Dual-Level Regulation of ACC Synthase Activity by MPK3/MPK6 Cascade and Its Downstream WRKY Transcription Factor during Ethylene Induction in Arabidopsis Guojing Li1,2., Xiangzong Meng2., Ruigang Wang1., Guohong Mao2¤, Ling Han 2 , Yidong Liu2, 2 Shuqun Zhang * 1 College of Life Sciences, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China, 2 Division of Biochemistry, Interdisciplinary Plant Group, and Bond Life Sciences Center, University of Missouri, Columbia, Missouri, United States of America Abstract Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea–induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea–induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/ MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction. Citation: Li G, Meng X, Wang R, Mao G, Han L, et al. (2012) Dual-Level Regulation of ACC Synthase Activity by MPK3/MPK6 Cascade and Its Downstream WRKY Transcription Factor during Ethylene Induction in Arabidopsis. PLoS Genet 8(6): e1002767. doi:10.1371/journal.pgen.1002767 Editor: Joseph Kieber, The University of North Carolina at Chapel Hill, United States of America Received January 6, 2012; Accepted April 26, 2012; Published June 28, 2012 Copyright: 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NSF grants MCB-0543109 and IOS-0743957 to SZ. GL was supported by the Innovative Research Group Fund NDPYTD2010-3 from Inner Mongolia Agricultural University and a fellowship award from the China Scholarship Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zhangsh@missouri.edu ¤ Current address: Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America . These authors contributed equally to this work. Introduction The gaseous phytohormone ethylene profoundly impacts plant growth, development, and response to environmental stimuli [1– 7]. Studies from a number of labs have defined a signaling pathway—from ethylene receptors to downstream signaling components to transcription factors—that alters gene expression and leads to ethylene-induced phenotypes (reviewed in [1,2,5,8,9]). Ethylene-regulated responses are suppressed in the absence of ethylene, and such suppression is released upon plant sensing of ethylene. As a result, all ethylene-regulated processes begin with the induction of ethylene biosynthesis [10]. Plants under stress, including wounding, flooding, drought, osmotic shock, ozone, and pathogen/insect invasion, produce elevated levels of ethylene [1,6,7,11]. For this reason, ethylene is also known as a plant stress hormone. The biosynthetic pathway of ethylene has been fully elucidated for over two decades. Two enzymatic steps are unique to ethylene biosynthesis: conversion of S-adenosyl-methionine (SAM), a common metabolic precursor, to 1-amino-cyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS) and oxidative cleavage of ACC to form ethylene by ACC oxidase (ACO) [1,4,12]. ACS activity is very low in tissues that do not produce a large amount of ethylene and is enhanced under conditions that promote ethylene formation [1,4,12–14]. In contrast, ACO is constitutively present in most vegetative tissues. As a result, ACS is believed to be the committing and generally rate-limiting enzyme in ethylene biosynthesis. ACS is encoded by a small gene family in plants. In Arabidopsis, there are nine ACS members. Based on the presence/absence of phosphorylation sites in their C-termini, ACS isoforms are classified into three types [15]. Type I ACS isoforms, which PLoS Genetics | www.plosgenetics.org 1 June 2012 | Volume 8 | Issue 6 | e1002767