Glyphosate Tolerance in Tobacco (Nicotiana tabacum L.)

A glyphosate-tolerant tobacco cell line, Nicotiana tabacum L. Indiana (I7), was selected from the glyphosate-sensitive Wisconsin 38 (W38) line through a single step exposure to the herbicide. Tolerance and growth characteristics of I7 cells were the same for cells maintained for more than 1 year in the presence or absence of glyphosate. Glyphosate tolerance levels were constant through the growth cycle. Tolerance is not due to reduced uptake of glyphosate. Shikimate levels in I7 and W38 cells maintained in glyphosate-free medium were similar, whereas W38 cells accumulated 46 times more shikimate than I7 cells, when cells of both lines were exposed to the herbicide. Glyphosate treatment caused increased levels of aromatic amino acids in W38 cells and slightly lower levels in I7 cells. Specific activities of dehydroquinate synthase, shikimate dehydrogenase, and shikimate kinase were similar in the two cell types, whereas DAHP synthase and EPSP synthase specific activities were elevated in I7 cells. Plants regenerated from I7 cells retained tolerance to glyphosate.     

Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp⁸⁸, Glu¹⁰⁸, Tyr¹³⁴, Asn¹³⁶, Leu²²⁶ and Gln²²⁷) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp⁸⁸, Tyr¹³⁴ or Asn¹³⁶ eliminated GlcNAc binding activity by GS-II, while those at Glut¹⁰⁸, Leu²²⁶ or Gln²²⁷ did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr¹³⁴ eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu²²⁶ and Gln²²⁷ did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.     

Moderately increased constitutive proline does not alter osmotic stress tolerance

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.     

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Summary 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

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 -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.     

Large quantities of recombinant PR-5 proteins from the extracellular matrix of tobacco: Rapid production of microbial-recalcitrant proteins

A procedure for the extraction of large quantities of PR-5 proteins that have been recalcitrant to microbial-based expression systems is described. Targeting of the recombinant proteins to the extracellular matrix allowed efficient protein extraction by a vacuum infiltration/centrifugation system. Approximately 1 kg of fresh leaves from transgenic tobacco plants overexpressing either truncated osmotin (Liu et al., 1996) or A9 fromAtriplex nummularia L. (Casas et al., 1991) yielded between 3 and 5 mg of purified proteins that fully retained their antifungal activity. The entire system of overexpression, extraction, and purification could be easily scaled up for the production of several grams of protein.     

First record of the grape leafhopper Erythroneura vulnerata Fitch (Hom., Cicadellidae) in Europe

Abstract:  The leafhopper Erythroneura vulnerata Fitch is native to North America, where it infests wild and cultivated grapes. In July 2004, E. vulnerata was recorded for the first time on Vitis vinifera L. (cv. Cabernet Sauvignon) in north-eastern Italy (Veneto region). This record is assumed to be the first in Europe. Preliminary observations on the pest distribution, seasonal abundance and the extent of symptoms in north-eastern Italy are reported.     

Alteration of the physical and chemical structure of the primary cell wall of growth-limited plant cells adapted to osmotic stress

Cells of tobacco (Nicotiana tabacum L.) adapted to grow in severe osmotic stress of 428 millimolar NaCl (−23 bar) or 30% polyethylene glycol 8000 (−28 bar) exhibit a drastically altered growth physiology that results in slower cell expansion and fully expanded cells with volumes only one-fifth to one-eighth those of unadapted cells. This reduced cell volume occurs despite maintenance of turgor pressures sometimes severalfold higher than those of unadapted cells. This report and others (NM Iraki et al [1989] Plant Physiol 90: 000-000 and 000-000) document physical and biochemical alterations of the cell walls which might explain how adapted cells decrease the ability of the wall to expand despite diversion of carbon used for osmotic adjustment away from synthesis of cell wall polysaccharides. Tensile strength measured by a gas decompression technique showed empirically that walls of NaCl-adapted cells are much weaker than those of unadapted cells. Correlated with this weakening was a substantial decrease in the proportion of crystalline cellulose in the primary cell wall. Even though the amount of insoluble protein associated with the wall was increased relative to other wall components, the amount of hydroxyproline in the insoluble protein of the wall was only about 10% that of unadapted cells. These results indicate that a cellulosic-extensin framework is a primary determinant of absolute wall tensile strength, but complete formation of this framework apparently is sacrificed to divert carbon to substances needed for osmotic adjustment. We propose that the absolute mass of this framework is not a principal determinant of the ability of the cell wall to extend.