Fig. 1. AtACS7 has ACS and C_β-S lyase dual enzymatic activities both in vitro and in planta.

(A) Chemical reactions catalyzed by ACSs (top) or C_β-S lyases (bottom). (B) Chromatograms of NH4⁺ produced from the C_β-S bond cleavage of l-cystine, which is catalyzed by either PpACL1 or AtACS7 in vitro. (C) In vitro pyruvate production assays of PpACL1- or AtACS7-containing C_β-S lyase reaction systems. (D) In vitro ACS activity assays of PpACL1 and AtACS7. (E to G) The prominent accumulation of AtACS7 protein resulted in a strong triple-response phenotype, an elevated ethylene production, and a significant increase in the content of pyruvate in the etiolated seedlings of two independent AtACS7-overexpressing lines (AtACS7-ox-3-4 and AtACS7-ox-7-5). (H) The C_β-S lyase activity of purified AtACS7 was PLP dependent. (I) Exogenous AVG totally suppressed the C_β-S lyase activity of purified AtACS7. (J and K) Determination of the optimal temperature and pH for C_β-S lyase activity of purified AtACS7. (L) Estimation of kinetic parameters of C_β-S lyase activity of purified AtACS7 under the optimal temperature and pH conditions. The C_β-S lyase activities were measured by the generation of pyruvate using l-cystine as substrate. Data represent means ± SE (n ≥ 3, biological replicates). The number of biological replicates for each experiment is indicated in Materials and Methods. Asterisks indicate statistically significant differences based on Student’s t test (α = 0.01). For negative control (NC), blank buffer was used instead of purified proteins.