We then crossed acs7-1 into the acs2-1/acs6-2 double mutant background and identified an acs2-1/acs6-2/acs7-1 triple mutant in the F3 generation. As shown in Figure 2, only about 10% residual ethylene production was observed in the acs2-1/acs6-2/ acs7-1 triple mutant, confirming the importance of ACS7 in B. cinerea-induced ethylene production. Residual ethylene induction in the acs2-1/acs6-2/acs7-1 triple mutant again points to involve- ment of additional ACS members. To identify them, we utilized the high-order acs mutants generated in Dr. Theologis’ lab [35]. We found that acs2-1/acs6-1 seedlings produced a lower level of ethylene than our acs2-1/acs6-2 double mutant after challenged with B. cinerea (Figure 3 and Figure S2), which is consistent with the knockdown nature of our acs6-2 allele. Additional mutation of ACS4, ACS5, and ACS9 genes, either one at a time or all three at once, in the acs2-1/acs6-1 background did not further reduce the ethylene induction. This finding is consistent with our previous conclusion that ACS5 and ACS9 are not involved in the ethylene induction triggered by B. cinerea infection [28]. In contrast, mutation of the ACS7 gene in the acs2-1/acs6-1/acs4-1/acs5-2/ acs9-1 background resulted in further reduction in the ethylene induction. In this sextuple mutant (acs2-1/acs6-1/acs4-1/acs5-2/ acs9-1/acs7-1) background, mutation of ACS11, but not ACS1, slightly reduced the ethylene production. The very low level of ethylene induction in the acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9- 1/acs7-1/acs11-1 plants also implicates the involvement of ACS8. In the absence of B. cinerea infection, seedlings of all genotypes produced less than 15 nL ethylene per gram of seedlings within 24 hours, a very low level in comparison to the B. cinerea-induced ethylene production (Figure S3). From this dataset, we can also conclude that ACS7 contributed the most to the basal level ethylene production. Seedlings without a functional ACS7 gene including the acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9-1/acs7-1/ acs11-1 failed to produce a detectable level of ethylene. As a result, the low-level ethylene production in this octuple acs mutant in response to B. cincerea infection is indeed a contribution of ACS8 gene. In summary, we can conclude that ACS2 and ACS6 are major contributors of ethylene induction and that ACS7, ACS8, and ACS11 contribute less, with a total of ,15% of the ethylene induction in B. cinerea-infected Arabidopsis. Among the minor contributors, the role of ACS7 and ACS8 in B. cinerea-induced 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 Arabi- dopsis 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). doi:10.1371/journal.pgen.1002767.g002 Dual-Level Regulation of Ethylene Biosynthesis 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. doi:10.1371/journal.pgen.1002767.g003 ethylene production is clear. In contrast, the contribution of ACS11 is somewhat uncertain because of the conclusion of its involvement is based on a small quantitative difference. One potential mechanism underlying each ACS isoform’s contribution to ethylene induction is through the up-regulation of their gene expression (Figure 1). There is a good correlation between the transcriptional activation of ACS gene expression (Figure 1) and involvement in B. cinerea-induced ethylene production (Figure 3). Induction of ACS2 and ACS6 gene expression is dependent on MPK3/MPK6 pathway Previously, we demonstrated the importance of phosphorylation regulation of ACS2 and ACS6 by MPK3 and MPK6 in Arabidopsis in response to pathogens/pathogen-associated molec- ular patterns (PAMPs) [16,28,29]. It is well known that the ACS6 gene is highly induced by stress, including wounding and pathogen infection [1,4,6,11,36,37]. However, the importance of ACS gene activation in pathogen-induced ethylene production remains unclear. After our discovery that phosphorylation of ACS2 and ACS6 proteins by MPK3/MPK6 is required for ACS2/ACS6 protein stabilization and accumulation, we started to explore the potential contribution of ACS gene activation. Theoretically, an increase in ACS transcript levels is likely to increase the rate of de novo ACS protein synthesis, which, in turn, will increase the net ACS protein/activity after MAPK phosphorylation and protein stabilization. To determine whether ethylene induction in the conditional gain-of-function GVG-NtMEK2DD (DD, for short) plants [16,28] is associated with ACS gene activation, we profiled the expression of ACS genes in DD plants after dexamethasone (DEX) treatment. As shown in Figure 4, the expressions of ACS2 and ACS6 were highly induced. Different from B. cinerea-infected seedlings, no induction in ACS7, ACS8, and ACS11 expression levels were observed. In addition, we noticed the kinetics of ACS6 induction in the DD plants were different from that in B. cinerea-infected plants (Figure 4 versus Figure 1). This difference is likely a result of the PLoS Genetics | www.plosgenetics.org 4 June 2012 | Volume 8 | Issue 6 | e1002767