A multi-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase (ACS6) in mature Arabidopsis leaves.

Physiological and biochemical studies have provided evidence that mechanical strain (touch)-induced modifications in plant growth and development may be due to ethylene. In order to better understand the involvement of ethylene in touch-induced responses, we identified and characterized an Arabidopsis cDNA (ACS6) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase which is an important regulatory enzyme in the ethylene biosynthetic pathway. Northern analysis showed that ACS6 was induced by touch in the leaves of 3-week old light-grown plants within 5 min and reached maximum transcription at 15 min. ACC, which is the product of ACC synthase and the immediate precursor to ethylene, exhibited a dramatic rise between 15 and 30 min after touch stimulation. Experiments with multiple touch treatments showed that a saturation in gene expression was obtained with one touch treatment and subsequent touch stimulations were progressively less effective in promoting ACS6 expression. Additional characterization of ACS6 gene expression indicated that the gene is also induced by wounding, and by treatment with LiCl, NaCl, CuCl2, auxin, cycloheximide (CHX), aminooxyacetic acid (AOA) and ethylene. ACC levels were also increased in response to each of these treatments with the exception of CHX and AOA which resulted in a decrease and no effect, respectively. Our results show that ACS6 is rapidly turned on in response to touch which is followed by an increase in ACC which is the immediate precursor to ethylene, thereby providing evidence that it is responsible for touch-inducible ethylene production in light-grown Arabidopsis plants. The identification and characterization of ACS6 now provides us with a tool to better understand the involvement of ethylene produced in response to external stimuli as well as during plant growth and development.

Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata).

Protein kinases are important in eukaryotic signal transduction pathways. In this study we designed degenerate oligonucleotides corresponding to two conserved regions of protein kinases and using the polymerase chain reaction (PCR) have amplified a 141 bp fragment of DNA from mungbeans (Vigna radiata Rwilcz cv. Berken). Sequence analysis of the PCR products indicates that they encode several putative protein kinases with respect to their identity with other known plant protein kinases. Using one of the six fragments (CPK3-8), we isolated a 2022 bp cDNA (VrCDPK-1) from a Vigna radiata lambda gt11 library. VrCDPK-1 has a 96 bp 5'-untranslated region and a 465 bp 3'-untranslated region and an open reading frame of 1461 bp. VrCDPK-1 contains all of the conserved regions commonly found in calcium dependent protein kinases (CDPK). VrCDPK-1 shares 24 to 89% sequence identity with previously reported sequences for plant CDPKs at the protein level. Southern analysis revealed the presence of several copies of the CDPK gene. VrCDPK-1 expression was stimulated when mungbean cuttings were treated with CaCl2, while treatment with MgCl2 had no effect. We are reporting for the first time a CDPK gene in mungbean which is inducible by mechanical strain. Cuttings treated with indole-3-acetic acid (IAA) or subjected to salt stress showed an increase in VrCDPK-1 expression. There was a dramatic stimulation in VrCDPK-1 expression 6 h after cuttings were treated with cycloheximide.

Identification of two new members of the 1-aminocyclopropane-1-carboxylate synthase-encoding multigene family in mung bean.

The key enzyme regulating ethylene biosynthesis in higher plants is 1-aminocyclopropane-1-carboxylate (ACC) synthase. In mung bean (MB), the existence of three genes encoding this enzyme has previously been reported [Botella et al., Plant Mol. Biol. 18 (1992) 793-797], one of which corresponds to a full-length indole-3-acetic acid-inducible cDNA [Botella et al., Plant Mol. Biol. (1992) 425-436]. In this paper we report the cloning of two new genomic sequences coding for ACC synthase in MB (MAC-4 and MAC-5). MAC-4 is 1340 bp long and encodes 388 amino acids (aa) while MAC-5 is 1393 bp long and encodes for 391 aa. Genomic Southern analysis suggests the existence of only one copy of each gene in the genome.

Identification and characterization of a full-length cDNA encoding for an auxin-induced 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments and expression of its mRNA in response to indole-3-acetic acid.

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. The identification and characterization of a full-length cDNA (pAIM-1) 1941 bp in length for indole-3-acetic acid (IAA)-induced ACC synthase is described in this paper. The pAIM-1 clone has an 87 bp leader and a 402 bp trailing sequence. The open reading frame is 1452 bp long encoding for a 54.6 kDa polypeptide (484 amino acids) which has a calculated isoelectric point of 6.0. In vitro transcription and translation experiments support the calculated molecular weight and show that the enzyme does not undergo processing. Eleven of the twelve amino acid residues which are conserved in aminotransferases are found in pAIM-1. The sequence for pMAC-1 which is one of the 5 genes we have identified in mung bean is contained in pAIM-1. pAIM-1 shares between 52 to 65% homology with previously reported sequences for ACC synthase at the protein level. There is little detectable pAIM-1 message found in untreated mung bean tissues; however, expression is apparent within 30 min following the addition of 10 microM IAA reaching a peak after approximately 5 h with a slight decrease in message after 12 h. These changes in message correlate with changes in ACC levels found in these tissues following treatment with 10 microM IAA.