Dual-Level Regulation of Ethylene Biosynthesis Figure 4. Activation of ACS gene expression after MPK3/MPK6 activation in the gain-of-function DD Arabidopsis seedlings. (A) Twelve-day-old conditional gain-of-function DD Arabidopsis seedlings grown in GC vials were treated with 1-mM DEX. Samples were collected at indicated times for total RNA preparation. After reverse transcription, expressions of all nine ACS genes were quantified by real-time PCR. Induction of ACS gene expression (fold of induction relative to the level before inoculation) was calculated by the double DCt method. (B) ACS transcript levels expressed as percentage of EF1a transcript, which allows comparison of expression levels among different ACS genes. In both calculations, the expression of EF1a was used as a reference. Error bars indicate standard deviations (n = 3). ND, not detectable. doi:10.1371/journal.pgen.1002767.g004 synchronous response in DD plants. In contrast, the infection process of B. cinerea is progressive. As more cells sensed the growing hyphae, higher levels of ACS6 transcript were induced at later time points. To confirm that MPK3 and MPK6 are responsible for induction of ACS2 and ACS6 expression levels in the gain-of- function DD plants, we examined the expression of ACS2 and ACS6 in DD/mpk3 and DD/mpk6 plants. As shown in Figure 5, induction of ACS2 and ACS6 was compromised in both the mpk3 and mpk6 single mutant backgrounds. This finding demonstrates that induction of ACS2 and ACS6 in DD seedlings after DEX treatment is indeed a result of MPK3/MPK6 activation. Based on the fact that MPK3 and MPK6 are highly activated after B. cinerea infection [28,38], we speculate that the MPK3/MPK6 cascade is involved in regulating the B. cinerea-induced ACS2/ACS6 gene expression and that induction of ACS7, ACS8, and ACS11 expression is regulated by pathway(s) other than MPK3/MPK6 cascade. To provide loss-of-function evidence to support the role of the MPK3/MPK6 cascade in B. cinerea-induced ACS2/ACS6 gene activation, we compared the induction of ACS2 and ACS6 gene expressions in wild type, mpk3 single mutant, mpk6 single mutant, and rescued mpk3/mpk6 double mutant. The rescued mpk3/mpk6 double mutant was obtained by transforming a DEX-inducible promoter-driven MPK6 (GVG-MPK6) into mpk32/2/mpk6+/2 plants. When the T3 mpk32/2/mpk6+/2/GVG-MPK6+/+ plants began to flower, DEX was sprayed every other day to rescue the embryo lethality of the mpk32/2/mpk62/2/GVG-MPK6+/+ zygotes. Progenies with mpk32/2/mpk62/2/GVG-MPK6+/+ genotype were called rescued mpk3/mpk6 double mutants [39], and were used for this experiment. As shown in Figure 6, B. cinerea-induced ACS2 and ACS6 expressions were little affected in either the mpk3 or mpk6 single mutant. In the rescued mpk3/mpk6 double mutant, the induction of both genes was dramatically reduced, which supports the conclusion that MPK3 and MPK6 regulate expressions of ACS2 and ACS6 based on the gain-of-function analysis. Different from B. cinerea-induced ACS2/ACS6 gene activation, gain-of-function DD-induced ACS2/ACS6 gene activation was compromised in either mpk3 or mpk6 single mutant background (Figure 5 and Figure 6). There are several potential explanations for this seemingly contradictory observation. First of all, MAPK- phosphorylation regulation of ACS2/ACS6 gene activation will be affected by both the phosphorylation of the downstream transcription factor(s) such as WRKY33 (more discussion below), and their dephosphorylation by the unidentified phosphatase(s). It is possible that in the gain-of-function DD plants, both MPK3 and MPK6 are needed to overcome the action of the phosphatase(s) to maintain the phosphorylation of there transcription factor(s) and the subsequent up-regulation of ACS2/ACS6 expression. In the absence of either MAPK, the signaling strength is below the threshold to counteract the phosphatases and the activation of ACS2/ACS6 expression is severely compromised. It is possible that, in addition to the activation of MPK3/MPK6 cascade, pathogen infection may also inactivate the dephosphorylation process as a mechanism to promote higher levels of ethylene production. In this situation, the absence of only one MAPK may not be sufficient PLoS Genetics | www.plosgenetics.org 5 June 2012 | Volume 8 | Issue 6 | e1002767