Elucidation Of Mechanisms Underlying The Post-transcriptional Regulation Of ACS7in Arabidopsis

The gaseous phytohormone ethylene is an important regulator of plant growth and development, and it also regulates plant responses to biotic/abiotic stresses. One important mechanism that modulates the production of ethylene in plant is the differential regulation of the expression and enzymatic activity of ACS (1-aminocyclopropane-1-carboxylate synthase), the rate-limiting enzyme of ethylene biosynthesis. ACS is encoded by a multigene family, and the expressions of ACS genes are highly regulated at both transcriptional and post-transcriptional levels. Based on their non-catalytic C-terminal sequences, ACS proteins are classified into type1, type2, and type3. The characteristics of their C-termini determine the mechanisms of their post-translational regulation. In the model plant, Arabidopsis thaliana, all types of ACS proteins have been demonstrated to be degraded through the ubiquitin/26S proteasome pathway. In type1and type2ACS isozymes, the C-terminal domains are involved in the regulation of proteins stability as the targeted sites for ubiquitination by E3ligases and phosphorylation through mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs). However, the sequences involved in the post-translational regulation of type3ACS proteins, have still not been identified. In this study, we used ACS7, the only type3ACS protein in Arabidopsis, to elucidate the molecular mechanism underlying the post-translational regulation of type3ACS proteins.Multiple sequence alignment of type3ACS proteins from different plant species with eight other functional ACS proteins from Arabidopsis revealed that the N-terminus of type3ACSs was longer than that of other ACSs. To investigate the longer N-terminal region role in the post-translational regulation of type3ACS proteins, we fused the N-terminal54residues of Arabidopsis ACS7to β-glucuronidase (GUS) reporter and analyzed the effect of this fragment on the accumulation of the GUS fusion protein in vivo. Both the level of accumulation and the stability of GUS fusion protein in the light-grown ACS7:N7(1-54)-GUS transgenic seedlings were significantly lower than those of GUS in the ACS7:GUS control. Among54residues,1-14conferred negative effect on the GUS fusion gene. Further analysis showed that deletion the N-terminal1-14residues had no effect on the enzyme activity of ACS7. However, the truncated version of ACS7was more stable than full-length ACS7, when transgenically expressed in Arabidopsis and led to a more severe ethylene-response phenotype in the light-grown transgenic seedlings, confirming the involvement of the N-terminal14residues in the post-translational regulation of ACS7. The proteasome inhibitor MG132suppressed the degradation of full-length ACS7in vivo, whereas little effect was observed on the N-terminal truncated ACS7. These results suggest that the N-terminus mediates the regulation of ACS7stability through the ubiquitin/26S proteasome pathway.The ACS7N-terminus-mediated protein degradation was also demonstrated to be regulated by developmental and environmental signals. Both the full-length ACS7and N7(1-54)-GUS fusion proteins were more stable in the etiolated transgenic seedlings. N7(1-54)-GUS fusion protein accumulation level analysis in ACS7:N7(1-54)-GUS transgenic Arabidopsis at different developmental stages revealed that it was also higher at the late-stages of seed germination and in the old rosette leaves, whereas in young seedlings and young rosette leaves, the level was very low, even could not be detected. Light-grown transgenic seedlings treated with ACC (1-aminocyclopropane-1-carboxylic acid), under heat shock, dehydration and salt stresses inhibited the N-terminus-mediated degradation of GUS fusion protein and increased its accumulation, whereas dark treatment promoted its degradation. However, ACS7:N7(1-54)-GUS transgenic seedlings treated with auxin specifically induced the accumulation of GUS fusion protein in roots.The negative effect of the N-terminus of ACS7was further supported by the observation that fusing the N-terminal1-14residues of ACS7to SSPP (SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE), a negative regulator of Arabidopsis leaf senescence, effectively rescued the SSPP-induced phenotypes in young transgenic Arabidopsis plants, and partly retained the delayed-senescence phenotype. These results suggest that the ACS7N-terminus-mediated protein degradation is not ACSs specific, and it can also negatively regulate the stability of other plant proteins.