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. 1998 Dec 8;95(25):15112-7.
doi: 10.1073/pnas.95.25.15112.

The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

R Chen  1 , P Hilson, J Sedbrook, E Rosen, T Caspar, P H Masson
Collaborators, Affiliations

The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

R Chen et al. Proc Natl Acad Sci U S A. .

Abstract

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

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Figures

Figure 1
Figure 1
Physiological analysis of agr1–5 mutant seedlings. Effects of 1-NAA (a), ACC (b), and TIBA (c) on wild-type and agr1–5 mutant root growth rates. The average root growth rate of wild-type Estland seedlings in media containing no supplements (12.3 mm for a and c, and 9.3 mm for b) was plotted as 100%. Each data point represents the average of the relative root growth rate (RRGR) for 6–16 wild-type Estland seedlings and for 10–12 agr1–5 mutant seedlings. (d) Relative retention (RR) of 3H-IAA by wild-type and agr1–5 mutant root tips. The average 3H-IAA retention at time 0 by wild-type and agr1–5 root tips (4,465 and 3,912 cpm, respectively) was plotted as 100%. Each data point represents the average of three replicates. In a–d, the SDs are represented by vertical bars. See Materials and Methods for experimental procedures.
Figure 2
Figure 2
Genetic and physical maps of the AGR1 region of chromosome 5. (a) Genetic map of the nga129 and LFY3 region of chromosome 5, showing the relative genetic positions of the nga129, m233, m558, and LFY3 markers, and the positions of the Ds 389–14 and Ds 392–13 insertions. (b) Mapping of AGR1 on a YAC contig of cloned genomic DNA fragments, showing the relative positions of specific RFLP markers (drawn as vertical shaded bars) and positions of individual YAC clones (drawn as horizontal black bars). (c) Fine mapping of AGR1 on a contig of cosmid (COS3A and COS46A) and lambda (L1, M1, N1, and R5) genomic clones, showing the number of recombinants identified in the mapping populations (see Materials and Methods) with recombination breakpoints between AGR1 and individual RFLP markers. Scales are in recombination units for a or kilobase pairs of DNA for b and c. (d) Wavy root-growth phenotype of segregating F2 plants (Right) derived from an agr1–4 plant (Left) transformed with the COS46A clone. Wild-type untransformed control plants are also shown (Center). Mutant seedlings are indicated by an asterisk.
Figure 3
Figure 3
The Arabidopsis AGR1 gene. (a) The structure of AGR1. The exons are shown in solid boxes. The initiation (ATG) and stop (TAA) codons are also shown, with their positions indicated above the nucleotide sequence. The first nucleotide of the initiation codon was arbitrarily chosen as 1. (b) Nature of the mutations found in the agr1–4, agr1–5, and agr1–6 alleles. Exon and intron sequences are shown in upper- and lowercase letters, respectively. The exon derived from the alternative splicing of the agr1–4 transcript is underlined. Deleted nucleotides are represented by dashes. Numbers in parentheses represent nucleotides omitted because of space limitation. (c) The amino acid sequence of the AGR1 protein. Predicted transmembrane domains are boxed (22). (d) Kyte–Doollitle hydropathy plot of the AGR1 protein sequence. (e) Alignment of the amino acid sequences of AGR1 and Methanococcus MdcF (24). The AGR1 (top) and the MdcF (bottom) sequences were aligned using the nih gapped blast search program (23). Identical and functionally conserved amino acids are shown in solid and shaded boxes, respectively. Sequence gaps are represented by dashed lines.
Figure 4
Figure 4
AGR1 mediates auxin transport in S. cerevisiae. (a) Relative retention of 3H-IAA by yeast cells transformed with pAGR1 (dotted bar) or with pANTI-AGR1 (open bar), after 0 and 60 min in the wash solution (see Materials and Methods). Total number of counts at time 0 (470 cpm for W3031A[pAGR1] and 752 cpm for W3031A[pANTI-AGR1]) are plotted as 100%. Each data point represents an average of two measurements. The vertical bars represent the range of the measurements. (b) Relative growth inhibition by 1 mM F-IAA of yeast cells transformed with pAGR1 (dotted bar) or with pANTI-AGR1 (open bar) after 0, 3, 7, 10, and 13 hr of treatment (see Materials and Methods). Each point represents an average of three independent measurements. SDs are shown by vertical bars.
Figure 5
Figure 5
The pattern of AGR1 gene expression. (a) Northern blot analysis of total RNA extracted from tissues of 7-day-old seedlings (lanes 1 and 2) or 24-day-old (lane 3 to 8) or 5-day-old etiolated (lanes 9 and 10) plants. Tissues from roots (lanes 1, 3, and 9), hypocotyls and cotyledons (lanes 2 and 10), rosette leaves (lane 4), cauline leaves (lane 5), inflorescence stems (lane 6), flowers (lane 7), and siliques (lane 8) were analyzed. A 900-bp cDNA corresponding to the 3′ end of AGR1 was used as the probe (see Materials and Methods). After exposure, the blots were stripped and rehybridized with an eIF4A probe to control for loading differences. (b) Whole-mount in situ hybridization analysis of 3-day-old wild-type root tips, using digoxygenin-labeled antisense (Left) and sense (Right) RNA probes derived from in vitro transcription of the 900-bp-long AGR1 cDNA template (see Materials and Methods). Photos were taken under a dark-field microscope. (Bar = 150 μm.)
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