Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth

Abstract

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

Figure 1.

Gravity Response Is Delayed in rcn1 Roots. Six-day-old seedlings were transferred to new agar plates and allowed to grow vertically for 24 hr, at which time the plate was rotated 90°. Curvature of roots plotted as a function of time after reorientation is shown. Each value represents the average ±se for at least 70 seedlings. Ws, Wassilewskija.

Figure 2.

Computerized Analysis Demonstrates the Reduced Rate of Gravitropic Bending in rcn1 and Cantharidin-Treated Roots. The absolute angles of the first 160 μm of seedling root tips were measured every minute. Wild-type, rcn1, cantharidin-treated wild-type, NPA-treated rcn1, and cantharidin- and NPA-treated wild-type roots were measured during the first 13 hr after reorientation. A representative sample of each genotype and treatment is shown. Ws, Wassilewskija; CT, cantharidin.

Figure 3.

Basipetal ³H-IAA Transport in rcn1 Roots Is Increased. An agar cylinder containing ³H-IAA was applied at the root tip of wild-type and rcn1 plants grown on control plates. After 5 hr of transport, the apical 1 mm of the root was excised and discarded, and the amount of ³H-IAA in each subsequent 2-mm segment back from the root tip was determined. Values shown are averages and se for at least 30 seedlings. The amount of auxin transported into each segment for wild type and rcn1 was compared by Student's t test with the P values indicated. **P < 0.01, ***P < 0.001. Ws, Wassilewskija.

Figure 4.

Reduced Phosphatase Activity Alters Basipetal and Acropetal Auxin Transport in Roots. (A) Basipetal IAA transport was measured by application of agar containing ³H-IAA with or without 100 μM NPA to the root tip of seedlings grown on control or 10-μM cantharidin plates. After 5 hr, the apical 1 mm of the root was excised and discarded, and the amount of ³H-IAA in the apical 1 to 6 mm of the root was determined. (B) NPA sensitivity of basipetal auxin transport was measured by placing seedlings on agar plates containing the NPA concentrations shown and assaying basipetal ³H-IAA transport as described above. (C) Acropetal auxin transport was measured by application of agar containing ³H-IAA with or without 100 μM NPA to the root/shoot junction of plants grown on control or 10-μM cantharidin plates. After 18 hr, the amount of ³H-IAA in the apical 10 mm of the root tip was determined. (D) NPA sensitivity of acropetal auxin transport was measured by placing seedlings on agar plates containing the NPA concentrations shown and assaying acropetal ³H-IAA transport as described above. Each data point represents the average and se for at least 40 plants from several experiments ([A] and [C]) or for at least 10 seedlings from a representative experiment presented as a percentage relative to the Wassilewskija (Ws) minus NPA value ([B] and [D]). The amount of auxin transported into each segment for wild type, rcn1, and cantharidin-treated wild type was compared by Student's t test with the P values indicated. **P < 0.01, ***P < 0.001.

Figure 5.

Expression of an Auxin-Responsive Reporter Is Increased on the Lower Side of Gravity-Stimulated Wild-Type Roots. GUS expression was visualized in 7-day-old seedlings homozygous for the DR5-GUS construct. Root tips are shown for a vertically grown seedling on control medium (A), a seedling 6 hr after gravity (g) stimulation on control medium (B), 1 μM NPA (C), 10 μM cantharidin (D), and 10 μM cantharidin and 0.5 μM NPA (F), and a seedling 14 hr after gravity stimulation on 10 μM cantharidin (E). Bar = 100 μm.

Figure 6.

Lateral Root Growth in rcn1 Shows Reduced NPA Sensitivity. Four-day-old wild-type and rcn1 seedlings were transferred to agar plates containing NPA at the concentrations indicated and allowed to grow vertically. After 6 days, the number of lateral roots emerging from each primary root was counted. Each data point shown gives the average ±se for 10 seedlings. Ws, Wassilewskija.