Process and DNA molecules for increasing the photosynthesis...

Details Process and DNA molecules for increasing the photosynthesis... Process and DNA molecules for increasing the photosynthesis...

1997-09-04

2001-06-12

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

73, 73, 73, 73, C536S023200, C536S023500, C536S023600, C536S023700, C800S284000, C800S288000, C800S290000, C800S289000

Reexamination Certificate

active

06245967

ABSTRACT:

The present invention relates to a process and DNA molecules for increasing the photosynthesis rate in plants as well as for an increased yield of plants. The photosynthesis rate and/or the yield is increased by the expression of a deregulated or unregulated fructose-1,6-bisphosphatase in the cytosol of transgenic plants. The invention also relates to the plant cells and plants obtainable by this process as well as to the use of DNA sequences coding for proteins having the enzymatic activity of a fructose-1,6-bisphosphatase for the production of plants exhibiting an increased photosynthesis rate. The invention furthermore relates to recombinant DNA molecules leading to the expression of a fructose-1,6-bisphosphatase in plant cells and plants and resulting in an increased photosynthesis rate.
Due to the continuously growing need for food which is a result from the ever-growing world population it is one of the objects of research in the field of biotechnology to try to increase the yields of useful plants. One possibility to attain this object is to genetically engineer the metabolism of plants. Respective targets are, e.g., the primary processes of photosynthesis that result in CO
2
fixation, the transport processes that participate in the distribution of the photoassimilates within the plant, but also the metabolic pathways that lead to the synthesis of storage substances such as starch, proteins or fats.
For example, the expression of a procaryotic asparagine synthetase in plant cells has been described which results in transgenic plants inter alia in an increase in biomass production (EP 0 511 979).
Another proposal has been to express a procaryotic polyphosphate kinase in the cytosol of transgenic plants. Such expression results in potato plants in an increase in yield in terms of tuber weight of up to 30%.
EP-A-0 442 592 describes the expression of an apoplastic invertase in potato plants which leads to a modified yield of transgenic plants so modified.
Further approaches have concentrated on a modification of the activities of enzymes that participate in the synthesis of sucrose, the most important transport metabolite in most plant species. In plants the CO
2
fixed in the course of photosynthesis is transported from the plastids to the cytosol in the form of triosephosphates (glyceraldehyde-3-phosphate and dihydroxyacetone phosphate). In the cytosol the enzyme aldolase forms a molecule of fructose-1,6-bisphosphate by condensation of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. This molecule is converted into a molecule of fructose-6-phosphate which in turn is the substrate for the synthesis of sucrose phosphate by the enzyme sucrose phosphate synthase according to the equation
fructose-6-phosphate+UDP glucose&rlarr2;sucrose phosphate+UDP.
The conversion of fructose-1,6-bisphosphate into fructose-6-phosphate is catalyzed by the enzyme fructose-1,6-bisphosphatase (in the following: FBPase; EC 3.1.3.11) which is regulated by various substances. For example, fructose-2,6-bisphosphate and AMP are potent inhibitors of said enzyme. AMP is an allosteric inhibitor, while fructose-2,6-bisphosphate binds to the active center of the enzyme (Ke et al., Proc. Natl. Acad. Sci. USA 86 (1989), 1475-1479; Liu et al., Biochem. Biophys. Res. Comm. 161 (1989), 689-695. Plant cells contain both a cytoplasmatic as well as a chloroplastic FBPase coded for by the nuclear genome. The reverse reaction (conversion of fructose-6-phosphate into fructose-1,6-bisphosphate) is catalyzed by the enzyme phospho-fructokinase (PFK) using ATP. Said enzyme is activated by fructose-6-phosphate, P
i
and fructose-2,6-bisphosphate and inhibited by glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Besides said enzymes another enzyme is present in plant cells, namely pyrophosphate:fructose-6-phosphate-1-phosphotransferase (PFP) which catalyzes both reactions according to the equation:
fructose-1,6-bisphosphate+P
i
&rlarr2;fructose-6-phosphate+PP
i
.
So far various attempts have been made to manipulate this step in the synthesis of sucrose such that the amount of CO
2
fixed is increased resulting in an increased biomass production. For example, it has been attempted to increase the production of fructose-1,6-bisphosphate by overexpressing a plant FBPase in the cytosol (Juan et al., Supplement to Plant Physiol., Vol. 105 (1994), 118). However, this does not lead to a measurable increase of sucrose synthesis. Antisense-inhibition of the PFP, too, failed to result in a detectable increase of sucrose synthesis in plant cells (Hajirezaei et al., Planta 192 (1994), 16-30). It has been furthermore attempted to influence the reaction catalyzed by FBPase by modifying the concentration of the allosteric inhibitor fructose-2,6-bisphosphate (Kruger and Scott, Biochemical Society Transactions, Transgenic Plants and Plant Biochemistry 22 (1994), 904-909). However, it has been found that an increase in the fructose-2,6-bisphosphate concentration has no effect on the photosynthesis rate and only a minor effect on the synthesis of starch or sucrose.
The problem underlying the present invention is to provide further processes generally useful in plants that allow an increase of the photosynthesis rate in plants und thus an increase in biomass production and yield.
The problem is solved by the provision of the embodiments characterized in the claims.
The invention relates to recombinant DNA molecules containing
(a) a promoter functional in plant cells and
(b) a DNA sequence linked with the promoter in sense orientation which codes for a polypeptide having the enzymatic activity of a fructose-1,6-bisphosphatase,
with the polypeptide having the enzymatic activity of a fructose-1,6-bisphosphatase being a deregulated or unregulated enzyme.
It has surprisingly been found that by expression of such DNA molecules in plant cells a dramatic increase in the photosynthesis rate in plants so modified can be achieved vis-à-vis wild type plants. The term “deregulated” means that said enzymes are not regulated in the same manner as the FBPase enzymes normally expressed in plant cells. Specifically, these enzymes are subject to other regulatory mechanisms, i.e., they are not inhibited to the same extent by the inhibitors or activated by the activators which normally inhibit or activate plant FBPases. For example, they are not inhibited by fructose-2,6-bisphosphate or AMP to the same extent as FBPases that are normally present in plants.
The term “unregulated FBPase enzymes” as used in the present invention relates to FBPase enzymes that are not subject to regulatory mechanisms in plant cells, specifically to those that are not regulated by AMP, ATP or fructose-2,6-bisphosphate.
An increased photosynthesis rate means that plants that have been transformed with a DNA molecule according to the invention which leads to the synthesis of a deregulated or unregulated FBPase in the plants exhibit an increased photosynthesis rate vis-à-vis non-transformed plants, preferably a photosynthesis rate that is increased by at least 10%, particularly a photosynthesis rate that is increased by at least 20%, most preferably a photosynthesis rate that is increased by 30-40%.
The promoter contained in the DNA molecules according to the invention in principle may be any promoter functional in plant cells. The expression of the DNA sequence coding for an unregulated or deregulated FBPase in principle may take place in any tissue of a transformed plant and at any point in time, preferably it takes place in photosynthetically active tissues. An example for an appropriate promoter is the 35S promoter of the cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812) which allows constitutive expression in all tissues of a plant. However, promoters may be used that lead to the expression of subsequent sequences only in a certain tissue of the plant, preferably in photosynthetically active tissue (see, e.g., Stockhaus et al., EMBO J. 8 (1989), 2245-2251) or at a point in time determinable by external influences (see, e.g.,

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