Dual-Level Regulation of Ethylene Biosynthesis 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. doi:10.1371/journal.pgen.1002767.g012 complex, which allows ERF104 to activate the expression of genes further downstream [50]. Recently, we identified the transcription factor WRKY33 as the substrate of MPK3 and MPK6 [34]. WRKY33 is involved in the induction of camalexin biosynthesis by promoting the expression of camalexin biosynthetic genes [34,51]. In this report, we demonstrate that WRKY33 is involved also in activation of ACS2 and ACS6 gene expression and ethylene induction. In the wrky33 mutant background, gain-of-function DD- induced ACS2 and ACS6 gene activation is essentially abolished. Furthermore, a ChIP-qPCR analysis demonstrated that WRKY33 directly binds to the promoters of ACS2 and ACS6 genes (Figure 9). These results reveal that WRKY33 regulates gene expression in multiple stress/defense responses and may function as a master transcriptional regulator downstream of the MPK3/MPK6 cascade. The expression of both ACS2 and ACS6 genes are regulated by the MPK3/MPK6 cascade and its downstream WRKY33. However, the induction kinetics of ACS2 and ACS6 genes are different in both gain-of-function DD transgenic plants (Figure 4) and wild type plants after pathogen treatment (Figure 1). One possibility is that one or more transcription factors, other than WRKY33, are involved. The differential involvement of these unknown transcription factors could result in the different kinetics observed in the induction of ACS2 and ACS6 genes. These transcription factors may or may not be regulated by the MPK3/ MPK6 cascade. The activation of MPK3 and MPK6 proteins in the gain-of-function DD plants is sufficient to induce the expression of ACS2 and ACS6 genes to levels similar to those observed in B. cinerea-infected plants (Figure 1 and Figure 4), suggesting that the transcriptional machinery controlling expression of ACS2 and ACS6 genes is fully turned on in DD plants. On the other hand, mutation of WRKY33 essentially blocks DD-induced ACS2/ACS6 gene activation (Figure 7B) but only partially blocks the induction of ACS2 and ACS6 genes in B. cinerea-infected plants (Figure 8B), suggesting the activation of additional components by B. cinerea that cannot be activated by MPK3/MPK6 cascade alone, possibly homologs of WRKY33 that can partially replace the function of WRKY33 in its absence. Contribution of MPK3/MPK6-independent pathway(s) to stress-/pathogen-induced ethylene biosynthesis The aforementioned discussions are focused on the role of the MPK3/MPK6 cascade in regulating ACS2 and ACS6, two major contributors of pathogen-induced ethylene production, as depicted in our working model (Figure 12). A similar level of reduction in ethylene induction in the mpk3/mpk6 and acs2/acs6 double mutants (,85%) is consistent with our conclusion that the MPK3/MPK6 signaling cascade only controls ACS2 and ACS6. Genetic evidence also supports the involvement of three additional ACS isoforms, ACS7, ACS8, and ACS11, in B. cinerea-induced ethylene production (Figure 2 and Figure 3). ACS7 and ACS11 are the two members in the Type III ACS group in Arabidopsis. ACS8 belongs to the Type II ACS group. Based on mutant analyses, these three ACS genes contribute about 15% of the total ethylene produced in B. cinerea-infected plants (Figure 3) [28]. Transcrip- tional activation of ACS7, ACS8, and ACS11 is not regulated by the MPK3/MPK6 cascade (Figure 4); the signaling pathway(s) involved in the activation of their expression is unknown. It is also unclear whether ACS7, ACS8, and ACS11 are regulated at the protein stability level. Since ACS7 and ACS11 do not have a typical putative phosphorylation site in their C-termini, they are likely to be regulated at the transcriptional level only. ACS8, similar to ACS5 and ACS9, has a putative CDPK phosphorylation site in its C-terminus. It is possible that phosphorylation by CDPK(s) is involved in its protein stability regulation, similar to ACS5 and ACS9 [32,33]. PLoS Genetics | www.plosgenetics.org 11 June 2012 | Volume 8 | Issue 6 | e1002767