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Summary A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5′ and 3′ ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1at the 5′ and 3′ sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1enhances the integration or expression of the transgene, or both. Clearly, TJ1is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed. [ABSTRACT FROM AUTHOR]

Summary: The emission of N[sub 2] and N[sub 2]O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO[sub 3][sup –], NO[sub 2][sup –] or NH[sub 4][sup +] -containing medium was investigated. [sup 15]N contents of gas sampled from gas-sealed pots, in which the plants were grown on [sup 15]N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC–MS). No emission of N[sub 2] was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO[sub 3][sup –]-containing medium. N[sub 2]O emission from clone 271 grown on NO[sub 3][sup –]-containing medium was detected, but not from the wild-type plants. The N[sub 2]O emission rate of clone 271 was 106 ng N[sub 2]O mg[sup –1] incorporated N week[sup –1] and the N[sub 2]O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N[sub 2]O was found from clone 271 or wild-type plants grown on medium containing NH[sub 4][sup +]. Emission of N[sub 2]O also was detected from clone 271 grown on NO[sub 2][sup –]-containing medium and its emission rate increased with increasing NO[sub 2][sup –] levels in plants. We speculate that NO[sub 3][sup –] is reduced to NO[sub 2][sup –] and that a part of NO[sub 2][sup –] is metabolized to N[sub 2]O in clone 271. [ABSTRACT FROM AUTHOR]

Summary The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism. [ABSTRACT FROM AUTHOR]

Indicates that all correspondence concerning 'The Plant Journal' paper titled 'Emission of nitrous oxide (N[sub 2]O) from transgenic tobacco expressing antisense NiR mRNA' should be addressed to Hiromichi Morikawa, Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.