Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-03-03
2004-05-25
Fredman, Jeffrey (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C536S023100
Reexamination Certificate
active
06740749
ABSTRACT:
This invention relates to new DNA sequences, a method for producing new plants which contain a new DNA sequence, the coding sequence thereof being expressed after ozone induction. The invention also relates to said new plants and the use of DNA sequences to produce ozone-responsive gene expression in plants and plant cells. Moreover, it relates to a new promotor, the specificity of which is increased by removal of the ozone response capacity thereof.
The ozone concentrations in the lower troposphere above the continents of the northern hemisphere have steadily increased over the past one hundred years as a result of greater industrial activities (Volz and Kley (1988) Nature 332, 240-242). Meanwhile, ozone values reach intermittent peak concentrations of 100 nL/L up to nL/L in Europe and North America (Krupa et al. (1995) Environ. Pollut: 87, 119-126).
The phytotoxicity of the air pollutant ozone has been well tested and documented, e.g., in Heagle (1989) Annu. Rev. Phytopathol. 27, 397-423; Heath (1994) in: Alscher, Wellburn (ed) “Plant responses to the gaseous environment”, pp. 121-145, Chapman & Hall, London. A decreased net photosynthesis and an increased early senescence are usually the result of such ozone impact which, consequently, results in diminished plant growth and a lower harvest yield.
Although ozone penetrates the plant cell through open stomata by means of diffusion, the ozone concentration in the intercellular spaces of the leaf is almost zero, irrespective of the environmental ozone concentration (Laisk et al. (1989) Plant Physiol. 90, 1163-1167). It is currently assumed that ozone reacts quickly with components of the cell walls and the plasmalemma and is converted into reactive oxygen species, such as peroxide-anions, hydroxyl radicals and hydrogen peroxide which were detected in ozone-treated plant material by using electron spin resonance spectroscopy (Mehlhorn et al. (1990) Physiologia Plantarum 79, 377-383). The so-called “oxidative burst”, i.e. the fast development of a relatively high quantity of reactive oxygen species, can lead to a dramatic disturbance of the normal cell function due to alteration of the permeability of the plant membrane, inactivation of redox-sensitive proteins and increased lipid peroxidation.
Recent tests conducted on ozone-treated plants showed an increased biosynthesis of non-specific, defensive enzymes, the function of which is to protect live cells against damage due to oxidative stress (Kangasjärvi et al. (1994 Plant, Cell and Environment 17, 783-794). Yet the signal-transduction chain, which is responsible for the ozone-induced gene activation, which transmits to the cell core the relevant information about the formation of apoplastic, reactive oxygen species, has not been understood up to now. Various factors, such as the increase of calcium concentration in cytosol (Price et al. (1994) The Plant Cell 6, 1301-1310), the formation of salicylic acid (Klessig and Malamy (1994) Plant Mol. Biol. 26, 1439-1458) and the phytohormone jasmon acid (Farmer (1994) Plant Mol. Bio. 26 1423-1437) and ethylene (Ecker (1995) Science 268, 667-674) are currently being discussed as possible signal connections, caused by oxidative stress, which generally play a part in defence reactions of plants.
Tests conducted on ozone-gassed tobacco plants showed that ozone causes an increased expression of various disease-resistant genes, namely a few PR-(pathogenesis-related) proteins (Ernst et al. (1992) Plant Mol. Biol. 20, 673-682; Ernst et al. (1996) J. Plant Physiol. 148, 215-221; Eckey-Kaltenbach et al. (1994) Plant Physiol. 104, 67-74). These results indicate that oxidative stress, caused by ozone, influences the expression and regulation of defensive genes of plants in a similar way as that described regarding pathogenic attack. Only very limited information on cis-regulatory elements and transcription factors, which possibly play a part in the control of gene expression of non-specific defensive genes as a response to various environmental influences, is available at present (Lee et al. (1994) Eur. J. Biochem. 226, 109-114). However, based on previous results, it can be assumed that with respect to the genes coding for PR-proteins, separate or at least only partly overlapping ways of signal transduction exist (Somssich (1994) in: Nover (ed) “Plant promoters and transcription factors”, pp. 163-179, Springer Publishing House, Berlin; Dolferus et al. (1994) Plant Physiol. 105, 1075-1087).
With respect to the activity of the stilbene synthase (STS), which takes part in the phytoalexin synthesis, it is known that in adult plants it is induced by environmental stress factors, such as, e.g., pathogenic attack (Langcake (1981) Physiol. Plant Pathol. 18, 213-226), ultraviolet light (Fritzemeier and Kindl (1981) Planta 151, 48-52) and ozone (Rosemann et al. (1991) Plant Physiol. 97, 1280-1286. Contrary to this a constitutive expression pattern was observed in embryos (Sparvoli et al. (1994) Plant Mol. Biol. 24, 743-755).
Stilbene synthase enzymes catalyze the synthesis of stilbenes such as resveratrol or pinosylvin from one molecule of p-cumaroyl-CoA or cinnamoyl-CoA and three units of malonyl-CoA. Resveratrol as well as pinosylvin have photoalexin properties and an antifungal activity, and perform, as phytoalexins in combination with other stilbenes derived from the phenylpropane metabolism, an important function in the defence against pathogens (Hart (1981) Annu. Rev. Phytopathol. 19, 437-458).
STS genes are found in some non-related plant species such as, e.g., peanut (Schröder et al. (1988) Eur. J. Biochem. 172, 161-169), grapevine (Hain et al. (1993) Nature 361, 153-156) and pine (Fliegmann et al. (1992) Plant Mol. Biol. 18, 489-503) and are organized in larger gene families, comprising six or more genes (Lanz et al. (1990) Planta 181, 169-175; Wiese et al. (1994) Plant Mol. Biol. 26, 667-677).
Experiments with transgenic tobacco cells indicate that the expression of the stilbene synthase is regulated mainly at a transcription level, and that the stress-induced signal transduction chain has been preserved in various plant species during the course of evolution (Hain et al. (1990) Plant Mol. Biol.15, 325-335.
STS genes from peanut (
Arachis hypogaea
) and grapevine (
Vitis vinifera
) have already been isolated (Schröder et al. (1988) supra or Hain et al. (1993) supra) and expressed in transgenic plants (Hain et al. (1990) supra or Hain et al. (1993) supra).
DNA sequences coding for stilbene synthase are known, e.g., from European Patent EP 0 309 862, German Patent Application DE-A-41 07 396, European Patent Application 0 464 461, as well as U.S. Pat. No. 5,500,367. These documents describe the isolation of stilbene-synthase genes and their use to produce transgenic plants. The resulting transgenic plants show greater resistance to various plant pests such as fungi, bacteria, insects, viruses and nematodes. Plasmids containing STS genes have been deposited with the German Collection of Microorganisms (DSM), Mascheroder Weg 1B, D-38124 Braunschweig. Also included in the deposition is the VstI-gene from grapevine in the pVstI plasmid, under deposit number DSM 6002 (DE-A-41 07 396, EP-A-0 464 461, U.S. Pat. No. 5,500,367).
While in the meantime also the use of STS coding sequences to produce male, sterile plants and altered blossom colour has been described (German Patent Application DE-A-44 40 200), a possible relation between STS gene expression and ozone induction has remained completely unexplored up to now.
Meanwhile there are various indications that in order to produce an effective as possible resistance to disease, based on the expression of STS genes in transgenic plants, it is advantageous if the expression of the heterologous STS gene (or the heterologous STS genes) in the plant is stimulated first of all by the attacking pathogen, i.e. if it is stimulated first of all by the interaction of plant and pathogen (Fischer and Hain (1994) Current Opinion in Biotechnology 5, 125-130; Fischer (1994) “Optimization of the heterological Expression of
Ernst Dietrich
Fischer Regina
Hain Rudiger
Sandermann Heinrich
Schubert Roland
Fredman Jeffrey
GSF-Forschungszentrum fur Umwelt
Merchant & Gould, P.C. (23552)
Switzer Juliet C.
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