The Arabidopsis transcription factor MYB77 modulates auxin signal transduction

Abstract

Auxin is a key plant hormone that regulates plant development, apical dominance, and growth-related tropisms, such as phototropism and gravitropism. In this study, we report a new Arabidopsis thaliana transcription factor, MYB77, that is involved in auxin response. In MYB77 knockout plants, we found that auxin-responsive gene expression was greatly attenuated. Lateral root density in the MYB77 knockout was lower than the wild type at low concentrations of indole-3-acetic acid (IAA) and also under low nutrient conditions. MYB77 interacts with auxin response factors (ARFs) in vitro through the C terminus (domains III and IV) of ARFs and the activation domain of MYB77. A synergistic genetic interaction was demonstrated between MYB77 and ARF7 that resulted in a strong reduction in lateral root numbers. Experiments with protoplasts confirmed that the coexpression of MYB77 and an ARF C terminus enhance reporter gene expression. R2R3 MYB transcription factors have not been previously implicated in regulating the expression of auxin-inducible genes. Also it was previously unknown that ARFs interact with proteins other than those in the Aux/IAA family via conserved domains. The interaction between MYB77 and ARFs defines a new type of combinatorial transcriptional control in plants. This newly defined transcription factor interaction is part of the plant cells' repertoire for modulating response to auxin, thereby controlling lateral root growth and development under changing environmental conditions.

Figure 1.

Arabidopsis MYB77-ox Plants Exhibit a Phenotype Similar to Wild-Type Plants Grown with Auxin. (A) MYB77-ox lines grown in soil develop poorly and appear stunted compared with wild-type Columbia (Col-0) or the knockout myb77-1. (B) Col-0, MYB77-ox, and the myb77-1 knockout plants on medium without additional IAA and Col-0 plants on medium containing 1 μM IAA.

Figure 2.

MYB77 Is Expressed in Lateral and Primary Roots. ProMYB77:GUS activity in wild-type roots is localized to lateral roots throughout development ([A] to [G]) and is also expressed in the primary root tip (H). Bars = 1 mm.

Figure 3.

MYB77 Alters Auxin Response and Lateral Root Growth in the Presence of Auxin. The intensity of expression and localization of the DR5:GUS reporter gene construct in Arabidopsis plants is altered by MYB77 expression. (A) The wild type expressing DR5:GUS. (B) MYB77-ox line expressing DR5:GUS. (C) myb77-1 knockout line expressing DR5:GUS. (D) Free IAA content in 5-d-old seedlings. Values are mean ± se from 300 seedlings per replicate (n = 3 replicates). (E) The number of lateral roots and the length of primary roots were measured under control conditions and with various concentrations of IAA on low salt medium in the wild type and in myb77-1 lines (n = 25 to 35 ± se). Mean lateral root density (number of lateral roots·cm⁻¹ of primary root) was calculated.

Figure 4.

Lateral Root Density as Affected by Nutrient Deprivation. (A) Mean lateral root density decreased significantly more in the myb77-1 and myb77-2 knockout than the wild type under potassium-deprived conditions but not under phosphorus and nitrogen deprivation. (B) Lateral root density decreased more significantly in myb77-1 and tir1-1 than in the wild type under potassium deprivation. Full nutrients (+NPK), with low (10 μM) K⁺ (−K), low (12 μM) phosphorus (−P), and low (100 μM) nitrogen (−N) (n = 50 plants ± se). Bars that have different letters at the top are significantly different (P < 0.05; Tukey's honestly significant difference).

Figure 5.

The Expression of Auxin-Regulated Genes Is Attenuated in the myb77-1 Knockout and Increased in MYB77-ox Lines. RNA gel blots of Arabidopsis wild type, knockout myb77-1, and MYB77-ox-1, -2, and -3 lines grown for 7 d and treated with 10 μM IAA for 0, 0.5, 1, 2, 6, and 24 h. The MYB77-ox lines were grown without IAA treatment. rRNA loading control is shown at the bottom.

Figure 6.

MYB77 Interacts with ARF Proteins in Vitro. (A) The 500-bp region of IAA19 that is upstream of the translational start is shown. AuxRE, MRE, and MYBcore elements are underlined. (B) Diagram showing the main structural features of the ARFs and IAA19. Regions of ARF7 were deleted to determine where ARF7 and MYB77 may interact, including ARF7-N, in which the DNA binding domain is present (truncated after amino acid 380), ARF7-AC containing both the activation domain and C-terminal domain (amino acids 1 to 379 were truncated), and ARF7-C (truncated from amino acids 1 to 956), in which both the DNA binding domain and the AC or the activation region have been deleted (C-terminal domain only). (C) In vitro pull-down assay using MYB77:His-purified protein and in vitro–translated targets, including ARF7, ARF1, IAA19, and truncated versions of ARF7. HIS:MYB66 and HIS:MYB123 proteins were used to test for specificity between MYBs and ARF7.

Figure 7.

The MYB77 Interacts with ARF7 in Protoplasts. (A) BiFC assay for MYB77 and ARF7, and ARF7 and IAA19 interaction in Arabidopsis roots cells and onion cells. YFP signal (green) indicates the interaction between proteins, and Hoescht signal (red) indicates the nucleus. YFP signal merged with Hoescht (yellow) shows localization in the nucleus. (B) Diagram of constructs used for protoplast transfections. (C) Wild-type Arabidopsis protoplast transfection assays with transfected 2XD0 reporter and Pro35S:MYB77 effector genes. NAA, 1-naphthalene acetic acid. (D) Arabidopsis protoplasts, isolated from plants containing a stably integrated Gal4-D1-3:GUS reporter gene, were transfected with 35S effector genes encoding truncated versions of ARF proteins fused to a yeast Gal4 DBD (GD) and MYB77. GD-1RC contains the repression domain and C terminus from ARF1, GD-5AC contains the activation domain and the C terminus from ARF5, GD-5A contains the activation domain from ARF5 only. ** indicates a significant difference from control at P < 0.01 and * a difference at P < 0.05 that were determined using a t test.

Figure 8.

ARF7 Interacts with the C-Terminal Domain of MYB77. (A) Full-length and deletion constructs of MYB77 are shown. R2R3 domain of MYB77 (R2R3; amino acids 1 to 110) and N-terminal deletion of MYB77 (ΔN; amino acids 111 to 301). (B) In vitro coimmunoprecipitation of ARF7 and MYB77. The [³⁵S]Met-labeled in vitro–translated proteins of HA:ARF7, MYB77, R2R3, and ΔN were incubated and then analyzed on an SDS-PAGE gel.

Figure 9.

Lateral Root Density of myb77-1, arf7/nph4-1, and the Double myb77-1 arf7-nph4-1 Mutant. Mean lateral root density was significantly lower in the double knockout plants of myb77-1 and arf7/nph4-1 than the wild type, myb77-1, and nph4-1 single knockout (n = 66 to 70 plants ± se). Bars that have different letters at the top are significantly different (P < 0.05; Tukey's honestly significant difference).