Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells1

Abstract

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [ 14 C]2,4-dichlorophenoxyacetic acid and [ 3 H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [ 3 H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [ 14 C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turnedover proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.

Gravity-regulated differential auxin transport from columella to lateral root cap cells

“…We further analyzed the effect of 20 M BFA on DR5-GFPm expression. BFA was recently shown to interfere with auxin efflux by abolishing polar localization of efflux carrier AtPIN proteins (19)(20)(21). In vertically grown roots, the signal pattern on BFA (Fig.…”

Flavonoid Accumulation in Arabidopsis Repressed in Lignin Synthesis Affects Auxin Transport and Plant Growth

“…The tir3/doc1 mutant that is affected both in light responses and in auxin transport defines a calossin-like gene named BIG and displays a reduction in the number of NPA binding sites in the microsomal fraction (Ruegger et al, 1997;Gil et al, 2001). A role of intracellular trafficking has been proposed because treatment with brefeldin A, an inhibitor of vesicular transport, prevents normal membrane localization of PIN1 and blocks auxin efflux (Delbarre et al, 1998;Geldner et al, 2001;Muday and Murphy, 2002;Geldner et al, 2003). Finally, several NBPs occur that differ in their ligand affinity (Lomax et al, 1995;Murphy et al, 2000Murphy et al, , 2002Muday and Murphy, 2002), suggesting multiple physiological functions for the recognition of the NBP complex.…”

ALTERED RESPONSE TO GRAVITY Is a Peripheral Membrane Protein That Modulates Gravity-Induced Cytoplasmic Alkalinization and Lateral Auxin Transport in Plant Statocytes

“…Staurosporine, a protein kinase inhibitor, was shown to reduce auxin efflux in tobacco (Delbarre et al, 1998). Also, a mutation in Arabidopsis RCN1 , which encodes a subunit of protein phosphatase 2A, resulted in increased basipetal auxin transport in the root tip and reduced gravitropic curvature (Garbers et al, 1996;Rashotte et al, 2001).…”