A Link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis

Abstract

The plant hormone ethylene participates in the regulation of a variety of developmental processes and serves as a key mediator of plant responses to biotic and abiotic stress factors. The diversity of ethylene functions is achieved, at least in part, by combinatorial interactions with other hormonal signals. Here, we show that ethylene-triggered inhibition of root growth, one of the classical effects of ethylene in Arabidopsis thaliana seedlings, is mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE alpha1 (WEI2/ASA1) and WEI7/ANTHRANILATE SYNTHASE beta1 (ASB1) genes that encode alpha- and beta-subunits of a rate-limiting enzyme of Trp biosynthesis, anthranilate synthase. Upregulation of WEI2/ASA1 and WEI7/ASB1 by ethylene results in the accumulation of auxin in the tip of primary root, whereas loss-of-function mutations in these genes prevent the ethylene-mediated auxin increase. Furthermore, wei2 and wei7 suppress the high-auxin phenotypes of superroot1 (sur1) and sur2, two auxin-overproducing mutants, suggesting that the roles of WEI2 and WEI7 in the regulation of auxin biosynthesis are not restricted to the ethylene response. Together, these findings reveal that ASA1 and ASB1 are key elements in the regulation of auxin production and an unexpected node of interaction between ethylene responses and auxin biosynthesis in Arabidopsis. This study provides a mechanistic explanation for the root-specific ethylene insensitivity of wei2 and wei7, illustrating how interactions between hormones can be used to achieve response specificity.

Figure 1.

wei2 and wei7 Are Root-Specific Ethylene-Insensitive Mutants. (A) Phenotypes of 3-d-old etiolated Col-0, wei2-1, wei2-2, wei2-3, wei7-1, wei7-2, and wei7-4 seedlings grown on AT plates in the presence of hydrocarbon-free air or air supplemented with 10 ppm ethylene. (B) and (C) Dose–response curves of hypocotyls (left) and roots (right) of 3-d-old etiolated Col-0, wei2-1, and wei7-4 seedlings grown in AT medium supplemented with 0, 0.2, 0.5, or 10 μM ACC (B) or with 0, 0.1, 1, or 10 μM IAA (C). Relative organ length (expressed as a percentage of the length observed in unsupplemented medium) is plotted on the y axis, and hormone concentrations are plotted on the x axis on a logarithmic scale. Asterisks indicate significant difference (P < 0.0001 in a two-way analysis of variance) between the wild-type and mutant responses at a particular concentration of ACC.

Figure 2.

WEI2 and WEI7 Function Downstream of CTR1 in the Ethylene Signaling Pathway. Phenotypes of 3-d-old etiolated ctr1-1, wei2-1 ctr1-1, and wei7-4 ctr1-1 seedlings grown in unsupplemented AT medium in the presence of hydrocarbon-free air are shown.

Figure 3.

Schemes of the WEI2 and WEI7 Genes. (A) and (B) The exon–intron structures of WEI2 (A) and WEI7 (B) are shown by boxes versus lines, respectively. The positions and types of mutations are as indicated. (C) Alignment of a WEI7 protein fragment of Arabidopsis with the corresponding regions of ASB from Oryza sativa (OsASB) and Bacillus cereus (BcASB). The conserved Gly-177 that is mutated to Glu in the wei7-2 mutant is marked by an arrow.

Figure 4.

Rescue of the Root Defect of wei2 and wei7 by Anthranilate, Trp, and IAA. Col-0, wei2-1, and wei7-4 seedlings were grown for 3 d in the dark in AT versus AT plus 10 μM ACC, with or without supplementation with 10 μM anthranilate (ANT) (A), 10 μM Trp (B), or 10 nM IAA (C). Average and sd of ≥20 seedlings are shown.

Figure 5.

WEI2 and WEI7 Show Ethylene-Inducible Expression in Root Tips of Etiolated Seedlings. GUS activity of the transcriptional reporters ASA1-GUS and ASB1-GUS was analyzed in Col-0 and ein2-5 seedlings grown in AT medium for 3 d in the dark in the presence of hydrocarbon-free air or air supplemented with 10 ppm ethylene. Plants were stained for GUS for 1 h, and representative seedlings were photographed. Bar = 0.1 mm.

Figure 6.

Ethylene Stimulates the Expression of the Synthetic Auxin Reporter DR5-GUS in Root Tips of Wild-Type Etiolated Seedlings. GUS activity was monitored in roots of Col-0, ein2-5, wei2-1, and wei7-4 seedlings grown in AT medium for 3 d in the dark in the presence of hydrocarbon-free air or air supplemented with 10 ppm ethylene. (A) Plants were stained for GUS overnight, and representative seedlings were photographed. Bar = 0.1 mm. (B) Seedlings were dissected into hypocotyls and roots, and MUG assay was performed. Pools of 100 to 140 seedlings per genotype per treatment per experiment were analyzed. Averages and sd values of three independent experiments are shown. The ethylene effects in Col versus wei2 or wei7 were significantly different (P < 0.05 by analysis of variance). MU, 4-methylumbelliferone.

Figure 7.

wei2 and wei7 Suppress the High-Auxin Phenotypes of sur1 and sur2. (A) and (B) Col-0, wei2-1, wei7-4, sur1, sur2, wei2-1 sur1, wei7-4 sur1, wei2-1 sur2, and wei7-4 sur2 were germinated on horizontal AT plates in the light for 5 d (large insets) or for 7 d followed by 4 d on vertical AT plates (small insets) (A) or grown in soil for 4 weeks (B) and then photographed. (C) Double mutant wei2-1 sur1 plants were grown in soil for 8 weeks and then photographed.

Figure 8.

Schemes of the Ethylene Biosynthetic, Signaling, and Response Pathways. Key elements of the pathways are displayed in a linear manner and are connected by arrows. A previously established link between auxin and ethylene biosynthesis is indicated. The new branch in the ethylene response pathway that connects ethylene with auxin biosynthesis is highlighted by a gray box. SAM, S-adenosyl-Met.