Auxin responsiveness of a novel cytochrome p450 in rice coleoptiles

Abstract

An early auxin-induced gene was isolated from rice (Oryza sativa L. subsp. japonica cv Nihonmasari) coleoptiles by a fluorescent-labeled differential display screen. The full-length gene contains conserved domains characteristic for the cytochrome p450 superfamily. This gene, designated as CYP87A3, was weakly expressed in dark-grown coleoptiles but was up-regulated rapidly and transiently when coleoptile segments were incubated in 5 microm indole-3-acetic acid. This induction by auxin could not be suppressed by cycloheximide. Depletion of segments from endogenous auxin reduced the amount of CYP87A3 transcripts. The CYP87A3 transcript level was rapidly, although transiently, up-regulated in response to light as well. The observed pattern of gene regulation might indicate a role in the suppression of auxin-induced coleoptile growth. The role of CYP87A3 is discussed with respect to auxin signaling in the regulation of coleoptile growth.

Figure 1.

Fluorescence differential display screen for auxin-inducible transcripts. A, Scheme of the experiment: 1, coleoptiles were grown for 6 d in darkness; 2, coleoptile segments were excised; and 3, segments were predepleted in water for 1 h and then incubated for 1 more h in IAA or in water. B, A part of an FDD gel image showing the CYP87A3 expression pattern in WT and the mutant Yin-Yang. Total RNA was isolated from coleoptile segments incubated for 1 h either in distilled water (lanes 1 and 3) or in 5 μm IAA (lanes 2 and 4).

Figure 2.

Multiple sequence alignment of CYP87A3 with related CYP proteins. CYP87A3 exhibits homology to the 23α-hydroxylase CPD (CYP90A1), the 22α-hydroxylase DWF4 (CYP90B1), ROTUNDIFOLIA (CYP90C1), DWF3 (CYP88A1), and DWARF (CYP85). The locations of conserved domains are indicated above the sequences. Amino acid residues that are identical between the compared sequences are indicated by reverse font; 80% conserved, by white letters in gray boxes; 60% conserved, by gray boxes.

Figure 3.

Subcellular localization of 35S:CYP87A3:CFP fusion protein. The transient expression of CYP87A3 with in-frame C-terminally fused CFP was analyzed in coleoptile epidermal cell by confocal laser scanning microscope. co, Control, 35S::CFP; a through c, sequential confocal sections through the same cell expressing CYP87A3::CFP, beginning from the outer cell surface. White bar = 20 μm; N, nucleus.

Figure 4.

Kinetics of CYP87A3 expression in response to exogenous IAA. Total RNA (15 μg per lane) was isolated from coleoptile segments that either had been excised directly from seedlings grown for 6 d in darkness (lane 9) or that after excision had been incubated in water (lanes 1-4) or in 5 μm IAA (lanes 5-8), respectively. The same membrane was hybridized with probes specific to CYP87A3 and 28S rRNA (loading control).

Figure 5.

The response of CYP87A3 transcripts to different concentrations of IAA. Total RNA (15 μg per lane) was isolated from coleoptile segments that had been incubated for 1 h in IAA of different concentrations directly after excision. Top row, CYP87A3-specific probe was used; bottom row, loading control shown by hybridization with 28S rRNA-specific probe.

Figure 6.

CYP87A3 transcripts are elevated after cycloheximide treatment. Total RNA (15 μg per lane) was isolated from coleoptiles that have been treated with cycloheximide and/or auxin. When coleoptiles were treated by cycloheximide in combination with auxin (lane 7), cycloheximide was applied 30 min before IAA to ensure inhibition of protein synthesis before the exogenous auxin could enter the cell. The working concentrations were 5 μm for IAA and 70 μm for cycloheximide. For details, refer to the legend of Figure 4. CHX, cycloheximide.

Figure 7.

CYP87A3 transcripts are transiently induced by red light. Total RNA (14 μg per lane) was isolated from coleoptiles after irradiation for different time intervals. A, Weak red light (RL), 3.8 μmol m^-2 s^-1. B, Strong red light, 38.9 μmol m^-2 s^-1. The irradiation time is indicated. For details refer to the legend of Figure 4.