Author Summary Plant immunity, similar to that in animals, also involves mitogen-activated protein kinase (MAPK) cascades. How- ever, plants use unique MAPK substrates and secondary signaling molecules in the process. Among them, ethylene, a gaseous plant hormone, plays critical roles. Ethylene- regulated responses begin with the induction of ethylene biosynthesis. 1-amino-cyclopropane-1-carboxylic acid syn- thase (ACS) catalyzes the committing and rate-limiting step in ethylene biosynthetic pathway. The Arabidopsis genome encodes nine different ACS isoforms. Two of them, ACS2 and ACS6, were previously shown to be phosphorylated and stabilized by MPK3 and MPK6, two Arabidopsis pathogen-responsive MAPKs. Using a genetic approach, we identified additional ACS isoforms including ACS7, ACS8, and ACS11 that also contribute to pathogen- induced ethylene production. In addition to direct phos- phorylation modification and stabilization of ACS2 and ACS6 proteins, MPK3 and MPK6 also regulate the expression of ACS2 and ACS6 genes through another MPK3/MPK6 substrate, WRKY33, a member of the plant- specific WRKY transcription factor family. Regulations of ACS isoforms at both transcriptional and post-translational levels contribute to the high-level ethylene production in plants challenged by invading pathogens. These findings shed light on our understanding of the regulation of the kinetics and magnitude of ethylene induction under different stress conditions. include Arabidopsis ACS1, ACS2, and ACS6, have phosphory- lation sites by both mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs) [16,17]. Type II ACS isoforms, which include Arabidopsis ACS4, ACS5, ACS8, and ACS9, only have putative CDPK phosphorylation sites. In contrast, Type III ACS isoforms have shorter C-terminal extension and lack both phosphorylation sites. ACS7 and ACS11 are the two Type III ACS isoforms in Arabidopsis. ACS1 has a short deletion with the highly conserved tripeptide Thr-Asn-Pro (TNP) missing. It is enzymatically inactive as a homodimer, but can form functional heterodimers with other Type I isoforms and may contribute to ethylene biosynthesis [18,19]. ACS isoforms show cell- and tissue-specific expression and are developmentally regulated. In addition, expression of some members is highly responsive to extracellular stimuli [1,20]. More recent studies have highlighted the importance of ACS protein stability regulation by protein phosphorylation and dephosphorylation. MAPK cascades are signaling modules down- stream of sensors/receptors that transduce extracellular stimuli into intracellular responses in eukaryotes. A basic MAPK cascade is composed of three interconnected kinases. MAPKs function at the bottom of the three-kinase cascade and are activated by MAPK kinases (MAPKKs) through phosphorylation on the Thr and Tyr residues in their activation motif between the kinase subdomain VII and VIII. The activity of MAPKKs is, in turn, regulated by MAPKK kinases (MAPKKKs) via phosphorylation of two Ser/Thr residues in the activation loop of MAPKKs. MAPKKKs receive signals from upstream receptors/sensors, most of the time indirectly with additional components involved [21,22]. The outputs of a MAPK cascade are dependent on the substrates of the MAPK(s) in the cascade. A subset of MAPKs in plants, represented by tobacco SIPK/Ntf4/WIPK and Arabidop- sis MPK3/MPK6, is activated under various stress conditions that elevate ethylene production (reviewed in [21,23–26]). A gain- of-function analysis in tobacco revealed that activation of Dual-Level Regulation of Ethylene Biosynthesis SIPK/WIPK induces high levels of ethylene production [27]. More detailed analyses in Arabidopsis have demonstrated that ACS2 and ACS6, two Type I ACS isoforms, are substrates of MPK3 and MPK6 [16,28]. Phosphorylation of ACS2/ACS6 by MPK3 and MPK6 stabilizes the ACS protein in vivo, resulting in increases in cellular ACS activity and in ethylene production. The degradation machinery targets the C-terminal, non-catalytic domain of ACS6 and possibly ACS2 because of their sequence similarity [29]. Phosphorylation of ACS6 introduces negative charges to its C-terminus, which reduces the turnover of ACS6 by the ubiquitin-proteasome degradation machinery. In addition to protein phosphorylation, protein dephosphory- lation also plays critical role in ACS stability regulation. Recently, it was demonstrated that protein phosphatase 2A dephosphory- lates ACS2/ACS6 and destabilizes them, a critical process that counteracts with MAPK phosphorylation [30]. Members of the Type II group, including ACS5 and ACS9, are also regulated at protein stability levels, possibly by protein phosphorylation as well [15,31–33]. However, the kinase(s) involved remain unidentified. Because of the complex regulation of ACS protein/activity at multiple levels, many details about the up-regulation of ethylene biosynthesis remain unclear, including the specific ACS isoforms involved in the ethylene induction in response to a specific stimulus, the regulatory pathways that control the expression of ACS genes, and the components involved in the regulation of ACS protein stability. It has been known for decades that a subset of ACS genes, including Arabidopsis ACS6, is transcriptionally activated in plants under stress or pathogen attack. However, the importance of this transcriptional activation and the under- lying regulatory mechanism are not known. Furthermore, ethylene induction by different stimuli exhibits different kinetics and magnitude. The underlying molecular mechanism of such differential induction is also unclear. We are interested in the regulation of ethylene biosynthesis in plants infected by pathogens. ACS2 and ACS6, two Type I ACS isoforms, are involved in Botrytis-induced ethylene production [28]. The residual levels of ethylene induction in the acs2/acs6 double mutant suggest involvement of additional ACS isoforms. In this study, we investigated (1) the potential involvement of all ACS isoforms in ethylene induction triggered by B. cinerea infection, (2) the importance of transcriptional activation of ACS gene expres- sion, (3) the signaling pathways involved in the ACS gene activation, and (4) the molecular mechanism underlying the differential kinetics and magnitude of ethylene induction by different stimuli. We found that members in all three ACS groups are involved in pathogen-induced ethylene production, with ACS2, ACS6, and ACS7 contributing the most to B. cinerea- induced ethylene production. Based on analyses of an ACS6 knockdown mutant and of conditional gain-of-function ACS6 transgenic lines, we also can conclude that the transcriptional activation of the ACS6 gene plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only function in the phosphorylation-induced stabilization of ACS2/ACS6 proteins, but also signal the ACS2 and ACS6 gene activation after B. cinerea infection. WRKY33, a MPK3/MPK6 substrate that regulates camalexin biosynthesis [34], is also responsible for turning on ACS2/ACS6 expression downstream of MPK3/MPK6 cascade. WRKY33 binds to the W-boxes in the ACS2/ACS6 promoters in vivo and is directly involved in MPK3/ MPK6-induced ACS2/ACS6 gene expression. The duration and magnitude of MPK3/MPK6 activation vary with different stimuli and correlate well with the duration and magnitude of ethylene induction. Regulation of ACS activity at multiple levels by the MPK3/MPK6 cascade is an important mechanism by which the PLoS Genetics | www.plosgenetics.org 2 June 2012 | Volume 8 | Issue 6 | e1002767