Dual-Level Regulation of Ethylene Biosynthesis 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-mM 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 EF1a transcript. Error bars indicate standard deviations (n = 3). doi:10.1371/journal.pgen.1002767.g007 able to partially compensate the loss of WRKY33. It is known that pathogen infection induces a large number of WRKY genes [40,41], some of which might be able to partially compensate the loss of WRKY33 gene in activating the expression of ACS2/ACS6. WRKY33 binds to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo Genetic analysis revealed that WRKY33 is essential for gain-of- function MPK3/MPK6-induced ACS2/ACS6 gene expression (Figure 7B). Examination of the ACS2 and ACS6 promoters 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 EF1a transcript. Error bars indicate standard deviations (n = 3). doi:10.1371/journal.pgen.1002767.g008 revealed the presence of eight and seven W-boxes, respectively (Figure 9A). To further substantiate the role of WRKY33 in the activation of ACS2 and ACS6 gene expression, we performed chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis to determine whether ACS2 and ACS6 genes are direct targets of the WRKY33 transcription factor. For this experiment, we used DD/wrky33 mutant plants complemented with a 35S promoter-driven WRKY33 transgene, which contains a four- copy myc epitope tag at the N-terminus (DD/wrky33/35S:4myc- WRKY33) [34]. The presence of the myc tag allowed us to immunoprecipitate the WRKY33-DNA complex by using a commer- cial anti-myc antibody. As shown in Figure 9B, immunoprecipitation PLoS Genetics | www.plosgenetics.org 7 June 2012 | Volume 8 | Issue 6 | e1002767