Figure 1.
Activation of ACS gene expression in Arabidopsis after B. cinerea infection.
(A) Twelve-day-old Arabidopsis seedlings grown in GC vials were inoculated with B. cinerea spores. Samples were collected at indicated times for total RNAs isolation. Expressions of all nine ACS genes were quantified by real-time PCR. Induction of ACS gene expression (fold of induction relative to the level before inoculation) was calculated by the double ΔCt method. (B) ACS transcript levels are expressed as percentage of EF1α transcript, which allows comparison of expression levels between different ACS genes. In both calculations, the expression of EF1α was used as a reference. Error bars indicate standard deviations (n = 3). ND, not detectable.
Figure 2.
ACS7 also contributes to B. cinerea-induced ethylene production in Arabidopsis.
Twelve-day-old wild type (Col-0), acs2-1/acs6-2 double mutant, and acs2-1/acs6-2/acs7-1 triple mutant Arabidopsis seedlings grown in GC vials were inoculated with B. cinerea. Ethylene levels in the headspace were determined at indicated times. Error bars indicate standard deviations (n = 3).
Figure 3.
Ethylene induction in high-order acs mutants after B. cinerea infection.
Twelve-day-old wild type (Col-0) and acs mutants generated in Dr. Theologis' lab were inoculated with B. cinerea. Ethylene levels in the headspace were determined at 24 hrs after spore inoculation. Error bars indicate standard deviations (n = 3). The allele numbers are omitted for easy labeling. They are acs1-1, acs2-1, acs4-1, acs5-2, acs6-1, acs7-1, acs9-1, and acs11-1.
Figure 4.
Activation of ACS gene expression after MPK3/MPK6 activation in the gain-of-function DD Arabidopsis seedlings.
(A) Twelve-day-old conditional gain-of-function DD Arabidopsis seedlings grown in GC vials were treated with 1-µM DEX. Samples were collected at indicated times for total RNA preparation. After reverse transcription, expressions of all nine ACS genes were quantified by real-time PCR. Induction of ACS gene expression (fold of induction relative to the level before inoculation) was calculated by the double ΔCt method. (B) ACS transcript levels expressed as percentage of EF1α transcript, which allows comparison of expression levels among different ACS genes. In both calculations, the expression of EF1α was used as a reference. Error bars indicate standard deviations (n = 3). ND, not detectable.
Figure 5.
Activation of ACS2 and ACS6 gene expressions in gain-of-function DD is dependent on downstream MPK3 and MPK6.
Twelve-day-old DD, DD/mpk3, and DD/mpk6 seedlings grown in GC vials were treated with 1-µM DEX. Samples were collected at indicated times. Total RNAs were extracted and treated with DNase to remove trace genomic DNA contamination. After reverse transcription, expressions of ACS2 (A) and ACS6 (B) genes were quantified by real-time PCR. ACS transcript levels were calculated as a percentage of the EF1α transcript. Error bars indicate standard deviations (n = 3).
Figure 6.
B. cinerea induced ACS2 and ACS6 gene activation is dependent on functional MPK3 and MPK6.
Twelve-day-old wild type (Col-0), mpk3, mpk6, and rescued mpk3/mpk6 double mutant seedlings grown in GC vials were inoculated with B. cinerea spores. Samples were collected at indicated times. Total RNAs were extracted and treated with DNase to remove trace genomic DNA contamination. After reverse transcription, expressions of ACS2 (A) and ACS6 (B) genes were quantified by real-time PCR. ACS transcript levels were calculated as a percentage of the EF1α transcript. Error bars indicate standard deviations (n = 3).
Figure 7.
WRKY33 functions downstream of the MPK3/MPK6 cascade in inducing the expression of ACS2 and ACS6 genes in the gain-of-function DD seedlings.
(A) Mutation of WRKY33 compromises ethylene induction in DD seedlings. Twelve-day-old DD and DD/wrky33 seedlings grown in GC vials were treated with 1-µM DEX. Ethylene accumulation in GC vials was monitored at indicated times, and then seedlings were collected for gene expression analysis. Error bars indicate standard deviations (n = 3). (B) MPK3/MPK6-induced ACS2 and ACS6 gene expression in DD plants is dependent on WRKY33. Total RNA was extracted from seedlings collected in (A). Expressions of ACS2 (upper panel) and ACS6 (lower panel) genes were quantified by real-time PCR. ACS transcript levels were calculated as a percentage of the EF1α transcript. Error bars indicate standard deviations (n = 3).
Figure 8.
Induction of ACS2 and ACS6 gene expression after B. cinerea infection was partially inhibited in wrky33 mutant.
(A) Mutation of WRKY33 partially blocks ethylene induction in Arabidopsis infected by B. cinerea. Twelve-day-old wild type (Col-0) and wrky33 seedlings grown in GC vials were inoculated with B. cinerea spores. Ethylene accumulation in GC vials was monitored at indicated times, and seedlings were collected for gene expression analysis. Error bars indicate standard deviations (n = 3). (B) B. cinerea-induced ACS2 and ACS6 gene expression is compromised in the wrky33 mutant. Total RNA was isolated from the seedlings collected in (A). Expressions of ACS2 (upper panel) and ACS6 (lower panel) genes were quantified by real-time PCR. ACS transcript levels were calculated as a percentage of the EF1α transcript. Error bars indicate standard deviations (n = 3).
Figure 9.
WRKY33 transcription factor binds to the promoter of ACS2 and ACS6 genes in vivo.
(A) The promoters of ACS2 and ACS6 genes are rich in W-boxes, the cis-element binding sites of the WRKY transcription factor. A diagram indicates the number and relative position of the W-boxes in the promoters of the ACS2 and ACS6 genes. Line arrows indicate the position of primers used for qPCR after chromatin immunoprecipitation (ChIP). Positions of the predicted transcriptional starting sites are indicated by arrows with turning lines and negative numbers. The T-DNA insertion site in the SALK_090423 acs6-2 allele, which locates in the promoter region of ACS6 gene, is also indicated. (B) ChIP-qPCR analysis was performed using DD/4myc-WRKY33WT plants generated from the cross of wrky33/4myc-WRKY33WT with DD lines. Input chromatin was isolated from two-week-old seedlings 12 hr after DEX treatment. Epitope-tagged WRKY33-chromatin complex was immunoprecipitated with an anti-myc antibody. A control reaction was processed side-by-side using mouse IgG. ChIP- and input-DNA samples were quantified by real-time qPCR using primers specific to the promoters of ACS2 (left panel) and ACS6 (right panel) genes. ChIP results are presented as percentage of input DNA. Error bars indicate standard deviations (n = 3).
Figure 10.
Conditional expression of the ACS6 gene in the acs2-1/acs6-2/acs7-1 mutant background restores ethylene induction triggered by B. cinerea infection.
(A) Induction of ACS6 gene expression in the acs2-1/acs6-2/acs7-1/GVG-ACS6 seedlings restores B. cinerea-induced ethylene production. Twelve-day-old seedlings of wild type, acs2-1/acs6-2/acs7-1, and two independent lines of the GVG-ACS6 transgene in the acs2-1/acs6-2/acs7-1 background (#5 and #12) were inoculated with B. cinerea spores. Two groups of acs2-1/acs6-2/acs7-1/GVG-ACS6 seedlings were included with one treated with 1-µM DEX and the other treated with an equal volume of ethanol, the solvent for the DEX stock solution, at the time of B. cinerea spore inoculation. Ethylene accumulation in GC vials was monitored at indicated times. Error bars indicate standard deviations (n = 3). (B) Induction of ACS6 transgene expression in acs2-1/acs6-2/acs7-1/GVG-ACS6 seedlings after DEX treatment. Seedlings were collected before (0 hr) and 6 hr after DEX treatment. Induction of the ACS6 from the GVG-ACS6 transgene was quantified by real-time PCR. ACS6 transcript levels were calculated as a percentage of the EF1α transcript. Error bars indicate standard deviations (n = 3).
Figure 11.
The acs6-2 mutant allele is a knockdown mutant.
(A) B. cinerea-induced ethylene production in wild type, acs6-2 (SALK_090423), and acs6-1 (SALK_025672) plants. Twelve-day-old seedlings grown in GC vials were inoculated with B. cinerea spores. Ethylene accumulation in GC vials was monitored at indicated times, and seedlings were collected for gene expression analysis. Error bars indicate standard deviations (n = 3). (B) Induction of ACS6 expression in wild type (Col-0), acs6-2, and acs6-1 seedlings after B. cinerea inoculation. Total RNA was isolated from the seedlings collected in (A). Expression of the ACS6 gene was quantified by real-time PCR. ACS6 transcript levels were expressed as fold of induction relative to the zero time point (upper panel) and as a percentage of the EF1α transcript (lower panel). Error bars indicate standard deviations (n = 3).
Figure 12.
A model depicting the dual-level regulation of ACS activity by MPK3/MPK6-dependent and independent pathways during pathogen-induced ethylene production.
Members of all three types of ACS isoforms are involved in pathogen-induced ethylene production. In B. cinerea-infected plants, Type I (ACS2/ACS6) isoforms contribute the most (∼85%). ACS2 and ACS6 are regulated by the MPK3/MPK6 cascade at both transcriptional and protein stability levels. The transcriptional up-regulation is mediated by WRKY33, a MPK3/MPK6 substrate. Type II (ACS8 and ACS11) and Type III (ACS7) isoforms are activated at the transcriptional level although the regulatory pathway(s) involved is not clear at present. Increase in total cellular ACS activity drives the elevated ethylene production, which triggers downstream responses.