NPH4, a conditional modulator of auxin-dependent differential growth responses in Arabidopsis

Abstract

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein.

Figure 1

Time course of hypocotyl growth of wild-type and nph4 seedlings in darkness. At the indicated times after induction of germination, seedlings were removed and hypocotyl lengths were measured to the nearest millimeter. Data represent the mean response of a minimum of 60 seedlings from two replicate experiments. The vertical error bars represent the se values. Because the symbols often overlap, some individual symbols and error bars are not visible. The dotted line represents a regression (r² = 0.942) calculated for combined wild-type and nph4 data during the period of linear growth (d 2, 3, and 4). Col, Wild-type Columbia ecotype.

Figure 2

Cellular morphology of wild-type (A), nph4-1 (B), nph4-2 (C), and nph4-3 (D) hypocotyls. All sections were taken from a region just below the apical hook of 3-d-old dark-grown seedlings, where phototropism is initiated (Orbovic and Poff, 1991), stained with periodic acid-Schiff's reagent, and viewed with bright-field optics. Morphologies are similar to those reported previously for etiolated Arabidopsis seedlings (Gendreau et al., 1997). Bar = 100 μm.

Figure 3

Morphogenesis of etiolated wild-type and nph4 seedlings grown in air (A) and 50 μL/L ethylene (B). Photographs were taken after 4 d of growth in darkness. The ethylene receptor mutant etr1-1 is shown as a negative control for ethylene responsiveness. Col, Wild-type Columbia ecotype.

Figure 4

Time course of apical hook opening in dark-grown wild-type and nph4 seedlings. Seedlings were grown in darkness on vertical plates. At the indicated times (after induction of germination), apical hook angles were determined (see Methods). Data represent the mean response of a minimum of 21 seedlings from two replicate experiments. The vertical error bars represent the se values. Because the symbols often overlap, some individual symbols and error bars are not visible. Col, Wild-type Columbia ecotype.

Figure 5

BL-dependent (A) and RL-dependent (B) hypocotyl growth inhibition in wild-type and nph4 seedlings. After 23 h of growth in darkness, seedlings were transferred to continuous light at the indicated fluence rates shown for an additional 96 h. Control seedlings were kept in darkness for the entire growth period. Hypocotyl lengths were measured from digitized images of seedlings (see Methods). Data represent the mean response of at least 33 seedlings from two replicate experiments. Vertical error bars represent the combined se values for dark- and light-grown seedlings. Because the symbols often overlap, some individual symbols and error bars are not visible. The response of cry1-deficient hy4-101 seedlings is presented as a negative control. The response of phyB-deficient phyB-9 seedlings is presented as a negative control. Col, Wild-type Columbia ecotype.

Figure 6

RL-dependent hypocotyl curvature in dark-grown wild-type and mutant seedlings. Sixty-hour-old seedlings were exposed to 20 h of continuous RL at the indicated fluence rates, and then curvatures were determined (see Methods). Data represent the mean response of a minimum of 33 seedlings from two replicate experiments. Vertical error bars represent the se values. Because the symbols often overlap, some individual symbols and error bars are not visible. Col, Wild-type Columbia ecotype.

Figure 7

Dose responses of wild-type and nph4 hypocotyls to exogenous IAA (A), 2,4-D (B), and 1-NAA (C). Three-day-old YL-grown seedlings were transferred to medium containing various concentrations of auxins (see Methods). Hypocotyl growth was measured 3 d later. Data represent the mean response (as a percent of controls) of a minimum of 90 seedlings from at least three replicate experiments. Vertical error bars represent the se values. Controls were seedlings transferred to plates containing only solvent (0.04% ethanol). Because the symbols often overlap, some individual symbols and error bars are not visible. Col, Wild-type Columbia ecotype.

Figure 8

Expression of auxin-induced genes in wild-type and nph4 seedlings. Total RNA was isolated from 7-d-old WL-grown seedlings that had been exposed to 100 μm IAA (+) or solvent (0.04% ethanol; −) for 1 h. Samples (20 μg each) were separated on a 1.0% agarose formaldehyde/Mops gel and then blotted to nylon. The blot was then hybridized with ³²P-labeled gene-specific probes against various auxin-induced genes: SAUR-AC1 (Gil et al., 1994); GH3 (Hagen et al., 1984); and IAA2, IAA4, IAA5, IAA6, IAA12, and IAA13 (Abel et al., 1995). The blot was also hybridized with a labeled actin probe (ACT7; McDowell et al., 1996) as a loading control. RNA from nph1-4 seedlings was used as an additional positive control. Similar overall results were observed in replicate experiments with both WL- and dark-grown seedlings (data not shown). The blot was rehybridized with multiple probes between strippings; thus, artificially flat upper and/or lower edges were generated on the IAA5, IAA6, and IAA13 transcripts when individual panels for these genes were cropped from the entire blot for photographs. Col, Wild-type Columbia ecotype; SAUR, SAUR-AC1.