Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells

Abstract

The phytohormone auxin is transported through the plant body either via vascular pathways or from cell to cell by specialized polar transport machinery. This machinery consists of a balanced system of passive diffusion combined with the activities of auxin influx and efflux carriers. Synthetic auxins that differ in the mechanisms of their transport across the plasma membrane together with polar auxin transport inhibitors have been used in many studies on particular auxin carriers and their role in plant development. However, the exact mechanism of action of auxin efflux and influx inhibitors has not been fully elucidated. In this report, the mechanism of action of the auxin influx inhibitors (1-naphthoxyacetic acid (1-NOA), 2-naphthoxyacetic acid (2-NOA), and 3-chloro-4-hydroxyphenylacetic acid (CHPAA)) is examined by direct measurements of auxin accumulation, cellular phenotypic analysis, as well as by localization studies of Arabidopsis thaliana L. auxin carriers heterologously expressed in Nicotiana tabacum L., cv. Bright Yellow cell suspensions. The mode of action of 1-NOA, 2-NOA, and CHPAA has been shown to be linked with the dynamics of the plasma membrane. The most potent inhibitor, 1-NOA, blocked the activities of both auxin influx and efflux carriers, whereas 2-NOA and CHPAA at the same concentration preferentially inhibited auxin influx. The results suggest that these, previously unknown, activities of putative auxin influx inhibitors regulate overall auxin transport across the plasma membrane depending on the dynamics of particular membrane vesicles.

Fig. 1.

Effects of auxin influx and efflux inhibitors on cell division activity and phenotype of tobacco BY-2 cells. The cells were grown (A) in a standard medium for 2 d, in the presence of auxin influx inhibitors (B) 1-NOA, (C) 2-NOA, (D) CHPAA, or (E) auxin efflux inhibitor NPA (10 μM each). Measurements of cell length (F) and cell density (G) showed that cells treated with 1-NOA were more elongated (F) and that cell division was reduced in contrast to cells treated with 2-NOA or CHPAA (G). Values in (F) and (G) represent means ±SE (n=10). (A–E) Scale bar, 50 μm.

Fig. 2.

The effect of the putative auxin influx inhibitors on intracellular accumulation of [³H]2,4-D and [³H]NAA in tobacco BY-2 cells. The kinetics of accumulation of 2 nM [³H]2,4-D (A) and [³H]NAA (B) in the presence of 10 μM 1-NOA (filled circles), 2-NOA (filled squares) or CHPAA (filled inverted triangles). Note the difference between the efficiency of 2-NOA, CHPAA, and 1-NOA in the inhibition of auxin influx as shown by reduced accumulation of [³H]2,4-D (A) compared with control cells. Increased accumulation of [³H]NAA (reflecting preferentially the activity of auxin efflux carriers) in the presence of 1-NOA is significantly higher than that in the presence of 2-NOA and CHPAA (10 μM each). (C) Concentration dependence of [³H]2,4-D (2 nM) in response to specified concentrations of 1-NOA (filled circles) or 2-NOA (filled squares) was determined after 20 min uptake period. Accumulation of [³H]2,4-D is suppressed more effectively with 2-NOA compared with 1-NOA. Error bars represent SE (n=4).

Fig. 3.

The effect of NPA on the intracellular accumulation of [³H]NAA in tobacco BY-2 cells treated with 1-NOA or 2-NOA. In-flight application of NPA (10 μM) to cells treated with 1-NOA (10 μM) (A) had very low impact on the accumulation of [³H]NAA (2 nM). In-flight application of NPA to cells treated with 2-NOA (10 μM) (B) increased auxin accumulation, suggesting that auxin efflux carriers were still active before NPA treatment (note also the higher absolute values of auxin accumulation in the case of 1-NOA in contrast to 2-NOA even before NPA treatment). Error bars represent SE (n=4). (C) Effects of the auxin influx and efflux inhibitors 1-NOA, 2-NOA, and NPA on intracellular accumulation of [³H]NAA in cells treated with the inhibitor of vesicle trafficking BFA. 2-NOA did not change the accumulation of [³H]NAA in cells treated with BFA, while both NPA and 1-NOA increased it. Values are percentages of control at 20 min after application of [³H]NAA and inhibitors. Error bars represent SE (n=4).

Fig. 4.

The effects of the putative auxin influx inhibitors on subcellular distribution of PIN1:GFP, EYFP:AUX1, and ABCB4:GFP fusion proteins in tobacco BY-2 cells. (A, E, I) non-treated controls. The effects of 1-NOA (20 μM, 3 h) (B), 2-NOA (20 μM, 3 h) (C), and CHPAA (20 μM, 3 h) (D) on the subcellular distribution of PIN1:GFP. The effects of 1-NOA (20 μM, 3 h) (F), 2-NOA (20 μM, 3 h) (G), and CHPAA (20 μM, 3 h) (H) on the subcellular distribution of EYFP:AUX1. The effects of 1-NOA (20 μM, 24 h) (J), 2-NOA (20 μM, 24 h) (K), and CHPAA (20 μM, 24 h) (L) on the subcellular distribution of ABCB4:GFP. Single confocal sections through the cortical cytoplasm (C, Apochromat ×40 1.2 W water immersion objective with a 1 Airy Unit pinhole). Scale bars, 10 μm.

Fig. 5.

Long-term treatments of EYFP:AUX1 or PIN1:GFP tobacco BY-2 cells with the putative auxin influx inhibitors. (A, E) non-treated controls. The effects of 1-NOA (20 μM, 24 h) (B), 2-NOA (20 μM, 24 h) (C), and CHPAA (20 μM, 24 h) (D) on cell division in BY-2 cells transformed with the EYFP:AUX1 construct. The effects of 1-NOA (20 μM, 24 h) (F), 2-NOA (20 μM, 24 h) (G), and CHPAA (20 μM, 24 h) (H) on cell division in BY-2 cells transformed with the PIN1:GFP construct. Single confocal sections through the perinuclear region (C, Apochromat ×40 /1.2 W water immersion objective with a 1 Airy Unit pinhole). Scale bars, 20 μm.

Fig. 6.

Molecular structure of synthetic auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA. Blue, carbon; red, oxygen; white, hydrogen; green, chlorine.