A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis

Abstract

In Arabidopsis thaliana, tryptophan pathway genes are induced in response to starvation, wounding, and pathogen attack, resulting in increased production of tryptophan and secondary metabolites important for development and defense. The Arabidopsis tryptophan pathway therefore provides an ideal system for elucidating how environmental stimuli are transduced into changes in plant gene expression. To characterize the factors that regulate the first gene in the pathway, ASA1, which is the key point of control, we have isolated altered tryptophan regulation (atr) mutants with deregulated expression of ASA1. One of these mutants, atr1D is dominant for increased transcription of ASA1 in specific seedling tissues. We have used atr1D to clone the ATR1 gene based on its map position. ATR1 encodes a Myb-like transcription factor that modulates ASA1 expression. The ATR1 transcript also includes a 5' regulatory region with three short ORFs, one of which is prematurely terminated by the atr1D mutation. Thus, ATR1 defines the first characterized tryptophan gene regulator in plants, and the atr1D mutation defines a sequence important for ATR1 expression.

Figure 1

atr1D regulatory phenotypes. (A) The trp1–100 atr1D double mutant is fluorescent on high-tryptophan medium. One-week-old seedlings of wild-type Col (TRP1/TRP1 ATR1/ATR1), Col trp1–100 gl1–1 (trp1/trp1 ATR1/ATR1), Col trp1–100 gl1–1 atr1D homozygotes (trp1/trp1 atr1D/atr1D), and Col trp1–100 gl1–1 atr1D/ATR1 F1 heterozygotes made by crossing Col trp1–100 gl1–1 atr1D with Col trp1–100 gl1–1 ATR1D (trp1/trp1 atr1D/ATR1) are shown under UV light grown on PNS medium containing 100 μM tryptophan. (B) The atr1D mutation confers dominant 5MT resistance. Seedlings of wild-type Col (ATR1/ATR1), a Col atr1D homozygote (atr1D/atr1D) made by crossing the Col trp1–100 atr1D isolate to Col and identifying homozygous 5MT-resistant plants, and a Col atr1D/ATR1 F1 heterozygote made by crossing Col atr1D with wild-type Col are shown after being grown on PNS medium containing 15 μM 5MT under yellow long-pass-filtered light for 10 days. (C) The atr1D mutation activates ASA1 expression in hypocotyls and lateral root junctions. Representative 2-week-old F1 seedlings of Arabidopsis made by crossing a transgenic No-O strain carrying the ASA1-GUS reporter fusion with either wild-type Col (ASA1-GUS/o ATR1/ATR1) or Col atr1D (ASA1-GUS/o atr1D/ATR1) are shown after growth on unsupplemented PNS medium followed by staining for GUS activity. Arrows indicate tissues with increased GUS expression in the atr1D mutant relative to the ATR1 control.

Figure 2

Cloning of the atr1D mutation. (A) Genetic analysis of F2 progeny from a three-factor cross between Col CER3 atr1D YI and Ler cer3 ATR1 yi localizes ATR1 between the visible markers. (B) A YAC clone contig spanning from the LFY gene northward over the ATR1 locus was constructed by hybridization and mapping analysis of end clones from LFY-containing YACs. Vertical lines represent end clones used for hybridization or detection of polymorphisms between Col and Ler. (C) λ clones spanning the ATR1 region between the left end of YAC CIC10A10 and the left end of YAC yUP17C2 were isolated from an atr1D genomic library. Genes within the ATR1 region were identified by sequence analysis and comparison with the GenBank database. Arrows indicate the direction of transcription. B, BamHI; S, SalI.

Figure 3

ATR1 transcript levels are elevated in the atr1D mutant. Total RNA was prepared from 2-week-old seedlings of wild-type Col (ATR1) or Col atr1D (atr1D) grown on unsupplemented PNS medium. Duplicate Northern blots of these samples were probed with an ATR1 cDNA probe (ATR1, 1-week exposure), an ASA1 cDNA probe (ASA1, 1-week exposure), or an RLK1 probe (RLK1, 1-week exposure). The blots were then stripped and reprobed with an α tubulin probe (TUB, 24-hr exposure) as a normalization control. The ATR1 blot reprobed with TUB is shown here. The quantitated ratio of transcripts detected in atr1D relative to ATR1 after normalization to TUB is given as the average of three independent experiments.

Figure 4

ATR1 is a Myb-like transcription factor with three ORFs in its 5′ leader region. (A) Alignments of the predicted ATR1 amino acid sequence with Myb transcription factor homologues from Arabidopsis and maize. Sequences of ATR1, C1 (20), P (21), and GL1 (22) were aligned manually. Residues shared by at least three sequences are boxed. Positions of the two ATR1 introns are indicated by arrows. (B) The 5′ regulatory leader region of the ATR1 transcript. The nucleic acid sequence upstream of the ATR1 start codon in the longest ATR1 cDNA isolate is shown. All of 14 other cDNAs analyzed that include the entire ATR1 ORF ended within this sequence at least 15 bp 5′ of the first upstream ATG. Three out-of-frame upstream start codons are underlined, and the conserved Myb start codon is underlined and in bold. Predicted protein sequences encoded by each ORF are shown below the nucleic acid sequence. A 4-bp polymorphism present in the Col 5′ leader but absent in the Ler 5′ leader is shown in lowercase. A second 9-bp-length polymorphism internal to the coding sequence deletes from Ler the residues encoded at positions 134–136 (QTG) in Col.