The non-catalytic N-terminal domain of ACS7 is involved in the post-translational regulation of this gene in Arabidopsis

Abstract

Post-transcriptional control of the expression of the 1-aminocyclopropane-1-carboxylate synthase (ACS) gene family is important for maintaining appropriate levels of ethylene production. However, the molecular mechanism underlying the post-transcriptional regulation of type 3 ACS proteins remains unclear. Multiple sequence alignment revealed that the N-terminus of type 3 ACSs was longer than that of other ACSs. Fusing the N-terminal 54 residues of ACS7, the sole type 3 ACS in Arabidopsis, to the β-glucuronidase (GUS) reporter significantly decreased the stability of N(7(1-54))-GUS fusion protein. Among these 54 residues, residues 1-14 conferred this negative effect on the GUS fusion gene. Consistently, a truncated form of ACS7 lacking residues 1-14 was 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. Interestingly, the ACS7 N-terminus had no effect on the stability of N(7)-GUS and ACS7 proteins at the etiolated seedling stage. Both exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) treatment and salt stress could rescue the levels of accumulation of N(7)-GUS fusion protein in light-grown seedlings. These results suggest that the non-catalytic N-terminus of ACS7 is involved in its own post-translational regulation. The proteasome inhibitor MG132 suppressed degradation of full-length ACS7 in vivo, but had little effect on the N-terminal truncated form of ACS7, indicating that the N-terminus mediates the regulation of ACS7 stability through the ubiquitin-26S proteasome pathway.

Producing the Ethylene Signal: Regulation and Diversification of Ethylene Biosynthetic Enzymes

“…hormone levels, developmental transitions). Light affects ACS stability both positively and negatively; for instance, turnover of ACS5 is decreased (Yoon and Kieber, 2013a), whereas ACS7 turnover is increased (Xiong et al, 2014) by light. Exogenous cytokinin and brassinosteroid treatments increase ethylene production via posttranslational stabilization (Woeste et al, 1999;Hansen et al, 2009), and pathogen infection increases ACS abundance at both transcriptional and posttranslational levels (Han et al, 2010;Li et al, 2012).…”

“…Predicted target sites for regulators are present (*) or absent (gray shading) but untested in several ACS isozymes. Protein half-lives have been measured for ACS6, SlACS2, ACS7, and ACS5 (Chae et al, 2003;Joo et al, 2008;Kamiyoshihara et al, 2010;Xiong et al, 2014); for epitopetagged reporters, the tag is indicated (myc, HA, or Flag). C, A complex overlay of motifs surrounds the TOE sequence in type 2 isozymes.…”

“…N-terminal sequences of Arabidopsis ACS7 (AtACS7) and type 3 isoforms from several other species were aligned manually; the alignment shown ends at the first residue of the conserved Box1 motif (Yamagami et al, 2003), and the consensus sequence (ConS) shows invariant (underlined), strongly conserved ($9/11, uppercase), and weakly conserved ($6/11, lowercase) residues. The first 14 amino acids (bold face) in ACS7 constitute an N-terminal stability determinant (ACS7-N 14 ; Xiong et al, 2014). Sequences in the N-terminal extension (pink shading) are not conserved, and similarity to the ACS7-N 14 motif is found only in sequences from cruciferous species.…”

“…Similar to the recognition of TOE motifs by ETO/ EOL proteins, XBAT recognition of ACS7 can be reconstituted in a yeast two-hybrid assay and in vitro, and has been assayed using N-terminally tagged ACS7 constructs (Prasad et al, 2010;. Interestingly, though, turnover of a C-terminally epitopetagged ACS7 requires a motif contained within the 14 N-terminal amino acids (ACS7-N 14 ; Xiong et al, 2014). Neither the XBAT32 dependence of ACS7-N 14 -mediated turnover nor the ACS7 dependence of ethylene overproduction in xbat32 mutants has been directly tested, thus it is unclear whether these constitute a single regulatory mechanism or two different turnover systems for ACS7.…”

“…Neither the XBAT32 dependence of ACS7-N 14 -mediated turnover nor the ACS7 dependence of ethylene overproduction in xbat32 mutants has been directly tested, thus it is unclear whether these constitute a single regulatory mechanism or two different turnover systems for ACS7. The N-terminal sequence of type 3 isozymes is very poorly conserved (Xiong et al, 2014), and sequences resembling the ACS7-N 14 motif are found only in type 3 isozymes from species within the Brassicaceae (Fig. 2D).…”