The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis

Abstract

Plants derive a number of important secondary metabolites from the amino acid tryptophan (Trp), including the growth regulator indole-3-acetic acid (IAA) and defense compounds against pathogens and herbivores. In previous work, we found that a dominant overexpression allele of the Arabidopsis (Arabidopsis thaliana) Myb transcription factor ATR1, atr1D, activates expression of a Trp synthesis gene as well as the Trp-metabolizing genes CYP79B2, CYP79B3, and CYP83B1, which encode enzymes implicated in production of IAA and indolic glucosinolate (IG) antiherbivore compounds. Here, we show that ATR1 overexpression confers elevated levels of IAA and IGs. In addition, we show that an atr1 loss-of-function mutation impairs expression of IG synthesis genes and confers reduced IG levels. Furthermore, the atr1-defective mutation suppresses Trp gene dysregulation in a cyp83B1 mutant background. Together, this work implicates ATR1 as a key homeostatic regulator of Trp metabolism and suggests that ATR1 can be manipulated to coordinately control the suite of enzymes that synthesize IGs.

Figure 1.

Trp metabolism in Arabidopsis. The pathway for Trp biosynthesis and its subsequent conversion into IAA or IGs via IAOx is shown, with key enzymatic steps discussed in the text as well as the chemical structures for Trp and selected Trp metabolites. Trp feedback inhibition of AS activity is indicated by a line with a perpendicular bar at ASA1. The dashed arrow indicates that there are different possible routes proposed from IAOx to IAA (Ljung et al., 2002).

Figure 2.

Effects of ATR1 overexpression or loss-of-function on seedling Trp gene expression and 5MT resistance. A, Seedling RNA prepared from the indicated strains grown on unsupplemented PNS agar medium was used in RNA gel-blot analysis with the indicated probes. B, Seedlings of the indicated strains were tested for resistance on the indicated concentrations of 5MT, with a representative individual for each strain shown. Both Col and Ws controls are included because atr1-2 is in the Ws background, whereas atr1D and 35S-ATR1 are in the Col background.

Figure 3.

ATR1 overexpression results in elevated IG accumulation mediated by CYP79B2 and CYP79B3. Total desulfoglucosinolates from 10-d-old seedlings or 3-week-old rosette leaves were analyzed by HPLC as described in “Materials and Methods.” For these two growth conditions, indolyl-3-methyl desulfoglucosinolate levels were normalized to a similarly grown Col control. Relative IG levels are shown as the mean ± se from three independent samples. For the atr1D cyp79B2 cyp79B3 triple mutant and the cyp79B2 cyp79B3 double mutant, IG levels were below detection, indicating that in these strains IGs are at least 100-fold reduced compared to Col.

Figure 4.

The atr1-2 loss-of-function mutation impairs expression of IG synthesis genes in adult leaves. RNA prepared from 3-week-old rosette leaves of Ws or the atr1-2 insertion mutant strain was used in RNA gel-blot analysis with the indicated probes.

Figure 5.

Effects of the atr1-2 loss-of-function mutation on IG accumulation. IG measurements were performed as described in Figure 3 and “Materials and Methods” on 3-week-old rosette leaves of Ws or the atr1-2 mutant, with Ws used as the normalization control.

Figure 6.

The atr1-2 mutation suppresses elevated Trp gene expression and 5MT resistance of the cyp83B1 mutant. A, Seedling RNA prepared from the indicated strains grown on unsupplemented PNS agar medium was used in RNA gel-blot analysis with the indicated probes. B, Seedlings of the indicated strains were tested for resistance on the indicated concentrations of 5MT, with a representative individual for each strain shown. 83B1 indicates the cyp83B1 mutation.

Figure 7.

The atr1-2 mutation and the cyp79B2 cyp79B3 double mutation suppress formation of adventitious roots in the cyp83B1 mutant. The indicated strains were grown on PNS agar medium under low-pass yellow filters in 18 to 30 μE m⁻² s⁻¹ light at 21°C, and representative seedlings were photographed at 14 d postgermination. Both Col and Ws controls are included because the atr1-2 mutation is in the Ws background, whereas cyp83B1 and the cyp79B2 cyp79B3 mutations are in the Col background.

Figure 8.

The cyp79B2 cyp79B3 double mutant displays elevated message levels for ATR1 and IG synthesis genes. Seedling RNA prepared from the indicated strains grown on unsupplemented PNS agar medium was used in RNA gel-blot analysis with the indicated probes. B2 indicates cyp79B2 and B3 indicates cyp79B3.