ACS is an enzyme with extremely high specific activity and its protein level is very low in plant cells even at induced state. Making things even more difficult, the molecular weight of ACS is similar to that of the large subunit of Rubisco, the most abundant protein in plants. These make it very difficult to visualize the ACS protein from green plant tissues by immunoblot analysis. In this paper, we described the immune-complex ACS assay, which is an ACS activity assay coupled to the immunoprecipitation with a member-specific ACS antibody. By taking advantage of the specificity of a member-specific antibody and the high specific activity of ACS enzyme, this method allows the detection of a specific ACS member in plant cells. In Arabidopsis, ACS is encoded by a small gene family with at least nine different members.

Ethylene, a gaseous plant hormone, plays vital roles in plant growth, development, and response to stress. Recent genetic studies revealed a number of important signaling components in the pathways downstream of ethylene. As Dr. Hans Kende pointed out, "Ethylene-regulated processes are mostly initiated by an increase in ethylene synthesis. Ethylene synthesis is under the control of environmental and/or endogenous signals. Thus, to understand ethylene responses, it will be necessary to identify the exogenous and endogenous factors that control ethylene synthesis and to elucidate the signal transduction pathways that lead to an induction of ethylene biosynthesis (Kende, H. 2001. Plant Physiol. 125: 81-84)." This paper revealed one such pathway.

The major discovery in this paper is a result of good detective work. We first noticed that selected lines of the gain-of-function NtMEK2 transgenic tobacco plants showed ethylene-induced morphology. By following this phenotype, we determined how MAPK activation induced ethylene biosynthesis. In collaboration with Dr. Robert Sharp's lab (Division of Plant Sciences, University of Missouri-Columbia), we first demonstrated that the ethylene production is greatly enhanced after the activation of SIPK/WIPK, the two downstream MAPKs of NtMEK2. By following the biosynthetic enzymes in the ethylene biosynthetic pathway, we found that a subset of ACS isoforms can be directly phosphorylated by MPK6. The phosphorylation stabilizes the ACS protein and leads to ethylene induction.