The AFB4 auxin receptor is a negative regulator of auxin signaling in seedlings

Abstract

The plant hormone auxin is perceived by a family of F box proteins called the TIR1/auxin-signaling F box proteins (AFBs). Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling. In this report, we demonstrate a unique role for the AFB4 clade. Both AFB4 and AFB5 function as auxin receptors based on in vitro assays. However, unlike other members of the family, loss of AFB4 results in a range of growth defects that are consistent with auxin hypersensitivity, including increased hypocotyl and petiole elongation and increased numbers of lateral roots. Indeed, qRT-PCR experiments show that afb4-2 is hypersensitive to indole-3-acetic acid (IAA) in the hypocotyl, indicating that AFB4 is a negative regulator of auxin response. Furthermore, we show that AFB4 has a particularly important role in the response of seedlings to elevated temperature. Finally, we provide evidence that the AFB4 clade is the major target of the picloram family of auxinic herbicides. These results reveal a previously unknown aspect of auxin receptor function.

Figure 1. AFB4 and AFB5 Interact with ASK1 and Interact with the Aux/IAAs in an Auxin-Dependent Manner Revealing their Role as Auxin Receptors Pull-down experiments were carried out using crude plant extracts prepared from [tir1-1] GVG-TIR1-myc, [afb5-5] AFB5-AFB5-myc, and [afb5-5] AFB5-AFB4-myc seedlings and recombinant GST-IAA3

(A) TIR1-myc, AFB4-myc, and AFB5-myc were immunoprecipitated with anti-myc antibody coupled to agarose beads, and ASK1 was detected with an anti-ASK1 antibody. The lower band corresponds to ASK1 protein. (B) Pull-down reactions were incubated for 45 min in the presence or absence of 50 μM indole-3-acetic acid (IAA). GST-IAA3 was immunoprecipitated with glutathione agarose beads, and AFB4-myc and AFB5-myc protein were detected with anti-c-myc-peroxidase antibody.

Figure 2. AFB4 and AFB5 Are Required for the Picloram Response

(A and B) Five-day-old wild-type (WT) and tir1/afb mutant seedlings grown on Murashige-Skoog medium were transferred to media containing 0, 1, 5, or 10 μM picloram (pic, A) or 50 nM 2,4-dichlorophenoxyacetic acid (2,4-D) or IAA (B) for an additional 3 days. (C) Four-day-old WT and tir1/afb seedlings were transferred to 5 μM picloram for an additional 2 days. In (A)–(C), root and hypocotyl length were expressed as a percent elongation based on no-auxin control growth, and error bars represent standard error. (D and E) Pull-down reactions were carried out as in Figure 1 with 50 μM of the indicated auxin. See also Figure S1.

Figure 3. The afb4-2 Mutant Shows Stronger Auxin-Related Phenotypes Than afb5-5 and Has Much Lower Expression Levels

(A and B) Petiole (A) and hypocotyl (B) lengths of 6-day-old WT and tir1/afb mutant seedlings. (C) Lateral root number divided by primary root length (mm) in 10-day-old WT and tir1/afb mutant seedlings grown under long-day (LD) photoperiods (16 hr light:8 hr dark). Measurement values are shown in Figure S2F. (D) Images of 7-day-old WT and tir1/afb mutant seedlings grown under LD photoperiods. Arrows point to lateral roots emerging from root-shoot junction. (E) Hypocotyl lengths of 6-day-old WT and mutant seedlings grown at 22°C. (F) qRT-PCR of TIR1, AFB4, and AFB5 in hypocotyl tissue from 4-day-old WT seedlings grown under short-day (SD) conditions. Error bars represent standard error. *p < 0.05 versus WT, **p < 0.05 versus WT and other single and double mutants by Student's t test. See also Figure S2.

Figure 4. The afb4-2 Mutant Is Hypersensitive to Endogenous IAA

(A) IAA measurements from 4-day-old dissected hypocotyls and cotyledon + hypocotyls combined. Error bars represent standard deviation. (B) qRT-PCR of IAA marker genes in hypocotyl tissue from 4-day-old WT and afb4-2 mutant seedlings following 2 hr treatment with 1 μM IAA. Expression values are normalized to the PP2AA3 reference gene [20]. (C) IAA dose-response curve for WT and afb4-2. Seedlings grown for 4 days under SD photoperiod were transferred to the indicated IAA concentration for 3 days. (D) Four-day-old WT and mutant seedlings were transferred to 29°C for 2 days. Control plates were maintained at 22°C. Hypocotyl length is expressed as a percent elongation based on the control plates. Error bars represent standard error. See also Figure S3.