Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2001-03-30
2003-08-19
Horlick, Kenneth R. (Department: 1637)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S287000, C800S288000, C800S295000, C536S023200, C536S023600, C435S113000, C435S193000, C435S320100
Reexamination Certificate
active
06608239
ABSTRACT:
The present invention relates to a recombinant DNA molecule comprising a nucleic acid molecule encoding a protein having serine acetyltransferase (SAT) activity and optionally a nucleic acid molecule encoding a protein having cysteine-&ggr;-synthase (C&ggr;S) activity; wherein said nucleic acid molecule(s) are operably linked to regulatory elements allowing the expression of the nucleic acid molecule(s) in plant cells. The present invention also provides vectors comprising said recombinant DNA molecules as well as plant cells, plant tissues and plants transformed therewith. The present invention further relates to the use of the aforementioned recombinant DNA molecules and vectors in plant cell and tissue culture, plant breeding and/or agriculture. Furthermore, the present invention involves the production of food, feed and additives therefor comprising the above-described plant cells, plant tissue and plants.
Higher plants use inorganic sulfate in the soil as the major sulfur source for synthesizing the sulfur-containing amino acids cysteine and methionine. Cysteine biosynthesis in plants has been postulated to play an essential role in the sulfur cycle in nature. Reduced sulfur in the form of cysteine is needed for many different functions in plants (Rennenberg, 1990; Schmidt, 1992). It is essential for the normal plant metabolism because of connecting serine and methionine metabolism by carrying the reduced sulfur necessary for methionine biosynthesis (Giovanelli, 1990; Ravanel, 1997; Brunold and Rennenberg, 1997). Additionally, cysteine serves as substrate for other sulfur containing molecules like certain co-factors, membrane compounds, and as an essential amino acid for protein synthesis (Giovanelli, 1980; Schmidt, 1992). Cysteine is also essential as a precursor for the production of glutathione (GSH) and other stress related metabolites. The demand for cysteine varies during plant development and is also dependend on changes in the environment, including light, sulfate availability and some kinds of stress, abiotic or biotic (von Arb and Brunold 1986; Nussbaum, 1988; Delhaize, 1989; Rauser, 1991; Ghisi, 1993; Hell, 1994).
For cysteine biosynthesis, first L-serine has to be activated by transfer of an acetyl-group from acetyl coenzyme A to form the intermediate O-acetyl-L-serine (OAS). This important reaction is catalized by serine acetyltransferase (SAT). The activation of serine, a key reaction in the cysteine biosynthetic pathway, has been investigated at the molecular level only in prokaryotes (Breton, 1990; Monroe, 1990; Evans, 1991; Lai and Baumann, 1992). The synthesis of cysteine in plants is accomplished by the sulfhydrylation of O-acetyl-L-serine in the presence of free or bound sulfide, catalized by O-acetylserine(thiol)-lyase (OAS-TL, cysteine synthase, CSase; E C 4.2.99.8.) (Schmidt and Jäger, 1990). This reaction has been extensively analysed (Saito, 1992, 1993 and 1994; Rolland, 1993 and 1996; Youssefian, 1993; Noji, 1994; Hell, 1994; Kuske, 1994 and 1996; Takahashi and Saito, 1996). In bacteria SAT and OAS-TL form a bifunctional complex called cysteine synthase. In this complex only a small proportion of the O-acetylserine(thiol)lyase (5%) is associated with all the SAT activity (Kredich, 1969). In addition, studies on the regulation of cysteine biosynthesis in bacteria revealed that serine acetyltransferase is sensitive to feedback inhibition by L-cysteine, and that O-acetylserine (or N-acetylserine) is involved in the transcriptional activation of several of the cys operon promotors (Ostrowski and Kredich, 1989; Kredich, 1993).
Plant cDNAs encoding serine acetyltransferases have recently been cloned from different species (Bogdanova, 1995; Murillo, 1995; Ruffet, 1995; Saito, 1995; Roberts and Wray, 1996). In plants, SAT also exists in a complex with OAS-TL, suggesting an efficient metabolic channeling from serine to cysteine by preventing the diffusion of the intermediate O-acetyl-L-serine (Nakamura, 1988; Nakamura and Tamura, 1990; Ruffet, 1994; Bogdanova and Hell, 1997; Hesse, 1997). Both SAT and OAS-TL have been reported to be localised in plastids, mitochondria and cytosol from several plants, suggesting that the ability to synthesize cysteine appears to be necessary in all cellular compartments with an endogenous protein biosynthetic capacity (Smith and Thompson, 1969; Smith, 1972; Brunold and Suter, 1982; Lunn, 1990; Rolland, 1992; Ruffet, 1994). In Pisum sativum for example, three different isoforms of SAT are existing, and each isoform seems to be specific for a given intracellular compartment (Ruffet, 1995). Beside these required cellular locations, the fact that cysteine biosynthesis is in complex interaction with uptake and reduction of sulfate, which itself is regulated by photosynthesis and nitrate assimilation (Anderson, 1990; Brunold, 1993), let suggest an important role of cysteine biosynthesis in sulfur metabolism in higher plants.
A very important feature of the reaction sequences of cysteine formation is the fact that SAT activity is much lower as compared to the activity of OAS-TL. In seeds and seedlings, OAS-TL is 10 to 20 times more active than SAT (Smith, 1971; Ngo and Shargool, 1974). In whole leaves the activity ratio of both enzymes is 100 to 300-fold (Nakamura, 1987), whereas in chloroplasts alone the ratio is up to 345-fold (Ruffet, 1994). As has been shown in Allium and spinach, SAT is in comparison to OAS-TL a low abundance enzyme (Nakamura and Tamura, 1990; Ruffet, 1994). Additionally, the availability of OAS was also discussed to be rate limiting for cysteine synthesis (Neuenschwander, 1991; Ghisi, 1990; Rennenberg 1983; Brunold, 1993; Saito, 1994). SAT activity is significantly regulated by feedback inhibition of cysteine in watermelon (Saito, 1995). Also on gene expression level SAT regulation takes place. In
Arabidopsis thaliana
in response to light and sulfur stress SAT mRNA accumulates by about twofold (Bogdanova, 1995). However, while the function and role of SAT, OAS and OAS-TL in the reaction cascade of cysteine biosynthesis have been subject to a lot of investigations previous attempts to alter the rate of cysteine synthesis failed (Saito, 1994). Hence, the precise regulation of the cysteine biosynthetic pathway is still not fully understood and part of controversial discussion. Therefore, means for the control the sulfur content in plants that may have applications in several aspects of agriculture were hitherto not available.
Thus, the technical problem underlying the present invention was to comply with the need for means and methods for modulating the content of sulfur compounds in plants.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the invention relates to a recombinant DNA molecule comprising
(a) a nucleic acid molecule encoding a protein having serine acetyltransferase (SAT) activity, and optionally
(b) a nucleic acid molecule encoding a protein having cysteine-&ggr;-synthase (C&ggr;S) activity;
wherein said nucleic acid molecule(s) are operably linked to regulatory elements allowing the expression of the nucleic acid molecule(s) in plant cells.
The term “protein having serine acetyltransferase (SAT) activity”, as used herein, means that said protein is able to transfer an acetyl-group from acetyl coenzyme A to L-serine to form the intermediate of the cysteine biosynthetic pathway O-acetyl-L-serine.
The term “protein having cysteine-&ggr;-synthase (C&ggr;S) activity” in accordance with the present invention denotes a protein capable of catalyzing the formation of L-cystathionine or L-homocysteine depending on the sulfur-containing substrate, L-cysteine or sulfide. This protein is also known as cystathionine &ggr;-synthase. The terms “cysteine-&ggr;-synthase” and “cystathionine &ggr;-synthase” are used interchangeable herein. In plants C&ggr;S usually catalyses the first reaction specific for methionine biosynthesis, namely the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. Thus, cysteine i
Harms Karsten
Hesse Holger
Hofgen Rainer
Horlick Kenneth R.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
Strzelecka Teresa E
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