A higher plant seven-transmembrane receptor that influences sensitivity to cytokinins

BACKGROUND: All organisms perceive and respond to a profusion of environmental and endogenous signals that influence growth, development and behavior. The G-protein signalling pathway is a highly conserved mechanism for transducing extracellular signals, and the superfamily of receptors that have seven transmembrane (7TM) domains is a primary element of this pathway. Evidence that heterotrimeric G proteins are involved in signal transduction in plants is accumulating, prompting speculation that plant 7TM receptors might exist. RESULTS: Using information in the dbEST database of expressed sequence tags, we isolated an Arabidopsis thaliana gene (GCR1) that encodes a protein with seven predicted membrane-spanning domains and other features characteristic of 7TM receptors. The protein shows 18-23% amino-acid identity (46-53% similarity) to, and good colinear alignment with, 7TM receptors from three different families. Its highest sequence identity is with the Dictyostelium cAMP receptors. GCR1 is expressed at very low levels in the roots, stems and leaves of Arabidopsis; it is a single-copy gene which maps close to the restriction fragment length polymorphism marker m291 on chromosome 5. Transgenic Arabidopsis expressing antisense GCR1 under the control of the constitutive cauliflower mosaic virus 35S promoter have reduced sensitivity to cytokinins in roots and shoots, yet respond normally to all other plant hormones. This suggests a functional role for GCR1 in cytokinin signal transduction. CONCLUSIONS: GCR1 encodes the first 7TM receptor homologue identified in higher plants and is involved in cytokinin signal transduction. This discovery suggests that 7TM receptors are ancient and predate the divergence of plants and animals.     

Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria

Selected nonpathogenic, root-colonizing bacteria are able to elicit induced systemic resistance (ISR) in plants. To elucidate the molecular mechanisms underlying this type of systemic resistance, an Arabidopsis-based model system was developed in which Pseudomonas syringae pv. tomato and Fusarium oxysporum f. sp. raphani were used as challenging pathogens. In Arabidopsis thaliana ecotypes Columbia and Landsberg erecta, colonization of the rhizosphere by P. fluorescens strain WCS417r induced systemic resistance against both pathogens. In contrast, ecotype RLD did not respond to WCS417r treatment, whereas all three ecotypes expressed systemic acquired resistance upon treatment with salicylic acid (SA). P. fluorescens strain WCS374r, previously shown to induce ISR in radish, did not elicit ISR in Arabidopsis. The opposite was found for P. putida strain WCS358r, which induced ISR in Arabidopsis but not in radish. These results demonstrate that rhizosphere pseudomonads are differentially active in eliciting ISR in related plant species. The outer membrane lipopolysaccharide (LPS) of WCS417r is the main ISR-inducing determinant in radish and carnation, and LPS-containing cell walls also elicit ISR in Arabidopsis. However, mutant WCS417rOA-, lacking the O-antigenic side chain of the LPS, induced levels of protection similar to those induced by wild-type WCS417r. This indicates that ISR-inducing bacteria produce more than a single factor that trigger ISR in Arabidopsis. Furthermore, WCS417r and WCS358r induced protection in both wild-type Arabidopsis and SA-nonaccumulating NahG plants without activating pathogenesis-related gene expression. This suggests that elicitation of an SA-independent signaling pathway is a characteristic feature of ISR-inducing biocontrol bacteria.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, from Arabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plant Arabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designated AtUBC1-3 and AtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of the AtUBC1-3 and AtUBC4-6 genes by the histochemical analysis of transgenic Arabidopsis containing the corresponding promoters fused to the beta-glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between the AtUBC1-3 and AtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s in Arabidopsis.     

EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis

The Arabidopsis ethylene receptor gene ETR1 and two related genes, ERS1 and ETR2, were identified previously. These three genes encode proteins homologous to the two-component regulators that are widely used for environment sensing in bacteria. Mutations in these genes confer ethylene insensitivity to wild-type plants. Here, we identified two Arabidopsis genes, EIN4 and ERS2, by cross-hybridizing them with ETR2. Sequence analysis showed that they are more closely related to ETR2 than they are to ETR1 or ERS1. EIN4 previously was isolated as a dominant ethylene-insensitive mutant. ERS2 also conferred dominant ethylene insensitivity when certain mutations were introduced into it. Double mutant analysis indicated that ERS2, similar to ETR1, ETR2, ERS1, and EIN4, acts upstream of CTR1. Therefore, EIN4 and ERS2, along with ETR1, ETR2, and ERS1, are members of the ethylene receptor-related gene family of Arabidopsis. RNA expression patterns of members of this gene family suggest that they might have distinct as well as redundant functions in ethylene perception.     

Assessing the suitability of gastric carcinoma for limited resection: endoscopic prediction of lymph node metastases

We have isolated a cytokinin up-regulated cDNA clone, H13, from an early stage of cultured tobacco mesophyll protoplasts by a differential display method. The expression of this gene was specifically induced by natural and synthetic cytokinins including N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-type cytokinin, although the simultaneous presence of auxin was also required. It seems that the preceding treatment of the tobacco mesophyll protoplasts by auxin is necessary for the gene to respond to cytokinin. The addition of a cytokinin antagonist, compound 182, which suppressed the induction of cell division in tobacco mesophyll protoplasts, completely abolished the expression of this gene. Though the predicted gene product of H13 did not suggest us any sequences of defined functions, two domains of the predicted sequence had significant homology to several reported sequences in the data base. The gene product of H13 is proposed to have a role in regenerating cell wall in cultured protoplasts, since a cDNA clone E6, from cotton fiber cells, which has the most closely related structure to H13, has been isolated from cells which showed active cellulose synthesis. This supposition is supported by the evidence that in the absence of cytokinin, cell wall regeneration was significantly suppressed, resulting in failure of the induction of cell division. Thus, the gene product of H13 is supposed to have a role in regenerating cell walls and facilitating the progression of the cell cycle, resulting in the sustained cell division of tobacco mesophyll protoplasts.     

Differential expression of a novel gene in response to coronatine, methyl jasmonate, and wounding in the Coi1 mutant of Arabidopsis

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).     

Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria

A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotransferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hypersensitive reaction, as compared to virulent pathogens, which lead to disease.     

Characterization of cauliflower mosaic virus (CaMV) resistance in virus-resistant ecotypes of Arabidopsis

Two Arabidopsis ecotypes are resistant to systemic infection by cauliflower mosaic virus (CaMV), a plant para-retrovirus. Arabidopsis ecotype Enkheim-2 (En-2) is highly resistant to CaMV infection while Bla-14 is more weakly resistant. CaMV resistance in En-2 can be largely attributed to the action of a single semidominant gene called cauliflower mosaic virus resistance1 (CAR1), located at a locus on chromosome 1. Resistance in Bla-14 is tightly linked to CAR1 and may be due to a weak allele at the same locus or another gene in a gene cluster. A quantitative polymerase chain reaction assay in conjunction with replication- and movement-incompetent viral mutants was used to determine whether virus replication or movement is affected in the resistant ecotypes. The pattern of accumulation of the wild-type virus in the resistant ecotype, En-2, was similar to that of a movement-incompetent CaMV mutant, suggesting that CAR1 interferes with or fails to support CaMV movement. CaMV-inoculated En-2 plants do not show visible signs of a hypersensitive response. However, indicators of an induced defense response do appear in CaMV-infected En-2 plants, such as the activation of pathogenesis-related protein gene expression and the production of camalexin, an Arabidopsis phytoalexin. Defense responses induced chemically or by mutation in the susceptible ecotypes delayed and reduced the severity of a CaMV infection. These findings suggest that CAR1 acts either in the susceptible ecotype to support virus movement or in the resistant ecotype to signal a defense response.     

Wounding changes the spatial expression pattern of the arabidopsis plastid omega-3 fatty acid desaturase gene (FAD7) through different signal transduction pathways

The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids in membrane lipids. The mRNA levels of the Arabidopsis FAD7 gene in rosette leaves rose rapidly after local wounding treatments. Wounding also induced the expression of the FAD7 gene in roots. To study wound-responsive expression of the FAD7 gene in further detail, we analyzed transgenic tobacco plants carrying the -825 Arabidopsis FAD7 promoter-beta-glucuronidase fusion gene. In unwounded transformants, FAD7 promoter activity was restricted to the tissues whose cells contained chloroplasts. Activation of the FAD7 promoter by local wounding treatments was more substantial in stems (29-fold) and roots (10-fold) of transgenic plants than it was in leaves (approximately two-fold). Significant induction by wounding was observed in the overall tissues of stems and included trichomes, the epidermis, cortex, vascular system, and the pith of the parenchyma. Strong promoter activity was found preferentially in the vascular tissues of wounded roots. These results indicate that wounding changes the spatial expression pattern of the FAD7 gene. Inhibitors of the octadecanoid pathway, salicylic acid and n-propyl gallate, strongly suppressed the wound activation of the FAD7 promoter in roots but not in leaves or stems. In unwounded plants, exogenously applied methyl jasmonate activated the FAD7 promoter in roots, whereas it repressed FAD7 promoter activity in leaves. Taken together, wound-responsive expression of the FAD7 gene in roots is thought to be mediated via the octadecanoid pathway, whereas in leaves, jasmonate-independent wound signals may induce the activation of the FAD7 gene. These observations indicate that wound-responsive expression of the FAD7 gene in aerial and subterranean parts of plants is brought about by way of different signal transduction pathways.     

Towards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays

cDNA microarrays containing 1443 Arabidopsis thaliana genes were analyzed for expression profiles in major organs of Arabidopsis plants. Novel expression profiles were identified for many coding sequences with putative gene identifications. Expression patterns of novel sequences provided clues to their possible functions. The results demonstrate how microarrays containing a large number of Arabidopsis genes can provide a powerful tool for plant gene discovery, functional analysis and elucidation of genetic regulatory networks.     

A novel iron-regulated metal transporter from plants identified by functional expression in yeast

Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans. In Arabidopsis, IRT1 is expressed in roots, is induced by iron deficiency, and has altered regulation in plant lines bearing mutations that affect the iron uptake system. These results provide the first molecular insight into iron transport by plants.