ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis

Abstract

Lateral root formation in Arabidopsis thaliana is regulated by two related AUXIN RESPONSE FACTORs, ARF7 and ARF19, which are transcriptional activators of early auxin response genes. The arf7 arf19 double knockout mutant is severely impaired in lateral root formation. Target-gene analysis in arf7 arf19 transgenic plants harboring inducible forms of ARF7 and ARF19 revealed that ARF7 and ARF19 directly regulate the auxin-mediated transcription of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18 (LBD16/ASL18) and/or LBD29/ASL16 in roots. Overexpression of LBD16/ASL18 and LBD29/ASL16 induces lateral root formation in the absence of ARF7 and ARF19. These LBD/ASL proteins are localized in the nucleus, and dominant repression of LBD16/ASL18 activity inhibits lateral root formation and auxin-mediated gene expression, strongly suggesting that these LBD/ASLs function downstream of ARF7- and ARF19-dependent auxin signaling in lateral root formation. Our results reveal that ARFs regulate lateral root formation via direct activation of LBD/ASLs in Arabidopsis.

Figure 1.

Auxin-Mediated Lateral Root Initiation Is Severely Impaired in the arf7 arf19 Double Mutant. (A) Numbers of lateral root initiation sites in wild-type and arf7 arf19 roots. Lateral root primordia with End199 activity were counted. More than 19 samples from 4- to 7-d-old seedlings grown on agar plates were examined for each genotype. Each dot indicates the length of the primary root versus the number of initiation sites with End199 activity. (B) to (E) Expression of the Pro_CycB1;1:CycB1;1(NT)-GUS reporter in roots of wild-type ([B] and [C]) and arf7 arf19 ([D] and [E]) seedlings germinated on 10 μM naphthylphthalamic acid (NPA) and transferred 72 h after germination to 10 μM NAA for 12 h. GUS activity in the root tip was detected in all samples. (F) and (G) Nuclear localization of ARF7-GFP and ARF19-GFP fusion proteins. Cells were counterstained with propidium iodide. (F) Fluorescence in the root epidermal cells of transgenic arf7 arf19 plants expressing ARF7-GFP under the control of the cauliflower mosaic virus (CaMV) 35S promoter. (G) Fluorescence in the root epidermal cells of transgenic arf7 arf19 plants expressing ARF19-GFP under the control of the ARF19 promoter.

Figure 2.

Expression Profiles of Auxin-Responsive LBD/ASL Genes. (A) Phylogenetic analysis of 10 Arabidopsis LBD/ASLs and rice (Oryza sativa) CRL1/ARL1 based on the LOB/AS2 domain. An unrooted dendrogram was obtained using the neighbor-joining method. Bootstrap values (n = 1000) are indicated at the nodes of the tree. (B) to (J) Analysis of LBD promoter activity in roots of 6-d-old transgenic seedlings. Seedlings were treated with (+NAA) or without (control) 1 μM NAA for 2.5 h before GUS staining (incubation time, 90 min). Bars = 100 μm. (B) to (D) GUS expression patterns in early lateral root primordia and/or root vasculature of Pro_LBD16:GUS (B), Pro_LBD29:GUS (C), and Pro_LBD33:GUS (D). (E) to (J) Auxin-inducible expression of Pro_LBD16:GUS ([E] and [F]), Pro_LBD29:GUS ([G] and [H]), and Pro_LBD33:GUS ([I] and [J]). (K) Semiquantitative RT-PCR analysis of IAA5, LBD16, LBD29, LBD33, and NAC1. Total RNA was extracted from 7-d-old wild-type and arf7 arf19 seedlings treated with or without 1 μM NAA or 10 μM CHX for 2.5 h. Transcripts were amplified by 28 cycles of PCR with gene-specific primers. The expression of ACT8 was used as a control. (L) Kinetics of mRNA accumulation in response to exogenous auxin. Wild-type Arabidopsis seedlings (6-d-old) were treated with 1 μM NAA for the indicated durations. Total RNA was assayed by real-time RT-PCR for the accumulation of IAA5, LBD16, LBD29, and LBD33 relative to an internal ACT8 control. Data are presented as means ± sd from three independent amplification reactions. Note that different scales are used in the graphs.

Figure 3.

LBD16 and LBD29 Are Potential Direct Targets of ARF7. (A) Phenotype of Pro_ARF7:ARF7-GR/arf7 arf19 plants. DEX treatment rescues the phenotype of the arf7 arf19 double mutant in lateral root formation. (B) Effects of chemicals on the expression of LBD genes in the roots of Pro_ARF7:ARF7-GR/arf7 arf19 plants. The 7-d-old seedlings were transferred to the medium with or without the indicated chemicals (1 μM NAA, 2 μM DEX, and 10 μM CHX), and roots were harvested for expression analysis after 4 h of treatment. The transcripts were analyzed by real-time PCR. The levels of LBD16, LBD29, and LBD33 expression were normalized to ACT8 and compared with the control condition. Data shown are averages of three biological replicates, with error bars representing sd. Note that different scales are used in the graphs. (C) Electrophoretic mobility shift assays with recombinant ARF7 proteins. ³²P-labeled LBD16 and LBD29 promoter fragments containing AuxRE sequence were used as probes. Arrows indicate the ARF7/DNA complex.

Figure 4.

Overexpression of LBD16 and LBD29 Partially Suppresses the Lateral Root Phenotype of arf7 arf19. (A) Twelve-day-old wild-type, arf7 arf19, Pro_35S:LBD16/arf7 arf19, and Pro_35S:LBD29-GFP/arf7 arf19 seedlings. Numbers of lateral roots (means ± sd) for each genotype are shown (n > 14). (B) and (C) Auxin sensitivity of wild-type, arf7 arf19, and Pro_35S:LBD16/arf7 arf19 roots. Four-day-old seedlings were transferred onto plates with (gray bars) or without (white bars) 1 μM NAA, and numbers of lateral roots (B) and root length (C) were determined after 3 d of vertical growth (n > 20). Bars and error bars represent means + sd.

Figure 5.

Nuclear Localization of LBD-GFP Fusion Proteins, and Dominant Repression Phenotype of LBD16. (A) and (B) Nuclear localization of LBD16-GFP and LBD29-GFP fusion proteins. Longitudinal confocal images of Pro_35S:LBD16-GFP/arf7 arf19 (A) and Pro_35S:LBD29-GFP/arf7 arf19 (B) transgenic Arabidopsis roots are shown. (C) Twelve-day-old wild-type, arf7 arf19, and two independent lines of Pro_35S:LBD16-SRDX (lines 5 and 6) seedlings. Numbers of lateral roots (means ± sd) for each genotype are shown (n > 16). (D) and (E) Closer views of aerial parts of wild-type (D) and Pro_35S:LBD16-SRDX ([E]; line 6) seedlings. (F) Expression profiles of auxin-responsive genes in wild-type, arf7 arf19, and Pro_35S:LBD16-SRDX (line 6) seedlings. Six-day-old seedlings of the indicated genotypes were transferred to the medium with or without 1 μM NAA, and whole seedlings were harvested for real-time PCR analysis after 4 h of treatment. The levels of LBD29, LBD33, and IAA5 expression were normalized to ACT8 and compared with wild-type control levels. Data are presented as means ± sd from three independent amplification reactions.

Figure 6.

Model of the ARF7- and ARF19-Dependent Auxin Signaling Cascade for Lateral Root Formation. Auxin accelerates the degradation of Aux/IAA proteins such as IAA14/SLR, IAA3/SHY2, IAA19/MSG2, and IAA28, thereby derepressing ARF7 and ARF19 function as transcriptional activators. ARF7 and ARF19 activate the transcription of several LBD/ASLs, including LBD16/ASL18 and LBD29/ASL16. LBD16/ASL18 also may activate the downstream transcriptional network for lateral root (LR) initiation as a transcriptional regulator.