A modified protocol of the freeze-thaw method') for transformation of Agrobacterium tumefaciens (A.t.)

SUMMARYTrehalose 6-phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2-oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration-dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.

SummaryThe coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1.30), was introduced into the genome of potato plants under the control of the cauli¯ower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5¢-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.

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Glutamate 1-semlaldehyde otra se [(S)-4-amino-5-oxopentanoate 4,5-amiomutse, EC 5.4.3.8] catalyzes the last step In the conversion of glutamate to 8-aminolevulinate of which eight molecules are needed to synthesize a chlorophyll molecule. Two fill-length cDNA clones that probably represent the homeologous Gsa genes of the two tobacco (Nicotiana tabacum) genomes have been isolated. The deduced amino acid sequences of the 468-residue-long precursor polypeptides differ by 10 amino adds. The cDNA sequence of isoenzyme 2 was inserted in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative in an expression vector and was introduced by Agrobacteriummediated transformation into tobacco plants. Antisense (3), has been attributed to position effects arising from the random integration of antisense genes into the host chromosomes. Additionally, a lack of quantitative relationships between the steady-state level of antisense mRNA, the target RNA, and the amount of protein produced is commonly observed as well as a great variability of the antisense phenotype in response to temperature and light conditions. Cornelissen (13,14) has demonstrated with transformed tobacco cells and plants that the antisense gene can control the transcript level in the nucleus and the translation efficiency of the target mRNA in the cytoplasm independently. The target enzyme was phosphinotricine acetyltransferase produced constitutively with a cauliflower mosaic virus (CaMV) 35S promoter-driven bar gene. In a second transformation of these plants, an antisense bar gene was introduced, and the turnover of the bar mRNA in the cyto-