined to be approximately 90%, and the application of the probe to relaxation-edited difference spectroscopy for identifying protein-ligand interactions was demonstrated using glutathione and glutathione S-transferase binding protein.

Glutathione↗

PubMed2003

Arabidopsis AtGSTF2 is regulated by ethylene and auxin, and encodes a glutathione S-transferase that interacts with flavonoids.

Expression of the Arabidopsis glutathione S-transferase (GST) gene AtGSTF2 is induced by several stimuli, but the function of this GST remains unknown. We demonstrate that AtGSTF2 expression is also induced by glutathione, paraquat, copper, and naphthalene acetic acid (NAA) via a mechanism independent of ethylene perception, as determined by analysis of the ethylene-insensitive etr1 mutant. Deletion analyses identified two promoter regions important for regulation of AtGSTF2 expression in response to several of these inducers. Previous studies have suggested that AtGSTF2 interacts with indole-3-acetic acid (IAA) and the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). We show that recombinant AtGSTF2 directly binds IAA, NPA, and the artificial auxin NAA. As NPA may act as an endogenous flavonoid regulator of auxin transport, competition between NPA and flavonoids for binding to AtGSTF2 was examined. Both quercetin and kaempferol competed with NPA for AtGSTF2 binding, indicating that all three compounds bind AtGSTF2 at the same site. In transgenic Arabidopsis seedlings, AtGSTF2::GUS expression occurred at the root-shoot transition zone and was induced in this region, as well as at the root distal elongation zone, after treatment with IAA. In wild-type seedlings, AtGSTF2 is localized near the plasma membrane of cells in the root-shoot transition zone. However, both AtGSTF2::GUS expression and localization of AtGSTF2 protein were disrupted in flavonoid-deficient tt4 seedlings. Our results indicate that AtGSTF2 is involved not only in stress responses but also in development under normal growth conditions.

Arabidopsis↗