The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens

Abstract

Jasmonates (JAs) inhibit plant growth and induce plant defense responses. To define genes in the Arabidopsis JA signal pathway, we screened for mutants with constitutive expression of a luciferase reporter for the JA-responsive promoter from the vegetative storage protein gene VSP1. One mutant, named constitutive expression of VSP1 (cev1), produced plants that were smaller than wild type, had stunted roots with long root hairs, accumulated anthocyanin, had constitutive expression of the defense-related genes VSP1, VSP2, Thi2.1, PDF1.2, and CHI-B, and had enhanced resistance to powdery mildew diseases. Genetic evidence indicated that the cev1 phenotype required both COI1, an essential component of the JA signal pathway, and ETR1, which encodes the ethylene receptor. We conclude that cev1 stimulates both the JA and the ethylene signal pathways and that CEV1 regulates an early step in an Arabidopsis defense pathway.

Figure 1.

Phenotypes of cev1 Plants. (A) Low-light images of Col-gl1 and cev1 seedlings containing the VSP1::luciferase reporter transgene. Seedlings were grown for 12 days on MS (Murashige and Skoog, 1962) agar. The figure shows the positions of seedlings (left), and a low-light image of the same seedlings sprayed with luciferin solution reveals luciferase activity (right). (B) Four-week-old Col-gl1 (left) and cev1 (right) plants grown in soil. (C) Seven-day-old Col-gl1 and cev1 seedlings germinated on MS medium in the presence (+) and absence (−) of 20 μM MeJA. (D) Root phenotypes of 7-day-old seedlings of (left to right) Col-gl1 and the mutants cev1, etr1, the double mutant cev1/cev1;etr1/etr1, and cev1 grown in 17 μM Ag⁺.

Figure 2.

VSP1 Promoter Activity in Col-gl1 and cev1 Plants. Col-gl1 and cev1 plants containing the VSP1::luciferase transgene were harvested and assayed for luciferase activity 12 days after germination. Treated seedlings were wounded with blunt forceps 16 hr before harvest or were transferred to MS plates containing 20 μM MeJA for 48 hr before harvest. Seedlings then were assayed individually for luciferase activity in vitro. Error bars show standard deviations of five replicates for Col-gl1 (white bars) and cev1 (gray bars).

Figure 3.

RNA Gel Blot Analysis of Col-gl1 and cev1 RNA Extracted from 12-Day-Old Seedlings. Treated seedlings had been wounded with blunt forceps 16 hr before harvest or were transferred to MS plates containing 20 μM MeJA for 48 hr before harvest. Total RNA (0.5 μg) was electropho-resed, blotted, and probed with a ³²P-labeled DNA fragment of the VSP gene, the PDF1.2 gene, or the CHI-B gene.

Figure 4.

GUS Expression Driven by the VSP1, VSP2, and Thi2.1 Promoters in Col-gl1 and cev1 Seedlings. Seedlings were grown for 12 days on MS medium or for 10 days on MS medium and transferred to medium containing 20 μM MeJA for 2 days and then stained for GUS activity.

Figure 5.

Effect of the coi1-1 and etr1 Mutations on the cev1 Phenotype. (A) Ten-day-old seedlings of cev1/cev1 (left) showing anthocyanin in petioles (arrow) and the cev1/cev1; coi1-1/coi1-1 (right) double mutant. (B) VSP1 promoter activity in wild-type seedlings, the cev1/cev1 and coi1-1/coi1-1 mutants, and the cev1/cev1;coi1-1/coi1-1 double mutant. Ten-day-old seedlings containing the VSP1::luciferase transgene in these mutant backgrounds were transferred to MS medium or MS supplemented with 20 μM MeJA for 2 days. Seedlings were extracted individually, and luciferase activity was measured. Error bars show standard deviations of 10 replicates without MeJA (white bars) and with MeJA (gray bars).

Figure 6.

Resistance of cev1 to Powdery Mildews. Four-week-old Col-gl1 and cev1 plants were inoculated with E. cichoracearum UCSC1, E. orontii MGH, or O. lycopersicum Oxford. After 7 days (for E. cichoracearum) or 5 days (for E. orontii and O. lycopersicum), leaves were fixed and cleared in a lactophenol solution and stained with trypan blue, and the number of conidiophores per colony was recorded. Numbers are the means from 50 colonies for Col-gl1 (white bars) and cev1 (gray bars). An unpaired t test analysis indicated that for each fungus, differences between the means for the Col-gl1 and cev1 plants were statistically significant (P < 0.001).

Figure 7.

Model for the cev1 Mutant Phenotype. The cev1 mutation activates the JA and ethylene signal pathways, possibly by inducing the synthesis of JA and ethylene, or by regulating COI1 and ETR1 directly. The model accounts for the observed cev1 mutant phenotype through independent effects of the activated JA and ethylene signal pathways, and through cross-talk between these pathways causing activation and suppression of different responses. TR indicates triple response, arrows indicate activation, and bars indicate suppression.