Inhibitory effects of auxins and related substances on the activity of an Arabidopsis glutathione S-transferase isozyme expressed in Escherichia coli

Glutathione S-transferases (GSTs; EC 2.5.1.18) are encoded by a gene family. Some GSTs have the capacity to bind to indole-3-acetic acid (IAA), whereas the gene expression of other GSTs is regulated by auxin. In order to assess a possible physiological significance of the auxin binding of GST, we investigated effects of auxins on the activity of GST expressed in Escherichia coli. cDNA cloning was carried out for the fifth gene ( GST5 ) of GST in Arabidopsis. Although the deduced amino acid sequence of GST5 was remotely related to that of the other Arabidopsis GSTs (less than 20% identical), the GST5 protein (GST5) expressed in E. coli showed GST activity. Apparent K_m values of GST5 are 0.86 and 1.29 m M for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene, respectively. IAA, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (1-NAA) and 2-NAA inhibited the enzyme activity competitively with respect to GSH. The apparent K_i of IAA is 1.56 m M . Salicylic acid inhibited GST activity in a noncompetitive manner. 2,4-D was the most inhibitory among the tested chemicals. GST5 bound to GSH-immobilized agarose gel was effectively eluted by IAA. These results indicate that IAA and the related substances bind to GST5 at the GSH-binding site, and exclude the possibility that the compounds could be substrates for GST5. Although the K_i value of IAA is too high for any physiological consequences, it might be assumed that GST activity is modulated in vivo by an auxin-related substance(s). The steady-state level of the GST5 mRNA was increased by wounding, heat shock, and spraying buffer on the plant, but was not influenced by auxin treatment.     

Gene expression during induction of somatic embryogenesis in carrot cell suspensions

We report the identification, via their cDNAs, of genes which are temporarily transcribed during the initiation of somatic embryogenesis in carrot (Daucus carota L.) cells cultured in an auxin-free medium. Their expression is roughly associated with the first morphogenetic, or globular, stage. A cDNA library ( gt 10) was established using poly(A)⁺ -rich RNAs from cells deprived of auxin for 8 d. By differential screening a number of clones corresponding to early-induced embryogenic genes were identified. For several a temporary accumulation of the specific mRNA between 6 and 16 d after induction was observed. With regard to the nucleotide sequence and the respective deduced amino-acid sequence, two glycine-rich proteins and a polypeptide with a proline-rich domain were among the products of genes activated at the onset of somatic embryogenesis.Abbreviations b, bp bases, basepairs - 2,4-D 2,4-dichlorophenoxyacetic acid Sequence data reported here will appear in the EMBL Genbank and DDBJ Nucleotide Sequence Databases under the following accession numbers: X 15436 for clone DC 2.15 (proline-rich protein), X 15706 for clone DC 7.1 (glycine-rich protein, DCGRP) and X 14067 for clone DC 9.1 (glycine-rich protein, DCGRP)This research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance.     

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.     

Formation of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) embryogenic callus involves peroxide-generating germin-like oxalate oxidase

In wheat ( Triticum aestivum L.), embryogenic callus formation comprises suppression of precocious germination by the zygotic embryo and the initiation of dedifferentiated cellular proliferation within it. Embryogenic calli are induced by treating immature embryos with 2,4-dichlorophenoxyacetic acid (2,4-D). Upon withdrawal from 2,4-D, somatic embryos develop from the periphery of the callus. Prior to visible callus formation, there is a striking induction of "germin-like" oxalate oxidase ("gl-OXO": EC 1.2.3.4) gene expression. Accumulation of gl-OXO mRNA is rapidly stimulated upon auxin treatment, with a consequent development of apoplastic enzyme activity producing H(2)O(2) within the cell wall. Within the dedifferentiated calli, gl-OXO enzyme activity becomes widespread over the surface of embryogenic calli. Differentiation of somatic embryos is initiated in regions of densely cytoplasmic, meristematic cells that are marked by highly localised expression of gl-OXO activity within these embryogenic cell masses. We suggest that this localised generation of H(2)O(2) by gl-OXO promotes peroxidative cross-linking of cell wall components, thereby preventing cellular expansion and maintaining these cell masses in an embryogenically competent condition.     

Mutant analysis in Arabidopsis provides insight into the molecular mode of action of the auxinic herbicide dicamba

Herbicides that mimic the natural auxin indole-3-acetic acid are widely used in weed control. One common auxin-like herbicide is dicamba, but despite its wide use, plant gene responses to dicamba have never been extensively studied. To further understand dicamba's mode of action, we utilized Arabidopsis auxin-insensitive mutants and compared their sensitivity to dicamba and the widely-studied auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The mutant axr4-2, which has disrupted auxin transport into cells, was resistant to 2,4-D but susceptible to dicamba. By comparing dicamba resistance in auxin signalling F-box receptor mutants (tir1-1, afb1, afb2, afb3, and afb5), only tir1-1 and afb5 were resistant to dicamba, and this resistance was additive in the double tir1-1/afb5 mutant. Interestingly, tir1-1 but not afb5 was resistant to 2,4-D. Whole genome analysis of dicamba-induced gene expression showed that 10 hours after application, dicamba stimulated many stress-responsive and signalling genes, including those involved in biosynthesis or signalling of auxin, ethylene, and abscisic acid (ABA), with TIR1 and AFB5 required for the dicamba-responsiveness of some genes. Research into dicamba-regulated gene expression and the selectivity of auxin receptors has provided molecular insight into dicamba-regulated signalling and could help in the development of novel herbicide resistance in crop plants.     

Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells

A calmodulin like domain protein kinase (CPK) homologue wasidentified in alfalfa and termed MsCPK3. The full-length sequenceof cDNA encoded a 535 amino acid polypeptide with a molecularweight of 60.2 kDa. The deduced amino acid sequence showed allthe conserved motifs that define other members of this kinasefamily, such as serine-threonine kinase domain, a junction regionand four potential Ca²⁺-binding EF sites. The recombinant MsCPK3protein purified from E. coli was activated by Ca²⁺and inhibitedby calmodulin antagonist (W-7) in in vitro phosphorylation assays.The expression of MsCPK3 gene increased in the early phase ofthe 2,4-D induced alfalfa somatic embryogenesis. Heat shockalso activated this gene while kinetin, ABA and NaCl treatmentdid not result in MsCPK3 mRNA accumulation. The data presentedsuggest that the new alfalfa CPK differs in stress responsesfrom the previously described homologues and in its potentialinvolvement in hormone and stress-activated reprogramming ofdevelopmental pathways during somatic embryogenesis. Key words: Medicago sativa, CPK, stress, 2,4-D, phosphorylation, somatic embryogenesis.     

Toward the production of artemisinin through tissue culture: Determining nutrient-hormone combinations suitable for cell suspension cultures

Summary Artemisia annua L. is the source of a potent antimalarial, artemisinin. As part of a program to produce artemisinin through tissue culture, a series of 14 multifactorial experiments were conducted to determine suitable conditions for initiating and maintaining friable callus fromA. annua. In the first six experiments, three different nutrient formulations [Gamborg B5 (B5), Murashige and Skoog (MS), and Whetmore and Rier (WR)], each with 32 combinations of auxins and cytokinins [2,4-dichlorophenoxyacetic acid (2,4-D) with benzyladenine (BA), or 1-naphthaleneacetic acid (NAA) with 6-furfurylaminopurine (kinetin)], were tested. Both B5 and WR nutrients supported friable callus formation from leaf explants with some combinations of auxin and cytokinin. Inasmuch as friable callus seemed to be produced over a wider range of auxin and cytokinin concentrations in combination with B5, the remaining experiments were conducted solely with this nutrient formulation. In the remaining eight experiments, it was determined that friable callus formed when combinations of NAA with kinetin or 2,4-D and BA were used with B5 medium. Lighter colored, more friable callus formed in response to 2,4-D and BA than with NAA and kinetin. No single combination of concentrations of auxin and cytokinin seemed to be &#x201C;ideal&#x201D; for producing friable callus. Ranges of 2,4-D from 0.5 to 2.0 with BA between 0.025 and 0.1, or NAA between 0.5 and 2.0 with kinetin between 0.5 and 1.0 mg/liter, produced acceptable results.     

Cloning and characterization of Somatic Embryogenesis Receptor Kinase I (EgSERK I) and its association with callus initiation in oil palm

The somatic embryogenesis receptor kinase (SERK) gene has been extensively studied in many plant species due to its role in conferring embryogenic competence to somatic cells. The oil palm (Elaeis guineensis Jacq.) full-length SERK I (EgSERK I) cDNA was first isolated from cell suspension culture using RACE-PCR. Total length of EgSERK I cDNA was 2378 bp in length with a 5’UTR region (358 bp) longer than 3’UTR region (130 bp) and the ORF was 1890 bp (629aa). The deduced amino acid sequence of EgSERK I contained protein domains commonly present in reported SERK proteins, including the hallmark proline-rich region and C-terminal domains. EgSERK I was most highly expressed in leaf explants and also detected in all tested tissues, including vegetative tissues, reproductive tissues, embryogenic tissues, and non-embryogenic tissues, suggesting that it may have a broad role in plant growth and development. Expression of EgSERK I in leaf explant was upregulated by minimal auxin concentration at the initial 6 h of incubation in callus induction media. EgSERK I mRNA was detected in the adjacent cells of the vascular tissues in the midvein region of leaf explants which serves as the callus initiation point of callogenesis in oil palm. Collectively, our findings suggest that the EgSERK I gene is involved in the callus initiation stage of oil palm somatic embryogenesis by transducing the signal to switch on the dedifferentiation process, triggering cellular reprogramming to form callus.