PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.

Exposure of oilseed rape (Brassica napus) plants to increasing leaf temperatures between 15°C and 25°C increased photorespiratory NH 4 ϩ production from 0.7 to 3.5 mol m Ϫ2 s Ϫ1 . Despite the 5-fold increase in the rate of NH 4 ϩ production, the NH 4 ϩ concentration in root and leaf tissue water and xylem sap dropped significantly, whereas that in the leaf apoplastic fluid remained constant. The in vitro activity of glutamine synthetase (GS) in both leaves and roots also increased with temperature and in all cases substantially exceeded the observed rates of photorespiratory NH 4 ϩ production. The surplus of GS in oilseed rape plants was confirmed using GS2 antisense plants with 50% to 75% lower in vitro leaf GS activity than in the wild type. Despite the substantial reduction in GS activity, there was no tendency for antisense plants to have higher tissue NH 4 ϩ concentrations than wild-type plants and no overall correlation between GS activity and tissue NH 4 ϩ concentration was observed. Antisense plants exposed to leaf temperatures increasing from 14°C to 27°C or to a trifold increase in the O 2 to CO 2 ratio did not show any change in steady-state leaf tissue NH 4 ϩ concentration or in NH 3 emission to the atmosphere. The antisense plants also had similar leaf tissue concentrations of glutamine, glycine, and serine as the wild type, whereas glutamate increased by 38%. It is concluded that photorespiration does not control tissue or apoplastic levels of NH 4 ϩ in oilseed rape leaves and, as a consequence, that photorespiration does not exert a direct control on leaf atmosphere NH 3 fluxes.Photorespiration is a complex network of biochemical processes taking place in the chloroplasts and peroxisomes, eventually leading to the evolution of stochiometric amounts of CO 2 and NH 3 in the mitochondria of C 3 plants. Photorespiration is light dependent and usually considered to be an energywasting process (Wingler et al., 2000), which may prevent photooxidation (Osmond et al., 1997) and ensure a steady flow of certain amino acids and keto acids used in other plant metabolic pathways, including the biosynthesis of antioxidants such as glutathione. Photorespiration is recognized as the quantitatively most important process generating NH 4 ϩ in plants during vegetative growth. The produced NH 4 ϩ is in equilibrium with NH 3 , which easily penetrates membranes and may be lost in significant amounts to the atmosphere via the water film in the leaf apoplast embedding the mesophyll cells.The chloroplastic isoform of Gln synthetase (GS2) is responsible for the assimilation of the large amounts of NH 4 ϩ produced in the mitochondria during photorespiration. This has been demonstrated by the use of barley (Hordeum vulgare) mutants lacking GS2 (Wallsgrove et al., 1987). These plants were not able to grow under photorespiratory conditions (21% [v/v] O 2 ) because NH 4 ϩ rapidly accumulated to toxic levels. Also, the use of the selective GS inhibitor DL-Met-dl-sulfoximine has shown that NH 4 ϩ accumulates within a few hours (...

The investigation and improvement of nitrogen efficiency in oilseed rape (Brassica napus L.) are important issues in rapeseed breeding. The objective of this study was to modify ammonium assimilation in transgenic rapeseed plants through the expression of the Escherichia coli asparagine synthetase (AsnA, E.C. 6.3.1.1) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and to study its influence on amino acid composition in leaves and on seed traits related to nitrogen efficiency. In regenerated transgenic plants, the 37 kDa AsnA protein was detected by Western blot analysis, but was lacking in untransformed control plants of cv. Drakkar. In the transformants, in vitro asparagine synthetase activities ranged from 105 to 185 nmol asparagine mg−1 protein h−1, whereas, in untransformed control plants, only negligible asparagine synthetase activities of up to 5 nmol asparagine mg−1 protein h−1 were found. Despite these significant activities, no changes in the amino acid composition in the leaves or in the phloem of transgenic plants were detectable. In a pot experiment, two transgenic lines expressing the prokaryotic asparagine synthetase clearly performed inferiorly to control plants at limiting nitrogen (N) fertilizer supply. Although the seed N content was increased, the seed yield and the seed N yield were reduced, which was interpreted as an increased nitrate assimilation leading, at limiting N supply, to a reduced seed yield and seed N yield. At high N fertilizer supply, the differences were less pronounced for one transgenic line, whereas the other showed a higher seed N yield and an improved nitrogen harvest index. The results show that the expression of the E. coli asnA gene in oilseed rape could be of advantage at high N supply, but not at limiting N fertilizer supply.

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for Brassica napus. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannoseselective medium, was transferred to B. napus hypocotyl explants by Agrobacterium-mediated transformation. More than 350 transgenic plants from three rapeseed varieties were obtained with transformation frequencies up to 24.2% when a combination of 4.5 g l -1 mannose and 10 g l -1 sucrose was used in the selection medium. For early identification of transgenic plants, histochemical staining with 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-Gluc) was used. Stable integration of the transgene was confirmed by PCR and Southern blot analysis. Mannose can be used as selective agent to identify transgenic plants in progeny i.e. segregation analysis. These results indicate that the mannose-selection system can be successfully used for Agrobacterium-mediated transformation of rapeseed.

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