Ethylene plays an important role in plant disease resistance. Using a high-order acs mutant, Tsuchisaka et al. (2009) demonstrated that ethylene production is essential to plant resistance against B. cinerea. However, ethylene induction was not examined in the study. In this report, we demonstrate that ethylene induction in acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9-1/ acs7-1/acs11-1 mutant plants after B. cinerea infection is only at ,2% of that in the wild type (Figure 3). Plant sensing of abiotic stress stimuli or invading pathogens triggers a number of signaling events. Among them, the activation of MAPK cascades and calcium influx are two of the earliest [21,24,52]. Our research demonstrates the regulation of ACS2 and ACS6 by a specific MAPK cascade at both transcriptional and post-translational levels. This pathway contributes ,85% of the total ethylene induction in plants challenged by pathogens. Regulation of the remaining ACS isoforms is unclear at present. Additional studies, including identification of the signaling pathway(s) involved in regulation of ACS7, ACS8, and ACS11 expressions, protein phosphorylation, and protein stability, are needed to further our understanding of the complex regulation of ethylene induction during the plant stress/defense response. Materials and Methods Plant growth conditions and treatments Soil-grown plants were maintained at 22uC in a growth chamber with a 14-hr light cycle (100 mE/m22 sec21). For experiments, seeds were surface-sterilized. After imbibition at 4uC for 3–5 days, seeds were sown in petri dishes with liquid half-strength Murashige and Skoog (MS) medium and grown in a growth chamber at 22uC with continuous light (70 mE/ m22 sec21). Five-day-old seedlings were transferred to 20-ml GC vials with 6 ml of liquid half-strength MS medium (10 seedlings per vial) and maintained under the same growth conditions. Twelve- to fourteen-day-old seedlings grown in GC vials were used for experiments. Procedures for Botrytis cinerea (Strain: DSM 4709) maintenance and spore preparation were described previously [28]. Twelve- day-old seedlings grown in GC vials were inoculated with B. cinerea spores at a final concentration of 4.06105 spores/vial. Induction of DD and ACS6 expressions in GVG-NtMEK2DD and GVG-ACS6 transgenic plants was performed by the addition of DEX stock solution (5 mM in ethanol) to a final concentration of 1 mM. An equal volume of ethanol was used as a negative (2DEX) control. At least two independent repetitions were performed with similar results for experiments with multiple time points. For single time-point experiments, at least three independent repetitions were performed. Mutant lines and generation of transgenic plants Arabidopsis thaliana Columbia (Col-0) ecotype was used as the wild-type control, unless stated otherwise. T-DNA insertion mutant alleles of MPK3 (At3g45640), MPK6 (At2g43790), ACS1 (At3g61510), ACS2 (At1g01480), and ACS6 (At4g11280) were described previously [16,28,39]. The two ACS7 (At4g26200) mutant alleles, acs7-1 (FLAG_431D05) and acs7-2 (CSHL_ET5768), were obtained from INRA and Cold Spring Harbor Laboratory, respectively. High-order acs mutants gener- ated in Dr. Athanasios Theologis’ laboratory [35] were obtained from the Arabidopsis Biological Resource Center (ABRC). The stock numbers are CS16564 (acs2-1), CS16569 (acs6-1), CS16581 (acs2-1/acs6-1), CS16603 (acs2-1/acs6-1/acs4-1), CS16607 (acs2-1/ acs6-1/acs5-2), CS16609 (acs2-1/acs6-1/acs9-1), CS16644 (acs2-1/ acs6-1/acs4-1/acs5-2/acs9-1), CS16649 (acs2-1/acs6-1/acs4-1/acs5- Dual-Level Regulation of Ethylene Biosynthesis 2/acs9-1/acs7-1), CS16650 (acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/ acs9-1/acs7-1), and CS16651 (acs1-1/acs2-1/acs6-1/acs4-1/acs5- 2/acs9-1/acs7-1/acs11-1). Conditionally rescued mpk3/mpk6 dou- ble mutant was generated by transformation of DEX-inducible promoter driven MPK6 cDNA (GVG-MPK6) into mpk32/2/mpk6+/2 plants [39]. Double mutant seedlings were recovered from seeds of mpk32/2/mpk6+/2/GVG-MPK6 plants sprayed with 30 mM DEX during the flowering stage. GVG-NtMEK2DD (abbreviated as DD), DD/mpk3, and DD/mpk6 lines were previously described [28,38]. The DEX-inducible promoter driven ACS6 construct (GVG- ACS6) was generated by cloning the ACS6 ORF with a 4xmyc tag [29] into the Xho I/Spe I sites of the pTA7002 vector [53]. The binary vector was transformed into Agrobacterium tumefaciens strain GV3101. Arabidopsis transformation was performed by the floral dip procedure [54], and transformants were identified by screening for hygromycin resistance. Independent lines with ACS6 transgene induction were identified based on real-time qPCR analysis. Ethylene measurement GC vials with Arabidopsis seedlings were flushed and capped immediately after treatment. At indicated times, ethylene levels in the headspace of the GC vials were measured by gas chroma- tography as previously described [16]. Seedlings were then collected, weighed, frozen in liquid nitrogen, and stored at 280uC for future analysis. RNA extraction and real-time PCR analysis Total RNA was extracted using the Trizol reagent (Invitrogen). After DNase treatment, RNA (2 mg) was used for reverse transcription. Real-time PCR analysis was performed using an OpticonTM 2 real-time PCR machine as described previously [38]. The transcript of the EF1a gene was used to normalize the samples. Relative gene expression was calculated using two different methods. The first method is the commonly used double DCt method, which gives fold of gene induction relative to basal level before treatment (0 hr time point). The second method expresses the transcript level relative to that of the EF1a gene in the same sample, which is a better method when comparison of expression levels of different genes is necessary. The primers used for real-time PCR were ACS1 (At3g61510, 59-ACGCTTT TCTCGTCCCTACTC-39 and 59-GGCCTTAAGGTACGCT- GATTC-39), ACS2 (At1g01480, 59-GGATGGTTTAGGATTT GCTTTG-39 and 59-GCACTCTTGTTCTGGATTACCTG- 39), ACS4 (At2g22810, 59-AACAACCTTGTGCTCACTGCT-39 and 59-AGATCCCTATCAAACCCTGGA-39), ACS5 (At5g 65800, 59-GACTCTCATGTTTTGCCTTGC-39 and 59-TTGG AAGCCATTAGAGCTTGA-39), ACS6 (At4g11280, 59-GTTC CAACCCCTTATTATCC-39 and 59-CCGTAATCTTGAACC- CATTA-39), ACS7 (At4g26200, 59-ACGGTACGATACCATTG TGGA-39 and 59-GCTCGCCGTCTTTAGTTTTCT-39), ACS8 (At4g37770, 59-CCTTCCTTCCTTCAAGAATGC-39 and 59- GAGAGTCTCGTTAGCCGGAGT-39), ACS9 (At3g49700, 59- CATACCTCGACGAAAACCAGA-39 and 59-TCATGTCAA CCCAACAGAACA-39), ACS11 (At4g08040, 59-CAAACGATG- GAGGTTGCTATG-39 and 59-TTGGAGACCCATTTGTTGA TAAG-39), and EF1a (At5g60390, 59-TGAGCACGCTCT TCTTGCTTTCA-39 and 59-GGTGGTGGCATCCATCTTGT TACA-39). ChIP–qPCR analysis F1 plants generated from the cross of wrky33/4myc-WRKY33 and DD lines were used for the ChIP assay. Two-week-old PLoS Genetics | www.plosgenetics.org 12 June 2012 | Volume 8 | Issue 6 | e1002767