Domain Swapping between AtACS7 and PpACL1 Results in Chimeric ACS-like Proteins with ACS or Cß-S Lyase Single Enzymatic Activity.

The gaseous hormone ethylene plays a pivotal role in plant growth and development. In seed plants, the key rate-limiting enzyme that controls ethylene biosynthesis is ACC synthase (ACS). ACS has, for a long time, been believed to be a single-activity enzyme until we recently discovered that it also possesses Cß-S lyase (CSL) activity. This discovery raises fundamental questions regarding the biological significance of the dual enzymatic activities of ACS. To address these issues, it is highly necessary to obtain ACS mutants with either ACS or CSL single activity. Here, domain swapping between Arabidopsis AtACS7 and moss CSL PpACL1 were performed. Enzymatic activity assays of the constructed chimeras revealed that, R10, which was produced by replacing AtACS7 box 6 with that of PpACL1, lost ACS but retained CSL activity, whereas R12 generated by box 4 substitution lost CSL and only had ACS activity. The activities of both chimeric proteins were compared with previously obtained single-activity mutants including R6, AtACS7Q98A, and AtACS7D245N. All the results provided new insights into the key residues required for ACS and CSL activities of AtACS7 and laid an important foundation for further in-depth study of the biological functions of its dual enzymatic activities.

Dual activities of ACC synthase: Novel clues regarding the molecular evolution of ACS genes.

Ethylene plays profound roles in plant development. The rate-limiting enzyme of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), which is generally believed to be a single-activity enzyme evolving from aspartate aminotransferases. Here, we demonstrate that, in addition to catalyzing the conversion of S-adenosyl-methionine to the ethylene precursor ACC, genuine ACSs widely have Cß-S lyase activity. Two N-terminal motifs, including a glutamine residue, are essential for conferring ACS activity to ACS-like proteins. Motif and activity analyses of ACS-like proteins from plants at different evolutionary stages suggest that the ACC-dependent pathway is uniquely developed in seed plants. A putative catalytic mechanism for the dual activities of ACSs is proposed on the basis of the crystal structure and biochemical data. These findings not only expand our current understanding of ACS functions but also provide novel insights into the evolutionary origin of ACS genes.

N-Terminus-Mediated Degradation of ACS7 Is Negatively Regulated by Senescence Signaling to Allow Optimal Ethylene Production during Leaf Development in Arabidopsis.

Senescence is the final phase of leaf development, characterized by key processes by which resources trapped in deteriorating leaves are degraded and recycled to sustain the growth of newly formed organs. As the gaseous hormone ethylene exerts a profound effect on the progression of leaf senescence, both the optimal timing and amount of its biosynthesis are essential for controlled leaf development. The rate-limiting enzyme that controls ethylene synthesis in higher plants is ACC synthase (ACS). In this study, we evaluated the production of ethylene and revealed an up-regulation of ACS7 during leaf senescence in Arabidopsis. We further showed that the promoter activity of ACS7 was maintained at a relatively high level throughout the whole rosette development process. However, the accumulation level of ACS7 protein was extremely low in the light-grown young seedlings, and it was gradually restored as plants aging. We previously demonstrated that degradation of ACS7 is regulated by its first 14 N-terminal residues, here we compared the phenotypes of transgenic Arabidopsis overexpressing a truncated ACS7 lacking the 14 residues with transgenic plants overexpressing the full-length protein. Results showed that seedlings overexpressing the truncated ACS7 exhibited a senescence phenotype much earlier than their counterparts overexpressing the full-length gene. Fusion of the 14 residues to SSPP, a PP2C-type senescence-suppressed protein phosphatase, effectively rescued the SSPP-induced suppression of rosette growth and development but had no effect on the delayed senescence. This observation further supported that N-terminus-mediated degradation of ACS7 is negatively regulated by leaf senescence signaling. All results of this study therefore suggest that ACS7 is one of the major contributors to the synthesis of 'senescence ethylene'. And more importantly, the N-terminal 14 residue-mediated degradation of this protein is highly regulated by senescence signaling to enable plants to produce the appropriate levels of ethylene required.