Jeannette M. Arteca

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 λgt11 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 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

Abstract1-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 μM 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 μM IAA.

Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants

Inflorescence stalks produced the highest amount of ethylene in response to IAA as compared with other plant parts tested. Leaf age had an effect on IAA-induced ethylene with the youngest leaves showing the greatest stimulation. The highest amount of IAA-induced ethylene was produced in the root or inflorescence tip with regions below this producing less. Inflorescence stalks treated with IAA, 2,4-D, or NAA over a range of concentrations exhibited an increase in ethylene production starting at 1 μM with increasingly greater responses up to 100 μM, followed by a plateau at 500 μM and a significant decline at 1000 μM. Both 2,4-D and NAA elicited a greater response than IAA at all concentrations tested in inflorescence stalks. Inflorescence leaves treated with IAA, 2,4-D, or NAA exhibited the same trend as inflorescence stalks. However, they produced significantly less ethylene. Inflorescence stalks and leaves treated with 100 μM IAA exhibited a dramatic increase in ethylene production 2 h following treatment initiation. Inflorescence stalks showed a further increase 4 h following treatment initiation and no further increase at 6 h. However, there was a slight decline between 6 h and 24 h. Inflorescence leaves exhibited similar rates of IAA-induced ethylene between 2 h and 24 h. Light and high temperature caused a decrease in IAA-induced ethylene in both inflorescence stalks and leaves. Three auxin-insensitive mutants were evaluated for their inflorescences responsiveness to IAA. aux2 did not produce ethylene in response to 100 μM IAA, while axr1-3 and axr1-12 showed reduced levels of IAA-induced ethylene as compared with Columbia wild type. Inflorescences treated with brassinolide alone had no effect on ethylene production. However, when brassinolide was used in combination with IAA there was a dramatic increase in ethylene production above the induction promoted by IAA alone.

Identification of two new members of the l-aminocyclopropane-l-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.

Use of a Monoclonal Antibody for the Determination of Free Indole-3-Acetic Acid

Summary The purpose of this study was to produce a highly specific monoclonal antibody against free indole-3-acetic acid (IAA) and to develop a competitive enzyme linked immunosorbent assay (CELIA). Hybridomas were produced by fusion of NS 1 myeloma cells with spleen cells from BALB/cj mice immunized with IAA conjugated to rat serum albumin via the indole nitrogen. A highly specific cell line secreting IgG 1 chain specific kappa light chain for IAA was obtained. This cell line has little or no cross reactivity with 2,4-dichlorophenoxyacetic acid (2,4-D) and structurally related indole compounds (including amino-acid conjugates). The standard competitive ELISA assay provides sensitivity in the pmol range. Crown gall tumor derived from mung bean ( Vigna radiata cv. Berken) stem tissue extracts were passed through SepPak β-Bondapak C-18 cartridges, Sephadex G-10, carboxymethyl-cellulose (CMC), polyvinylpolypyr-rolidone (PVPP) and DEAE-cellulose columns individually and in combination in order to evaluate what degree of purification was required in order to avoid interference with the immunological assay. It was found that SepPak and PVPP in combination provided an adequate clean-up for use with plant samples.

A 1-aminocyclopropane-1-carboxylate oxidase cDNA sequence from Pelargonium.

Specialist propagators have the ability to rapidly produce high-quality, disease-free Pelargonium plants. However, obtaining uniform plants from cuttings and minimizing peta1 shattering in Pelargonium plants that have been shipped long distances or stored is a major practical problem because the plants are exposed to darkness, transit vibration, water stress, and temperature extremes (Marousky and Harbaugh, 198 1). Peta1 shattering and yellowing leaves reduce the acceptability of plants and cuttings to purchasing growers. Many of these factors are caused by the production of ethylene, which can accumulate in the storage/shipping container. Some storage techniques have been developed to delay senescence and maintain the quality of plant materials; however, these techniques had limited success maintaining the quality of Pelargonium plants during storage. Nonspecific inhibitors (eg. silver thiosulfate, aminoethoxyvinylglycine, or aminooxyacetic acid) usually cause side reaction problems such as reduced rooting, increased susceptibility to Pythium (White, 1992), and others. At the present time the best way to reduce ethylene injury on Pelargonium plants is to keep them at low temperatures. However, during shipment there is little control over temperatures and during routine storage temperature control may not always be practical. The development of antisense technology to produce transgenic Pelargonium plants may be a viable solution to this problem. Ethylene biosynthesis requires ACC synthase to convert Sadenosylmethionine to ACC and ACC oxidase to convert ACC to ethylene (Yang and Hoffman, 1984). Using published amino acid sequences of ACC oxidase, we designed two degenerate oligonucleotide primers from conserved amino acid regions (DWESTF and KEPRFE). PCR-based amplification was camed out using these oligonucleotides as primers and mung bean genomic DNA as a template (J.M. Arteca and R.N. Arteca, unpublished data). The PCR product was sequenced to confirm its homology to ACC oxidase as mentioned above and used to screen a stress-induced Pelargonium cDNA library in Xgtll (T.-W. Wang and R.N. Arteca, unpublished data). Three clones were isolated and found to be identical by sequence analysis. The longest cDNA insert (GEFE-1) was 1291 bp long and contained an open reading frame encoding 318 amino acids (Table I). Information on the expression of this gene and ACC synthase will allow us

Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants

The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.