Dual-Level Regulation of Ethylene Biosynthesis 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-mM 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 EF1a transcript. Error bars indicate standard deviations (n = 3). doi:10.1371/journal.pgen.1002767.g010 plant disease resistance [6,7,35]. In contrast, other stress stimuli, including wounding, only trigger a transient and low-level ethylene biosynthesis. Previously, we reported that MPK3/MPK6 phos- phorylation-induced stabilization of ACS2 and ACS6 proteins is an important mechanism in promoting ethylene induction in Arabidopsis [16,28,29]. It is also clear from data based on acs2-1/ acs6-2 double mutant that ACS2 and ACS6 are not the only contributors in pathogen-induced ethylene production [28]. In this report, we demonstrate the involvement of three additional members of the ACS gene family. Mutation of ACS2 and ACS6, two Type I ACS members, abolishes ,85% of the ethylene production induced by B. cinerea (Figure 2 and Figure 3) [28]. We failed to detect the transcript from ACS1, the third member of the Arabidopsis Type I ACS isoforms, in the seedlings, and its mutation does not reduce pathogen-triggered ethylene production (Figure 3) [28]. As a result, we believe that ACS1 contributes little, if any, to pathogen-triggered ethylene production. ACS7, a member of the Type III ACS, plays an intermediate role. Its mutation in the acs2- 1/acs6-2 background reduces ethylene induction to less than 5% of that observed in the wild type (Figure 2 and Figure 3). This isoform is also the major contributor of the basal level ethylene production in the absence of pathogen infection (Figure S3). The transcripts of ACS2, ACS6, and ACS7 are among the most abundant in B. cinerea- infected Arabidopsis (Figure 1B). The residual levels of ethylene induction in acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9-1/acs7-1 mu- tant are likely from ACS8, a Type II ACS isoform, and ACS11, a Type III ACS isoform. Dual-level regulation of ACS2 and ACS6 by the MPK3/ MPK6 cascade in plant stress/defense response In addition to post-translational regulation, we found that transcriptional activation of the ACS genes is also critical to the high-level of ethylene induction, as depicted in our working model (Figure 12). Stress- and pathogen-activation of ACS genes, such as Arabidopsis ACS6, is well established [1,36,42]. In this report, we delineated a signaling pathway involved in the transcriptional activation of ACS2/ACS6 in Arabidopsis after pathogen infection. It is interesting to find that MPK3 and MPK6 not only function in the phosphorylation-induced stabilization of ACS2/ACS6 proteins, but also regulate the expression of ACS2 and ACS6 genes. The MPK3/ MPK6 cascade-induced ACS2/ACS6 gene activation is mediated by WRKY33, another MPK3/MPK6 substrate [34]. WRKY33 binds to the W-boxes in the promoters of the ACS2 and ACS6 genes directly in vivo (Figure 9) and is involved in the MPK3/MPK6- induced ACS2/ACS6 gene expression (Figure 7). Mutation of WRKY33 resulted in a smaller reduction (,60%) in ACS2/ACS6 gene activation in response to B. cinerea infection (Figure 8), possibly due to the presence of other WRKY(s) that can partially compensate the loss of WRKY33. Conditional overexpression of ACS6 in the acs2-1/acs6-2/acs7-1 mutant background greatly enhances the ethylene induction (Figure 10). Furthermore, reduction in ethylene induction in the acs6-2 allele, a knockdown mutant (Figure 11), provides loss-of-function evidence that demonstrates the importance of ACS6 gene activation during pathogen invasion. Transcriptional activation of ACS2 likely has a similar role. Induction of ACS6 expression is associated with stress-induced ethylene production [1,20,36,42]. However, direct evidence supporting the role of ACS gene activation has been lacking. In Arabidopsis, overexpression of wild type ACS6 genes is not sufficient to elevate ethylene production because of the require- ment of protein phosphorylation and stabilization [16]. In addition, overexpression of the ACS6 gene in the wild type background fails to enhance ethylene production upon B. cinerea inoculation (Li, G., Liu, Y., and Zhang, S., unpublished data), a result of the high-level gene activation of the endogenous ACS genes (Figure 1). In this study, we expressed the ACS6 gene in an acs2-1/acs6-2/acs7-1 mutant background. The use of a DEX- inducible promoter and two independent lines with different levels of ACS6 gene induction after DEX treatment allowed us to demonstrate the importance of ACS6 gene activation (Figure 10). Our acs2-1/acs6-2 double mutant produces about 25% of the wild type level of ethylene after B. cinerea infection (Figure 2) [28]. In contrast, the acs2-1/acs6-1 line only produces ,15% of the wild type ethylene (Figure 3). The difference between these two double mutants is likely a result of different acs6 mutant alleles since both have the same acs2-1 mutant allele. The difference between PLoS Genetics | www.plosgenetics.org 9 June 2012 | Volume 8 | Issue 6 | e1002767