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. 2004 Dec;16(12):3460-79.
doi: 10.1105/tpc.104.025833. Epub 2004 Nov 17.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

Jonathan P Anderson  1 , Ellet Badruzsaufari, Peer M Schenk, John M Manners, Olivia J Desmond, Christina Ehlert, Donald J Maclean, Paul R Ebert, Kemal Kazan
Affiliations

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

Jonathan P Anderson et al. Plant Cell. 2004 Dec.

Abstract

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

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Figures

Figure 1.
Figure 1.
Exogenous ABA Suppresses PDF1.2 Expression in Arabidopsis. (A) Fold changes in relative transcript abundance of the PDF1.2 in MJ-, ethylene-, ABA-, MJ+ABA-, ethylene+ABA- (second to last column), and water stress–treated wild-type (Columbia-0 [Col-0]) plants. Total RNA was isolated from plants 48 h after each treatment, converted to cDNA, and used as template in RT-Q-PCR assays. Transcript levels of PDF1.2 were normalized to the expression of β-actin genes measured in the same samples and expressed logarithmically relative to the normalized transcript levels in mock-treated wild-type plants. Average data with error bars from two independent experiments are presented. The numbers on each bar show fold increase or fold decrease caused by each treatment in PDF1.2 transcript levels relative to those in mock-treated plants. (B) and (C) Three- to four-week-old homozygous plants transformed with the PDF1.2promoter:GUS construct were either mock treated or treated with ABA, MJ, or combination of ABA and MJ for 48 h. Histochemical GUS staining was performed overnight on 10 seedlings for each experiment (B). The leaves that display strong GUS activity after MJ treatment as evidenced by saturated blue color are indicated by arrows. GUS activity was also measured fluorometrically using at least 10 seedlings for each treatment (C). Average data from two separate experiments are shown. Error bars indicate standard deviation.
Figure 2.
Figure 2.
ABA Suppresses JA-Ethylene Responsive Defense Gene Expression in Arabidopsis. (A) Fold changes (induction or suppression) in relative transcript abundance of PDF1.2, CHI, HEL, and LEC in MJ- and MJ+ABA-treated wild-type (Col-0) plants relative to the mock-treated wild-type plants. (B) Fold changes in relative transcript abundance of PDF1.2, CHI, HEL, and LEC in mock-, MJ-, or ABA-treated aba2-1 mutant relative to the expression of the same genes in similarly treated wild-type plants. Total RNA was isolated from plants 48 h after each treatment, converted to cDNA, and used as template in RT-Q-PCR assays. Transcript levels in treated wild-type plants were normalized to the expression of β-actin genes measured in the same samples and expressed logarithmically relative to the similarly normalized transcript levels in mock-treated wild-type plants. The numbers on each bar show fold increase or fold decrease caused by each treatment in the transcript levels of genes relative to those in mock-treated plants (A). Transcript levels in treated aba2-1 plants were normalized relative to the expression of β-actin genes measured in the same samples and expressed logarithmically relative to the similarly normalized transcript levels in treated wild-type plants. The numbers on each bar show fold increase or fold decrease caused by each treatment in the transcript levels of genes in the aba2-1 background relative to those in similarly treated wild-type plants (B). Average data with error bars from two independent experiments are shown in both (A) and (B).
Figure 3.
Figure 3.
AtMYC2 Is Induced during Plant Defense Responses in Arabidopsis. AtMYC2 and PDF1.2 expression in wild-type (Col-0) plants were examined in time-course studies after inoculation with F. oxysporum (A) and after treatment with MJ (B). Total RNA was extracted from leaf tissue of 3- to 4-week-old plants (8 to 10 leaf stage) for each time point, converted to cDNA, and subjected to RT-Q-PCR analysis. The AtMYC2 and PDF1.2 transcript levels in treated/inoculated plants were normalized to the expression of β-actin measured in the same samples and expressed logarithmically relative to the similarly normalized expression levels in mock-inoculated/mock-treated plants. Each bar represents average data with error bars from two independent experiments. The numbers on each bar show fold increase or fold decrease caused by each treatment at transcript levels of AtMYC2 and PDF1.2 relative to those in mock-treated/inoculated plants.
Figure 4.
Figure 4.
JA-Ethylene Responsive Defense Genes Show Elevated Levels of Basal and Activated Transcription in the jin1-9/myc2 Mutant. Relative expression ratios of PDF1.2, CHI, and HEL transcripts in untreated (A), MJ- (B), and ethylene-treated (C) plants of the wild type (Col-0) and the homozygous plants of the jin1-9/myc2 mutant. Total RNA was extracted from leaf tissue of 3- to 4-week-old plants 24 h after MJ and ethylene treatments, converted to cDNA, and subjected to RT-Q-PCR analysis. The transcript levels in the jin1-9/myc2 mutant were normalized to the expression of β-tubulin measured in the same samples and expressed logarithmically relative to the normalized expression levels in similarly treated wild-type plants. Each bar represents average data with error bars from two independent experiments. The numbers on each bar show fold increase in defense gene transcript levels in the jin1-9/myc2 mutant relative to those in mock-treated plants.
Figure 5.
Figure 5.
Transient or Stable Overexpression of AtMYC2 in Arabidopsis Suppresses PDF1.2 Expression. (A) Transient overexpression of ERF1 in Arabidopsis protoplasts activates expression from the PDF1.2, whereas transient overexpression of AtMYC2 in Arabidopsis protoplasts suppresses the basal transcription from PDF1.2 as detected by RT-Q-PCR 48 h after transformation. AtMYC2 overexpression is not sufficient in suppressing PDF1.2 transcript levels when coexpressed with ERF1 in Arabidopsis protoplasts. Total RNA was isolated from the 35S:ERF1, 35S:AtMYC2, and 35S:ERF1+35S:AtMYC2 transformed protoplasts, converted to cDNA, and subjected to RT-Q-PCR analysis. The PDF1.2 transcript levels in the transformed samples were normalized to the expression of β-actin measured in the same samples and expressed logarithmically relative to the normalized expression levels measured in vector-only transformed protoplasts. Each bar represents average data with error bars from two independent experiments. The numbers on each bar show fold increase or fold decrease in PDF1.2 transcript levels caused by overexpression relative to the PDF1.2 levels in vector-transformed protoplasts. (B) The stable overexpression of AtMYC2 in Arabidopsis activates rd22 expression while suppressing PDF1.2 in plants either mock-treated or treated with ABA for 11 h. Total RNA was isolated from transformed protoplasts or treated plants, converted to cDNA, and subjected to RT-Q-PCR analysis. The AtMYC2, rd22, and PDF1.2 transcript levels in untreated and ABA-treated wild type and the 35S:AtMYC2 plants were normalized to the expression of β-actin (multiplied by 1000 for clarity) measured in the same sample and expressed logarithmically. Each bar represents average data with error bars from two independent experiments. The numbers on each bar show relative transcript abundance of AtMYC2, PDF1.2, and rd22, relative to the β-actin transcript levels measured in the same samples.
Figure 6.
Figure 6.
AtMYC2 Function Is Dispensable for Suppression of PDF1.2 by Exogenous ABA. Total RNA was isolated from mock- or ABA-treated wild-type and jin1-9/myc2 plants 24 h after treatment, converted to cDNA, and subjected to RT-Q-PCR analysis. The PDF1.2 transcript levels in mock- and ABA-treated wild-type and the jin1-9/myc2 mutant were normalized to the expression of β-actin genes measured in the same samples (multiplied by 1000 for clarity). Each bar represents average data with error bars from two independent experiments. The numbers on each bar show relative transcript abundance of PDF1.2 relative to the β-actin transcript levels measured in the same samples.
Figure 7.
Figure 7.
The jin1-9/myc2 and aba2-1 Mutants Show Enhanced Resistance to the Root-Infecting Fungal Pathogen F. oxysporum. (A) to (D) In three separate experiments, 140 each of the 3-week-old wild-type (Col-0) and myc2 plants were inoculated with F. oxysporum (A). The percentage of plants showing strong wilting phenotype (arrows) (B), and the total numbers of necrotic leaves (C) were scored 10 d after inoculation. The amount of fungal RNA present in the inoculated tissue is estimated (D) using all 30 plants of each of the inoculated wild-type and myc2 plants in experiment 3 (10 d after inoculation) by RT-Q-PCR with primers specific to F. oxysporum rRNA. (E) and (F) In two separate experiments, 60 plants of each of the wild-type (Col-0) and aba2-1 plants were inoculated with root-infecting fungal pathogen F. oxysporum (E). The plants showing strong wilting symptoms were counted 10 d after inoculation to assess the disease severity (F).
Figure 8.
Figure 8.
Analysis of Expression from ABA-Regulated Genes in abi1-1 and abi2-1 Mutants. Quantification of the relative abundance of the PDF1.2, KIN1, rd22, and VSP2 transcripts in mock- or MJ- and/or ABA-treated wild-type (Ler) and the abi1-1 and abi2-1 mutants. Total RNA was isolated from plants 48 h after each treatment, converted to cDNA, and used as template in RT-Q-PCR assays. The PDF1.2, KIN1, rd22, and VSP2 transcript levels were normalized to the expression of β-actin (multiplied by 1000 for clarity) measured in the same samples and expressed logarithmically. Each bar represents average data with error bars from two independent experiments. The numbers on each bar show transcript levels of PDF1.2, KIN1, VSP2, and rd22 relative to the β-actin transcript levels measured in the same samples.
Figure 9.
Figure 9.
Ethylene Insensitivity Enhances ABA-Responsive Gene Expression in Vegetative Tissues. Relative transcript levels of KIN1, rd22, VSP2, ABI1, and PDF1.2 in mock- and/or ABA-, MJ-, and/or ethylene-treated wild-type and jar1-1, ein2-1, etr1-1, and ein3-1 mutant plants. Total RNA was isolated from plants 48 h after each treatment, converted to cDNA, and used as template in RT-Q-PCR assays. The KIN1, rd22, VSP2, PDF1.2, and ABI1 transcript levels were normalized to the expression of β-actin (multiplied by 1000 for clarity) measured in the same samples and expressed logarithmically. Each bar represents average data with error bars from two independent experiments. The numbers on each bar show relative transcript levels of each gene relative to the β-actin transcript levels measured in the same samples.
Figure 10.
Figure 10.
ABA, JA, and Ethylene Signaling Pathways Interact for Regulation of Stress Responsive Gene Expression in Arabidopsis. Relative transcript levels of VSP2, rd22, and PDF1.2 in mock-, ABA-, MJ-, ethylene-, or combination of ABA-MJ and ABA-ethylene (Col-0 only)-treated wild-type and aba2-1 mutant plants. Total RNA was isolated from plants 48 h after each treatment, converted to cDNA, and used as template in RT-Q-PCR assays. The VSP2, rd22, and PDF1.2 transcript levels were normalized to the expression of β-actin (multiplied by 1000 for clarity) measured in the same samples and expressed logarithmically. Average data from two independent experiments with error bars are presented. The numbers on each bar show transcript levels of each gene relative to the β-actin transcript levels measured in the same samples.
Figure 11.
Figure 11.
Proposed Model of Genetic Interactions among ABA, JA, and Ethylene Signaling Pathways for Modulation of Stress-Responsive Gene Expression in Arabidopsis. Arrows indicate positive regulation, and blunt ends indicate negative regulation.
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