Figure 6.

ACX1A Catalyzes an Essential Step in JA Biosynthesis.

(A) In vitro metabolism of OPC8 and OPDA in a coupled ACS-ACX reaction. The indicated substrates (5 μL in 0.25% Triton X-100) or mock control were added to a reaction mixture containing yeast ACS, CoA, and ATP. This reaction proceeded for 0.5 h at 25°C, at which time the ACX reaction was initiated by addition of 1 μg of purified LeACX1A. ACX1A-catalyzed production of H₂O₂ was monitored spectrophotometrically as described in Methods.

(B) JA levels in response to wounding. Two-leaf-stage wild-type (closed circles) and acx1 (open circles) plants were mechanically wounded with a hemostat at the distal end of each leaflet. Leaf tissue was harvested for quantification of JA levels at various times after wounding. Unwounded plants were used for the 0 h time point. Data show the mean ± sd of three independent sample preparations and are representative of three independent experiments. FW, fresh weight.

(C) OPDA levels in response to wounding. Two-leaf-stage wild-type (closed bars) and acx1 (open bars) plants were mechanically wounded as described above, and OPDA levels were measured 1 h after wounding (wounded). OPDA was also extracted from leaves of unwounded plants (control). Data show the mean ± sd of three independent samples and are indicative of three independent experiments.