levels/kinetics of ethylene production are regulated during plant stress/defense response. Results Activation of ACS gene expression in B. cinerea–infected Arabidopsis Our previous research demonstrated involvement of ACS2 and ACS6 in ethylene induction in B. cinerea-infected Arabidopsis [28]. This research also implicated the involvement of additional ACS genes since there was still an approximately 25% residual level of ethylene induction in the acs2/acs6 double mutant. To identify the ACS isoforms involved, we profiled the expression of all nine ACS genes in Arabidopsis infected with B. cinerea. As shown in Figure 1A, transcripts of ACS2, ACS6, ACS7, and ACS8 accumu- lated approximately 1600, 200, 50, and 1200 fold, respectively, over their basal levels. ACS11 transcript also accumulated about 6 fold. ACS5 and ACS9 transcripts could be reliably detected, but no increases were observed. In contrast, ACS1 and ACS4 transcripts were not detectable. To better assess the potential contribution of each ACS gene to ethylene production, we also calculated their expression levels relative to that of EF1a (Figure 1B). This calculation allowed us to compare the relative levels of expression between different ACS genes. From this dataset, we found that the expression levels of ACS2, ACS6, and ACS7 were among the highest. ACS8 and ACS11 had lower levels of expression after induction, while ACS5 and ACS9 expression remained very low. The expression of ACS8 increased more than 1200 fold relative to its basal level (Figure 1A). However, because of its low basal level expression, the induced level of ACS8 transcript was still much Dual-Level Regulation of Ethylene Biosynthesis lower than those of ACS2, ACS6, and ACS7 (Figure 1B). If regulation at other levels is the same, ACS8 is likely a minor contributor despite the high-fold induction. In contrast, levels of ACS7 transcript were considerably elevated (Figure 1B), despite a relatively low fold induction (Figure 1A), a result of a relatively high basal level. Based on these results, we speculated that ACS7 might be a major contributor to ethylene induction after plant sensing of pathogen invasion besides ACS2 and ACS6. B. cinerea–induced ethylene production involves ACS isoforms in all three groups To establish the involvement of ACS7, we identified two mutant alleles of ACS7, acs7-1 (FLAG_431D05, in Ws-0 background) and acs7-2 (CSHL_ET5768, in Ler-0 background). In both mutant alleles, B. cinerea-induced ethylene production is slightly reduced (Figure S1), similar to that in the acs2 or acs6 single mutant [28]. This result suggests that ACS7 also contributes to B. cinerea-induced ethylene production. In our previous publications [16,28], we did not assign allele numbers to the acs2 and acs6 mutants. To be consistent with the nomenclature used in Dr. Theologis’s lab [35], the acs6 allele (Salk_090423) was given an allele number of acs6-2. This allele turned out to be a knockdown mutant (more discussion later). In contrast, the acs6-1 mutant allele (SALK_025672) in the study by Tsuchisaka et al. (2009) is a mutant with a T-DNA insertion in the open reading frame (ORF) [35]. We failed to identify any plant with a T-DNA insertion when we initially ordered this line from the Arabidopsis Biological Resource Center (ABRC) in 2003. The acs2 and acs7-1 mutant alleles we used [16,28](Figure S1) are the same as the acs2-1 and acs7-1 alleles, respectively, in Tsuchisaka et al. (2009) report [35]. Figure 1. Activation of ACS gene expression in Arabidopsis after B. cinerea infection. (A) Twelve-day-old Arabidopsis seedlings grown in GC vials were inoculated with B. cinerea spores. Samples were collected at indicated times for total RNAs isolation. 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 are expressed as percentage of EF1a transcript, which allows comparison of expression levels between 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.g001 PLoS Genetics | www.plosgenetics.org 3 June 2012 | Volume 8 | Issue 6 | e1002767