MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana

Abstract

We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation. Yet other aspects of growth and development of msg2 plants are almost normal. These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000). Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain II of an Auxin/Indole-3-Acetic Acid protein, IAA19. Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7. Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) two-hybrid assay and to the whole latter protein in vitro by pull-down assay. These results suggest that MSG2/IAA19 and NPH4/ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.

Figure 1.

Differential Growth Responses Observed in Hypocotyls of the Wild Type, msg2-1, and nph4-1. (A) IAA-induced growth curvature observed 16 h after unilateral application of lanolin paste containing the indicated concentrations of IAA to hypocotyls. Values shown represent the mean ± sd of at least seven seedlings. (B) Time course of gravitropic reorientation. Seedlings grown on vertically held plates for 3 d in darkness were turned 90° to a horizontal position, and the angle of hypocotyl curvature was measured at the indicated times thereafter; 90° represents complete reorientation upward. Values shown represent the mean ± se of four independent experiments, in which growth curvature of 11 seedlings was measured. (C) Time course of second-positive phototropism. Seedlings grown on vertically held plates for 3 d in darkness were subjected to unilateral blue light (0.1 μmol·m⁻²·s⁻¹), and the angle of hypocotyl curvature was measured. Values shown represent the mean ± se of six or seven independent experiments, in which growth curvature of 13 to 30 seedlings was measured. (D) Hook curvature observed in dark-grown seedlings after induction of germination by incubating at 4°C for 3 d and then at 23°C for 1 d under continuous white light condition. Values shown represent the mean ± se of three independent experiments, in which hook curvature of 18 to 25 seedlings was measured. Open circle, wild type; closed circle, msg2-1; open triangle, nph4-1.

Figure 2.

Effects of 2,4-D on Hypocotyl or Root Growth of the Wild Type, msg2-1, and nph4-1. For measurement of hypocotyl growth (A), the seedlings were grown hydroponically for 5 d in the presence of 2,4-D at 23°C in darkness after germination was induced in the absence of 2,4-D. 2,4-D was added to the medium before germination when root growth (B) was examined. Organ length is expressed relative to the mean organ length of the same genotype in medium without 2,4-D. Each value represents the mean ± se of three independent experiments, in which ∼20 seedlings were used. Open circle, wild type; closed circle, msg2-1; open triangle, nph4-1.

Figure 3.

Morphology of msg2-1 Mutants. (A) and (B) Six-week-old wild-type (A) and msg2-1 (B) plants grown under continuous white light at 23°C. Note the short infertile siliques in (B). Diameter of the pots was 5.5 cm. (C) and (D) Eight-day-old wild-type (C) and msg2-1 (D) plants grown under continuous white light at 23°C on vertically held agar plates. Grid is 0.5 inches wide.

Figure 4.

Time Course of Lateral Root Formation in the Wild Type, msg2-1, and nph4-1, and Its Induction by Exogenous Auxin. After induction of germination, seedlings were grown on vertically held agar plates under continuous white light condition. For examination of auxin-induced lateral root formation, 3-d-old seedlings were transferred to the medium containing 40 nM IAA. Data represent the mean ± se of three independent experiments, in which 8 to 13 seedlings were examined. Open circle, wild type; open triangle, msg2-1; open square, nph4-1; closed symbols, induction by exogenous auxin.

Figure 5.

The IAA19 Protein and msg2 Mutations. Dominant mutations in the conserved domain II of Aux/IAA proteins reported to date are also shown for comparison. Amino acid residues almost perfectly conserved in the 24 domain II–containing Aux/IAA proteins are indicated by asterisks.

Figure 6.

RNA Gel Blot Analysis of Expression of MSG2/IAA19 Gene. (A) and (B) Dose–response curve (A) and time course (B) of auxin induction of MSG2/IAA19 mRNA in 3-d-old etiolated seedlings of the wild type. Seedlings were grown hydroponically at 23°C in the dark and treated with IAA for 1 h in (A) and with 50 μM IAA in (B). (C) Quantitative estimation of MSG2/IAA19 mRNA in 3-d-old etiolated seedlings of the wild type and nph4-1 treated with 50 μM IAA for 1 h. Seedlings were grown hydroponically at 23°C. (D) Tissue specificity of MSG2/IAA19 expression. Etiolated seedlings (E) were grown hydroponically for 3 d. Etiolated hypocotyls (H) were obtained from 3-d-old seedlings grown on agar plates. Roots (R) were obtained from 9-d-old light grown seedlings grown on agar plates. Rosette (L) and cauline leaves (C), inflorescence stems (S), and flowers (F) were prepared from 5-week-old plants grown on soil under continuous light condition. Twenty micrograms of total RNA were electrophoresed in each lane. In (A) to (C), values represent the mean ± sd of three independent experiments. Signals of actin8 mRNA (bottom panel, inset of [A] to [C]) or rRNA bands stained with ethidium bromide (D) were shown as a loading control.

Figure 7.

GUS Staining of Wild-Type Seedlings Transgenic for the IAA19 Promoter:GUS Fusion. Seedlings were grown hydroponically in the dark ([A] and [B]) or under continuous white light condition ([C] and [D]) for 3 d at 23°C. They were then treated with ([B] and [D]) or without ([A] and [C]) 50 μM IAA for 3 h. A lateral root is indicated by arrow in (C). Staining in primary root tips (E) and lateral root primordia (F) after the IAA treatment was also shown. Arrows in (F) point to cell walls indicating anticlinal cell divisions. Bars = 2 mm in (D) (the same magnification from [A] to [D]) and 0.1 mm in (E) and (F).

Figure 8.

RNA Gel Blot Analysis of Expression of Auxin Early Genes IAA4, DFL1, SAUR-AC1, and MSG2/IAA19 in msg2-1 Background. Seedlings were grown hydroponically at 23°C in the dark and treated with 50 μM IAA for 1 h. Twenty micrograms of total RNA were electrophoresed in each lane, and actin8 (ACT8) mRNA bands were shown as a loading control.

Figure 9.

Molecular Interaction between ARF CTDs and Aux/IAA Proteins in a S. cerevisiae Two-Hybrid Assay. SFY526 host cells were transformed with plasmids derived from pGBT9 and pGAD424, and the transformants were then assayed for LacZ activity. Interaction between the fusion of the murine p53 protein and the GAL4 DBD (p53) and the fusion of the SV40 large T-antigen and the GAL4 AD (T-antigen) were measured as a positive control. Effects of the wild-type GAL4 protein were also checked. Data represent the mean ± sd of three to nine independent colonies. (A) Schematic diagram showing the intact and truncated ARF and Aux/IAA proteins used in S. cerevisiae two-hybrid analysis. Shaded boxes indicate CTD. A hatched box indicates the MR of ARF7. The bold numbers indicate the first amino acid residue of CTDs. The other numbers indicate the positions of deletions with regard to each full-length protein. (B) Molecular interaction between four Aux/IAA proteins and CTD of three ARF proteins.

Figure 10.

Pull-Down Assays with His-Tagged MSG2/IAA19 and FLAG-Tagged NPH4/ARF7. Crude extracts of E. coli cells expressing either fusion protein were mixed and immunoprecipitated with immobilized anti-FLAG antibody. Samples eluted with FLAG peptide were subjected to protein gel blot analysis. A plasmid, pBluescript II KS+ containing no inserts (pBS), was used as a negative control.