Li(+)-regulated 1-aminocyclopropane-1-carboxylate synthase gene expression in Arabidopsis thaliana.

In Arabidopsis thaliana, 1-aminocyclopropane-1-carboxylate synthase (ACS) is encoded by a multigene family consisting of at least five members whose expression is induced by hormones, developmental signals, and protein synthesis inhibition. Li+, known to interfere with the phosphoinositide (PI) second messenger system by inhibiting the activity of inositol-phosphate phosphatases, is one of the strongest inducers of ACC synthase activity in plants. Treatment of etiolated Arabidopsis seedlings with LiCl results in a rapid induction of the ACS5 gene. Also, LiCl represses the cycloheximide (CHX)-induced accumulation of the ACS2 mRNA. The effects of Li+ on the expression of ACS5 and ACS2 are specific, dose-dependent, and can be reversed by Ca2+ and mimicked by the protein kinase inhibitor K-252a. The results suggest that the regulation of some ACS genes by various inducers may involve protein kinase activity, which in turn may be controlled through an inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ mobilization. Since plants contain no Li+, the cation appears to unmask pre-existing biochemical capacity that may be utilized by various unknown transducers during plant growth and development.

Characterization of two members (ACS1 and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana.

The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined. The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1. The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome. The chromosomal locations of the five members of the At ACS multigene family have been determined. The results show that each family member resides on a different chromosome. This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication. The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast. The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205. Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity. The results suggest that TNP is crucial for expression of ACS activity in E. coli.

LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum). Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis.

ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene and is encoded by a highly divergent multigene family in tomato (Rottmann, W. H., Peter, G. F., Oeller, P. W., Keller, J. A., Shen, N. F., Nagy, B. P., Taylor, L. P., Campbell, A. D., and Theologis, A. (1991) J. Mol. Biol. 222, 937-961). Two members of the family, LE-ACS2 and LE-ACS4, are induced during fruit ripening and upon treatment of mature green fruits with exogenous ethylene (C2H4) in a dose-dependent manner. Both genes are superinduced by wounding of pericarp tissue during various stages of ripening. The wound-induced accumulation of LE-ACS2 mRNA is more rapid and greater than that of LE-ACS4. Both mRNAs accumulate in the absence of protein synthesis, suggesting that their induction is a primary response to the inducer. The LE-ACS4 gene was isolated and structurally characterized. The function of the LE-ACS4 protein (53,509 Da, pI 5.4) was verified by expression experiments in Escherichia coli. The promoters of LE-ACS2 and LE-ACS4 contain potential cis-acting regulatory elements responsible for induction by ethylene, wounding, and anaerobiosis. In addition, elements for binding the transcriptional factors EmBP1, GBF-1, and OCSBF-1 are also present. Phylogenetic analysis of 20 ACC synthases from dicots and monocots indicate that the LE-ACS2 and LE-ACS4 proteins belong to an unique sublineage that includes an additional member of the tobacco family, NT-ACS1. The divergence of this sublineage is a relatively recent event in the evolution of ACC synthase protein.

The nucleotide sequence of the 5' flanking region of the Arabidopsis ACS2 gene.

The ACS2 gene of Arabidopsis thaliana encodes 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.1.1.14) - the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The gene is unique among ACC synthase genes isolated thus far in that it is highly expressed in both reproductive (e.g. flower) and vegetative tissues (e.g. leaves and roots). In addition, the accumulation of ACS2 transcripts is greatly induced by protein synthesis inhibition (Liang et al 1992). We report here the DNA sequence of the 5' flanking region of the ACS2 gene and an analysis of the sequence with respect to the potential cis-acting regulatory elements that can be recognized by DNA-binding proteins from plants and other organisms.

The 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana.

Genomic sequences encoding five divergent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase polypeptides (ACC1, ACC2, ACC3, ACC4, and ACC5) have been isolated from Arabidopsis thaliana by using heterologous cDNAs and PCR fragments amplified from genomic DNA with degenerate oligonucleotide primers. Each gene is located on a different chromosome in the Arabidopsis genome. The genes are differentially expressed during development and in response to environmental stimuli. Protein-synthesis inhibition derepresses the expression of all genes but most dramatically derepresses that of ACC2, suggesting that their expression may be under negative control. The sequence of ACC2 was determined, and its transcription initiation site was defined. Authenticity of the polypeptide encoded by the gene was confirmed by expression experiments in Escherichia coli. The predicted size of the protein is 55,623 Da, and it contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. Comparative analysis of structural and expression characteristics of ACC synthase genes from Arabidopsis and other plant species suggests that the sequence divergence of the ACC synthase genes and possibly the distinct regulatory networks governing the expression of ACC synthase subfamilies arose early in plant evolution and before the divergence of monocots and dicots.

1-aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence.

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.1.1.14). It catalyzes the conversion of S-adenosylmethionine to ACC, the precursor of ethylene. We isolated complementary DNA sequences, ptACC2 and ptACC4, for two distinct and differentially regulated ACC synthase mRNAs expressed in ripe tomato fruit. The authenticity of the clones has been confirmed by expression experiments in E. coli. The predicted size of the encoded polypeptides (54,690 and 53,519 Da) is similar to that of the primary in vitro translation products and to the proteins found in vivo. The sequence of the gene encoding one mRNA, LE-ACC2, has been determined and its transcription initiation site defined. Four additional genes, LE-ACC1A, LE-ACC1B, LE-ACC3 and LE-ACC4, have also been identified and the sequence of their coding regions determined. The LE-ACC1A and LE-ACC1B genes are adjacent to each other and are convergently transcribed. Their encoded polypeptides are 96% identical; the identity of the other polypeptides to each other varies between 50 and 70%. The proteins predicted to be encoded by the ACC synthase genes so far cloned from tomato and zucchini contain 11 of the 12 conserved amino acid residues found in various aminotransferases involved in the binding of the substrate and the cofactor pyridoxal-5'-phosphate. The data indicate that ACC synthase is encoded by a divergent multigene family in tomato that encodes proteins related to aminotransferases.