Dual-Level Regulation of Ethylene Biosynthesis 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-mM DEX. Samples were collected at indicated times. Total RNAs were extracted and treated with DNase to remove trace genomic DNA contamination. After reverse transcription, expres- sions of ACS2 (A) and ACS6 (B) 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.g005 to block ACS2/ACS6 activation. Alternatively, it is possible that the activation of pathways other than MAPK cascade can compensate the weakened MAPK pathway in the single mpk3 or mpk6 mutant, making it necessary to mutate both MPK3 and MPK6 to see the loss- of-function phenotype in response to B. cinerea infection. Similar phenomenon was also observed in MPK3/MPK6-mediated camalexin induction in response to B. cinerea infection [38]. WRKY33 is involved in the MPK3/MPK6-regulated ACS2/ ACS6 gene activation WRKY33 is a substrate of MPK3/MPK6 in regulating the pathogen-induced phytoalexin biosynthesis [34]. WRKY33 func- tions as a transcriptional activator downstream of MPK3 and MPK6 in promoting the expression of camalexin biosynthetic genes. To determine whether WRKY33 also is involved in activation of ACS2 and ACS6 genes downstream of the MPK3/ MPK6 cascade, we quantified the expression of these two genes in DD and DD/wrky33 plants. As shown in Figure 7B, the induction of ACS2 and ACS6 mRNA by the gain-of-function DD transgene was compromised in wrky33 mutant background. Associated with this, the induction of ethylene biosynthesis was mostly inhibited (Figure 7A). Previously, we showed that DD protein induction and MPK3/MPK6 activation in DD/wrky33 plants are indistinguishable from those in DD plants [34], which strongly supports the conclusion that WRKY33 also functions downstream of MPK3/ MPK6 in promoting the expression of ACS2 and ACS6 genes. The low residual levels of ACS2 and ACS6 gene activation is likely to be a result of other WRKY transcription factors that can partially substitute for the loss of WRKY33. 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 EF1a transcript. Error bars indicate standard deviations (n = 3). doi:10.1371/journal.pgen.1002767.g006 In contrast to the gain-of-function DD plants, mutation of the WRKY33 gene had a minor impact on ethylene induction triggered by B. cinerea infection. As shown in Figure 8A, we observed only about a 20% decrease in ethylene production in both alleles of the wrky33 mutant. A comparison of ACS2 and ACS6 gene expressions in the wild type and in the wrky33 mutant revealed that about one-third of the induction in ACS2/ACS6 expression remained in the wrky33 mutant (Figure 8B). This suggests that ACS2 and ACS6 still could be partially activated in the absence of WRKY33. Because of the low residual ACS2/ACS6 gene activation in the mpk3/mpk6 mutant (Figure 6), we speculate that the residual levels seen in the wrky33 mutant are MPK3/MPK6- dependent but WRKY33-independent, again pointing to addi- tional transcription factors, possibly WRKY33 homologs that partially replace WRKY33 in its absence. No major reductions were observed in the induction of ACS7, ACS8, and ACS11 expression in wrky33 infected with B. cinerea (Figure S4), which is consistent with the conclusion that their activation is MPK3/MPK6 and WRKY33 independent. The normal activation of ACS7, ACS8, and ACS11 expressions, together with the residual level of ACS2/ACS6 gene activation and protein phosphorylation stabilization, may explain the observation that the induction of ethylene in the wrky33 mutant was reduced by only about 20% after B. cinerea infection (Figure 8A). In contrast, the wrky33 mutation almost completely blocked induction of ethylene biosynthesis in the gain-of-function DD plants (Figure 7A). B. cinerea can activate multiple signaling pathways in plants. It is possible that pathway(s) other than MPK3/MPK6 cascade are PLoS Genetics | www.plosgenetics.org 6 June 2012 | Volume 8 | Issue 6 | e1002767