Showing papers by "Ray A. Bressan published in 1997"

Molecular Aspects of Osmotic Stress in Plants

Abstract: Plant molecular responses to osmotic stress are complex as evidenced by the isolation of numerous OR (osmotic stress-regulated) genes. Although functions including osmolyte biosynthesis, membrane transport, signal transduction, and cellular protection have been predicted for OR genes, few of them have been established. Current efforts toward isolating and analyzing the expression of individual OR genes should be replaced by systematic approaches to analyze all OR genes simultaneously in selected plant species. Both transcriptional and posttranscriptional regulation of OR genes have been described. Cis-elements that respond to osmotic stress through abscisic acid (ABA)-dependent as well as ABA-independent pathways have been identified. Functional genetic approaches using yeast and plant model systems are expected to complement current molecular analysis of overexpression of OR genes in transgenic plants. These systems will help to establish functions of OR genes, to dissect osmotic stress-signalin...

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein

Abstract: Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity Pir proteins have been immunolocalized to the cell wall The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins

Cloning of a polycistronic cDNA from tomato encoding γ-glutamyl kinase and γ-glutamyl phosphate reductase

Abstract: We isolated from a tomato cDNA library the tomPRO1 locus, which encodes γ-glutamyl kinase (GK) and γ-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E. coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Abstract: Transgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 µM) 2,4-D, 0.5 mg/l (2.32 µM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T1 progeny. However, no β-glucuronidase (GUS) activity could be detected in T1 plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.

Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region.

Abstract: The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals In this study, tissue-specific and inducer-mediated expression of the reporter gene beta-glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum) The region of the promoter between -248 to -108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C2H4 and NaCl)-mediated expression of the reporter gene Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem The regulators ABA, C2H4 and NaCl exhibited tissue-specific induction of this promoter The promoter was specifically responsive to C2H4 in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C2H4 was unable to induce activity Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C2H4 However, significant C2H4-induced activity was obtained with a promoter fragment containing the AT and PR elements together

Induction of pathogen resistance and pathogenesis‐related genes in tobacco by a heat‐stable Trichoderma mycelial extract and plant signal messengers

Abstract: Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings (Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica. The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.

Moderately increased constitutive proline does not alter osmotic stress tolerance

Abstract: Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.

Soybean cysteine proteinase inhibitors, nucleotides encoding the same, and methods of use thereof

Abstract: The invention relates to the isolation, purification and use of three soybean cysteine proteinase inhibitors, designated L1, R1 and N2, which have strong inhibitory activity with respect to cysteine proteinases found in the digestive tracts of certain herbivorous insects. The invention further relates to three nucleotide sequences which have been cloned, isolated and sequenced, these nucleotide sequences coding for the three above-named proteins, as well as a vector having one or more of these sequences provided therein. The invention also relates to plants transformed by inventive vectors, transformed plants having an enhanced ability to resist predation by said insects, as well as microorganisms transformed using inventive vectors or plasmids.

Genetic and molecular control of seed dormancy uo° M

Abstract: The dormancy trait is generally governed by many genes, and in a few cases these genes have been mapped to specific chromosome regions. Many genes that are differentially expressed between dormant and nondormant seeds have been isolated. Although the identities and functions of many of these genes remain obscure, others are probably involved in desiccation tolerance and maintaining longevity in dormant seeds. The use of molecular genetics and model systems now seems to present the best way forward for research on seed dormancy.