Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

Abstract

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

Figure 1

Exogenous ATP induces root curling and growth of lateral roots. Shown are 1-week-old Arabidopsis plants (ecotype Wassilewskija) grown on a plate containing MS only (A), or MS +: 1 mm ATP (B), 2 mm ATP (C), 3 mm ATP (D), 2 mm ADP (E), 2 mm AMP (F), 2 mm ITP (G), and 2 mm ADP plus 2 mm phosphate, pH 4.1 (H). For these experiments, the pH of the ATP was not adjusted before adding it into the media.

Figure 2

Exogenous ATP inhibits root bending. A, Gravity response of the roots of seedlings grown on media containing MS only for 6 d, transferred to a new plate containing MS only (left) or MS + 5 mm ATP (right), and then turned 90° and grown for 2 more d. B, Bending pattern of the roots of plants grown horizontally 2 d after transfer to: a medium containing MS only or a medium containing MS + 5 mm ATP. Arrows show the start position of root tip after the transfer.

Figure 3

Exogenous ATP induces accumulation of GUS stain in the root tips of DR5-GUS seedlings. GUS-stained root tips of DR5-GUS seedlings grown 6 d in NPA at a concentration of: 0 μm (A), 0.1 μm (B), 1 μm (C), and 10 μm (D); or in 2 mm ATP (E), 2 mm ADP (F), 2 mm ITP (G), and 2 mm AMP (H). The pH of ATP was not pre-adjusted.

Figure 4

Exogenous ATP increases the sensitivity of roots to growth inhibition by exogenous auxin. Wassilewskija seeds were grown in MS medium with different concentrations of NAA in the presence (or absence) of 2 mm ATP. After 6 d, the seedlings were taken out, and the root length was measured. Each data point represents the average ± se (n > 60). The pH of the ATP stock was not pre-adjusted. a, Plants grown in 2.5 × 10⁻⁷ m NAA alone; b, plants grown in 2.5 × 10⁻⁷ m NAA with 2 mm ATP.

Figure 5

Exogenous ATP inhibits basipetal auxin transport in Arabidopsis and maize roots. A, Basipetal auxin transport in Arabidopsis roots was allowed to precede in darkness for 5 h, at which time the [³H]IAA in a 5-mm root section 1 mm back from the tip was quantified by scintillation counting. Values represent average ± se. B, Basipetal auxin transport in maize roots was allowed to proceed in darkness for 4 h, at which time the [³H]IAA amount in the receiver agar block was quantified by scintillation counting. The experiment was repeated at least four times. The data represent the average ± se (n > 16).

Figure 6

Exogenous ATP promotes retention of auxin in corn roots. Corn (yellow dent) root tips (3 mm) were excised and incubated in MS liquid medium (no agar) plus 10⁻⁷ m [³H]IAA, with or without ATP or ADP. After the root tips were washed with MS medium, the radioactivity in roots was measured by scintillation counting. The level of [³H]IAA retained in roots treated with 5 mm ATP is significantly different from that in roots treated with 5 mm ADP and in untreated roots. Values represent average ± se (n = 18; P < 0.001).