Figure 11. The acs6-2 mutant allele is a knockdown mutant. (A) B. cinerea-induced ethylene production in wild type, acs6-2 (SALK_090423), and acs6-1 (SALK_025672) plants. Twelve-day-old seedlings grown in GC vials were inoculated with B. cinerea spores. Ethylene accumulation in GC vials was monitored at indicated times, and seedlings were collected for gene expression analysis. Error bars indicate standard deviations (n = 3). (B) Induction of ACS6 expression in wild type (Col-0), acs6-2, and acs6-1 seedlings after B. cinerea inoculation. Total RNA was isolated from the seedlings collected in (A). Expression of the ACS6 gene was quantified by real-time PCR. ACS6 transcript levels were expressed as fold of induction relative to the zero time point (upper panel) and as a percentage of the EF1a transcript (lower panel). Error bars indicate standard deviations (n = 3). doi:10.1371/journal.pgen.1002767.g011 acs2-1/acs6-2 and acs2-1/acs6-1 also suggests that the acs6-2 allele is not a complete mutant, which is supported by the presence of ACS6 transcript in acs6-2 allele (Figure 11B). Since the T-DNA insertion in this allele is in front of the transcriptional starting site (Figure 9A), functional transcript is likely to be produced in this mutant allele. Nonetheless, the reduction of ethylene induction in the acs6-2 single mutant or in the acs2-1/acs6-2 double mutant [28](Figure 2) demonstrates the importance of high-level induction of ACS6 expression in pathogen-induced ethylene production. Interestingly, the reduction in ethylene induction in the acs2/acs6 double mutant is always more than the sum of the reduction in each single mutant (Figure 3) [28]. It is possible that the heterodimers of ACS2 and ACS6 are less active than each of homodimer. In the absence of one isoform, only the homodimer Dual-Level Regulation of Ethylene Biosynthesis can be formed, which partially compensates for the loss of the other isoform. Importance of dual-level regulation ACS isoforms in determining the levels and kinetics of ethylene induction Although very inefficient energy-wise, regulation of the ACS protein at the protein stability level by phosphorylation/ dephosphorylation allows rapid induction of ethylene biosynthesis, which can occur within minutes after plant sensing of external stimuli [43–45]. Such rapid response could be important to plant response to the stress/pathogen stimuli. However, even after phosphorylation stabilization, the ACS6 protein may not be very stable. The half-life of the phospho-mimicking ACS6DDD is only ,3 hours [29]. In the meantime, protein phosphatase 2A will counteract with the MAPKs by dephosphorylating the phospho- ACS protein [30]. Under this circumstance, transcriptional activation can provide another mechanism to further enhance the ethylene induction in response to pathogen infection. It is well known that stress-induced ethylene production follows different kinetics depending on the stimuli. However, the molecular mechanism underlying this difference is unclear. A good correlation exists between the kinetics of MPK3/MPK6 activation and ethylene induction. For instance, wounding induces a transient ethylene production, which is associated with a transient activation of MAPKs [46,47]. In contrast, infection of plants by pathogens, especially necrotrophic fungal pathogens, triggers a long-lasting and high-level induction of ethylene biosynthesis, which correlates with a long-lasting and high-magnitude activation of MPK3/MPK6 [28]. As depicted in Figure 12, MPK3 and MPK6 regulate ethylene induction via two different mechanisms: by direct phosphorylation and stabilization of ACS2 and ACS6 proteins [16,28,29] and by activation of ACS2 and ACS6 gene expression (this study). Transient activation of MPK3/MPK6 by wounding is also associated with the activation of ACS2/ACS6 gene expression [36]. However, due to the transient nature of MAPK activation, which returns to basal level within ,0.5 hr to 1 hr [47], the de novo synthesized ACS protein may not have the chance to be phosphorylated and will be degraded quickly in the absence of MAPK phosphorylation. In B. cinerea- infected plants, the induction of ACS2 and ACS6 gene expression will result in high rates of de novo protein synthesis. On top of this, the high-level and long-lasting activation of MPK3/MPK6 [28,38] can maintain de novo synthesized ACS2 and ACS6 proteins in a phosphorylated state and thereby stabilize the protein against proteasome-mediated degradation [16,29]. This dual-level regula- tory mechanism can maintain a greatly enhanced level of cellular ACS activity and ethylene production in pathogen-infected plants. Recently, it was shown that a PP2A protein phosphatase can counteract with MPK3/MPK6 by dephosphorylating ACS2/ACS6 and can destabilize the ACS protein [30]. In this situation, it is even more important to have the high-level, long-lasting activation of MPK3/MPK6 in order to maintain the ACS2/ACS6 protein in a phosphorylated state to ensure the high rate ethylene biosynthesis observed in plants challenged by pathogens. WRKY33 is a key transcriptional regulator downstream of MPK3/MPK6 in regulating gene expression in multiple pathways Activation of MPK3/MPK6 and their orthologs in other plant species induces the expression of large number of stress/defense related genes [48,49], suggesting the involvement of downstream transcription factors. ERF104 is a substrate of MPK6. Phosphor- ylation of ERF104 by MPK6 results in release of ERF104 from the PLoS Genetics | www.plosgenetics.org 10 June 2012 | Volume 8 | Issue 6 | e1002767