Auxin Resistance in the Apical Part Is Associated with a Defect in Adventitious Root Formation.
(A) Eight-day-old wild-type and ago1-33 siblings germinated and grown in the light in the presence or absence of 5 μM picloram.
(B) Wild-type and ago1-33 siblings (left) and sur2-3 and ago1-33 sur2-3 siblings (right). Seedlings were germinated and grown in the light for 8 d.
(C) and (D) Three-week-old ago1-3 (C) and wild-type (D) siblings grown in the light on media without or with increasing concentrations of 1-NAA. Arrows indicate the hypocotyl/root junction.
(E) Wild-type Col-0 (left) and sur1-3 (right) seedlings grown for 15 d in vitro.
(F) One-month-old sur1-3 ago1-3 double mutant.

Auxin Stimulates Adventitious Roots on Etiolated ago1 Seedlings but Never as in the Wild Type.
Emergent adventitious roots were scored 7 d after transfer to the light. At least 40 seedlings were used for each data point. This was repeated on three independent biological replicates.
(A) Siblings from a heterozygous ago1-3 mother plant were germinated and grown in the dark for different times, then transferred to the light. The proportion of seedlings forming one or more adventitious roots was determined after 1 week in the light.
(B) Proportion of wild-type and mutant seedlings forming one or more adventitious roots after transfer to the light for 1 week on a medium with or without 1-NAA.
(C) Average number of adventitious roots formed on wild-type or mutant seedlings in the absence or presence of 1-NAA. Error bars indicate se.
(D) Average number of adventitious roots formed on Col-0, sur2, the five ago1 mutants, and the five sur2 ago1 double mutants after etiolation and transfer to the light for 1 week. Error bars indicate se.

Free IAA Content in Col-0, ago1-3, sur2-1(hyb), and ago1-3 sur2-1(hyb) Seedlings.
The endogenous free IAA level was measured in seedlings germinated and grown for 2.5 d in the dark, then transferred to the light for 24, 48, or 72 h. FW, fresh weight.
(A) Entire seedlings. Free IAA content was lower in ago1-3 than in the wild type (P < 0.5). The sur2-1 ago1-3(hyb) double mutant contained significantly more auxin than the wild type (P < 0.01) but less than sur2-1(hyb) (P < 0.01).
(B) Root. The auxin content increased in all the genotypes after transfer to the light. No significant difference between ago1-3 and Col-0 or sur2-1(hyb) and sur2-1 ago1-3(hyb) could be detected.
(C) Apical part (cotyledons plus hypocotyl). The auxin content decreased significantly in ago1-3 seedlings after transfer to the light (P < 0.01) and was significantly lower than that in the wild type after 72 h in the light (P < 0.01). The auxin content increased significantly in sur2-1(hyb) after transfer to the light (P < 0.05) but decreased in sur2-1 ago1-3(hyb) (P < 0.05).
Three biological replicates were used for each data point. Error bars indicate sd. A t test was performed according to http://graphpad.com/quickcalcs/ttest1.cfm.

ago1 Is Hyperresponsive to Light.
(A) Hypocotyl length of the different ago1 mutants grown in various light conditions. Hypocotyl length was measured on wild-type and mutant siblings from the different alleles grown in vitro under different light conditions for 8 d. cW, constant white light (150 μE·m⁻²·s⁻¹); cR, constant red light (9 μE·m⁻²·s⁻¹); cFR, constant far-red light (0.25 μE·m⁻²·s⁻¹); cB, constant blue light (3.7 μE·m⁻²·s⁻¹). Error bars indicate sd.
(B) phyA is epistatic to ago1 in far-red light. Hypocotyl length of Col-0, ago1-3, phyA, phyA ago1-3, phyA ago1-33, and phyA ago1-35, 8 d after germination either in the dark or in cFR. Error bars indicate sd.

The Auxin Response Factor ARF17 Represses GH3 Genes and Adventitious Root Formation.
(A) Total RNAs were extracted from hypocotyls of Col-0 and ago1-3 siblings etiolated in the dark for 2.5 d and then transferred for 48 h into the light. The indicated mRNAs were quantified by real-time quantitative PCR using primers surrounding putative miRNA cleavage sites. ATHB2 and ARF7 were used as controls not targeted by miRNAs. Expression for each gene was normalized to that of ACTIN2. Error bars indicate se of two independent biological replicates.
(B) Scheme of the ARF17 OX T-DNA insertion line SALK 062511. Black boxes represent CDS, lines represent introns, untranslated regions, or promoters. The positions of the inverted repeat T-DNA insertion and left border primers (Lba1) are indicated. Approximate positions of the genomic primers used in genotyping are indicated with arrows below the transcript.
(C) Relative abundance of ARF17 transcript in ARF17 OX and Col-8 hypocotyls etiolated for 2.5 d and exposed to light for 2 d. Expression for the gene was normalized to that of ACTIN2. Quantification was made by real-time quantitative PCR using primers surrounding the putative miRNA cleavage site. Error bars indicate se of two independent biological replicates.
(D) Relative abundance of GH3-3, GH3-5, and GH3-6 transcripts in ARF17 OX and Col-8 hypocotyls etiolated for 2.5 d and exposed to light for 2 d. Quantification was performed using semiquantitative RT-PCR as described in Methods. Expression for the genes was normalized to that of 18S rRNA. Error bars indicate se of three independent RT-PCR replicates (P < 0.01). These experiments were repeated on two independent biological replicates.
(E) Proportion of wild-type and ARF17 OX siblings developing one or more adventitious roots after etiolation and transfer to the light for 1 week.
(F) Average number of adventitious roots formed on ARF17 OX and Col-8 after etiolation and transfer to the light for 1 week. Error bars indicate se (P < 0.01; n > 30). Observations were done on three independent biological replicates.

The Apical Part but Not the Root of ago1 Mutants Is Resistant to Auxin.
(A) Hypocotyl length of wild-type and ago1-3 siblings grown in vitro on increasing concentrations of picloram, 8 d after germination in the light.
(B) Hypocotyl length of wild-type and different ago1 mutants grown in vitro in the presence or absence of 5 μM picloram, 8 d after germination in the light.
(C) Root length of wild-type and ago1-3 siblings grown on increasing concentrations of picloram, 8 d after germination in the light.
(D) and (E) Root (D) and hypocotyl (E) length of Col-0 seedlings and siblings from a plant homozygous for the sur2-3 mutation and heterozygous for the different weak alleles of ago1, 8 d after germination in the light.
(F) Hypocotyl length of wild-type and ago1-3 siblings grown in vitro in the dark. The hypocotyl was measured at different time points.
Error bars indicate sd.

Adventitious Root Initiation, but Not Lateral Root Initiation, Is Affected in ago1-3.
(A) to (D) Siblings from a heterozygous ago1-3 plant expressing the CycB1:uidA marker gene were germinated and grown in the light for 5 d on a medium without auxin and then transferred to a medium containing 1 μM NAA for 6 d.
(A) and (B) Wild type.
(C) and (D) ago1-3.
(E) Siblings from a heterozygous ago1-3 parent plant were germinated and grown in the dark for 2.5 d before transfer to the light.
(F) Col-0 could make up to four adventitious roots after etiolation and transfer to the light for 1 week on a medium containing 1 μM 1-NAA. Arrows indicate adventitious roots.
(G) ago1-3 siblings could very rarely develop up to three adventitious roots in the same conditions as in (F). Arrows indicate adventitious roots.
(H) and (I) sur2-3 and sur2-3 ago1 etiolated siblings after 1 week in the light. ago1-32 is the intermediate allele mutant (H), and ago1-33 is one of the weakest alleles (I).

sur2 ago1 Double Mutants Initiate Adventitious Roots in the Dark.
(A) and (B) CycB1:uidA expression in etiolated siblings of sur2-1 and sur2-1 ago1-3(hyb), 2.5 d after germination in the dark (A) and after transfer to the light for 3 d (B). Arrows indicate GUS staining in the sur2-1 ago1-3(hyb) hypocotyl. Stars indicate the junction between the hypocotyl and the root.
(C) CycB1:uidA expression in etiolated siblings of sur2-1 and sur2-1 ago1-3(hyb) was monitored in seedlings germinated and grown in the dark for 2.5 d and transferred to light for 1 to 7 d. This allowed identification and scoring of the initiation of very early primordia as well as older emerging roots. Error bars indicate se.

IAA Biosynthesis Rate and Level of IAA Metabolites in Col-0, ago1-3, sur2-1(hyb), and ago1-3 sur2-1(hyb) Seedlings.
(A) IAA biosynthesis rate in the apical part of seedlings (hypocotyl plus cotyledons), 72 h after transfer to the light. Four replicates were used for each data point. Error bars indicate sd.
(B) to (E) Quantification of IAA metabolites. Measurements were performed on entire seedlings grown for 2.5 d in the dark for oxoindole-3-acetic acid (B), N-(indole-3-acetyl)-Asp (C), N-(indole-3-acetyl)-Ala (D), and N-(indole-3-acetyl)-Glu (E). Four replicates were used for each data point. Error bars indicate sd. FW, fresh weight.

Expression of the GH3:uidA Reporter Gene in ago1-3 Seedlings.
(A) and (B) GH3:uidA expression in wild-type (A) and ago1-3 (B) seedlings geminated and grown in the light for 8 d in the absence or presence of 1 μM 1-NAA. Bars = 2 mm.
(C) and (D) GH3:uidA expression in wild-type (C) and ago1-3 (D) seedlings that were etiolated for 2.5 d in the dark before transfer to the light for 44 h. Seedlings were then transferred to liquid culture medium without or with 10 μM 1-NAA for 4 h in the same growth conditions. GUS staining was overnight. From left to right in each panel: three nontreated seedling and three seedlings treated with NAA. Bars = 6 mm.