DE19700334A1 - Production of transgenic plants over-expressing phospho-enol pyruvate - Google Patents

Abstract

Methods for producing plants with increased growth rate and yield are characterised in that: (a) additional pyruvate phosphate di-kinase (PPDK) is expressed in the cytosol or plastids of the plants, whereby additional phospho-enol pyruvate (PEP) is produced; (b) additional PPDK is expressed in the roots of the plants, whereby additional PEP is produced; (c) additional PPDK is expressed in the tubers or fruits of the plants, whereby additional PEP is produced; (d) the plants contain the chimeric DNA construct B33-PPDK, or (e) the plants contain the chimeric DNA construct B33- DELTA PPDK. Also claimed are the chimeric genes B33-PPDK and B33- DELTA PPDK.

Description

Problem

During the 20th century, plant breeding and selection became remarkable significant improvements in the yield and quality of grain seeds (Borlaug and Dowswell 1988). However, such increases cannot be achieved continuously. Of the that’s a lot of serious problems that humans face Population growth the greatest. At the current speed of the population growth, the global population will double to 20 billion by 2030 to have. This is already leading to the loss of agricultural areas. To the Providing the world population with food even in 40 years will be all of ours Ingenuity needed. The urgent need to increase food production can perhaps be satisfied with the help of plant molecular biology. This allows direct access to an immense gene pool as it transfers useful ones Genes from any organism allowed in plants.

Plants are amazing organisms. You can use all of your energy needs Cover with the help of sunlight. In addition, they generate as photosynthesis known process, carbohydrates from carbon dioxide. These substances can be used both as energy memory, can be used for biosynthesis as well as structural components. But not all plant tissues are capable of photosynthesis. To the non-photosynthetic Active tissues belong to everyone who does not come into contact with light or others Have tasks such as the underground roots, the lead tissues and the ripening seeds. Of course, no photosynthesis can take place in the dark. The Photosynthetically active tissues therefore need enough energy during the light phases produce and store to supply the whole organism and if possible to drive growth and reproduction. Usually there are short-term ones Storage (starch, sucrose) and transfer (sucrose) of energy in the form of Sugar. The main customers of sucrose are growing leaves and not photosynthetically active parts of plants. The non-photosynthetically active parts of the plant also need all of their organic components from the transferred carbon Manufacture frameworks (Dennis and Turpin 1990; Mohr and Schopfer 1978; Strasburger et al. 1991; Taiz and Zeiger 1991).  

When breathing hexose, glucose and fructose from sucrose, can up to 38 ATP (adenosine triphophate (ATP) is the energy currency of cells. The change in free Energy (ΔG) for ATP hydrolysis is -50 to under physiological conditions -65 kJ / mol. A NADH can be converted to 3 ATP.) (Voet and Voet 1992). But at the same time the carbon dioxide fixed during photosynthesis is released again. Photosynthesis requires 18 ATP and 12 NADPH for the production of a hexose (Mohr and Schopfer 1978; Strasburger et al. 1991; Taiz and Zeiger 1991). This corresponds to an amount of energy of 54 ATP. A simple calculation shows that from photosynthesis to breathing 16 ATP loss too are listed.

Can Breathing Reduce Yield? The answer is yes. A 20% Reducing breathing can lead to a 10-20% increase in crop yield (Lambers 1985; Wilson and Jones 1982). Now it is difficult to stay in the field for longer Light periods, optimal growth temperatures, optimal air or soil moisture ensure. Therefore, other solutions to reduce carbon dioxide loss through breathing are needed being found.

Approach

How can the carbon dioxide released during breathing be fixed again regardless of photosynthesis? Fig. 1 shows a biochemical way of refixing carbon dioxide (Latzko and Kelly 1983). The fixation is carried out here by the phosphoenol pyruvate carboxylase (PEPCase; EC 4.1.1.31). PEPCase is a key enzyme in anaplerotic metabolism (filling up the tricarboxylic acid cycle). The vegetable pyruvate, phosphate dikinase (PPDK; EC 2.7.9.1) is a plastid enzyme which is suspected to provide phosphoenolpyruvate (PEP) for the PEPCase reaction in non-photosynthetically active tissues (Latzko and Kelly 1983; Meyer et al. 1978).

The product of the phosphoenolpyruvate carboxylation is oxaloacetate. This can be converted to the amino acid aspartic acid in a transaminase reaction. Alanine mostly serves as a substrate here. Pyruvate is formed from the alanine, which can be used again by the PPDK. The cycle shown in Fig. 1 can fix carbon dioxide at the expense of two high-energy phosphate groups (or the equivalent of two ATP). The Calvin cycle requires three ATP and two NADPH + H⁺ (or the equivalent of nine ATP) for the fixation of carbon dioxide (Taiz and Zeiger 1991). The fixation of carbon dioxide by PPDK and PEPCase can therefore help plants to save a lot of energy (seven ATP per refixed carbon dioxide) and, above all, protects the plant from part of the carbon dioxide released during breathing.

Another reason for the occurrence of PEPCase and PPDK in photosynthetically active tissues of C ₃ plants is proposed by Aoyagi and Bassham (1986) ( Fig. 2). PEPCase and PPDK serve for the intracellular carbon dioxide enrichment, which leads to increased carbon dioxide fixation by the ribulose-1,5-bisphosphate carboxylase (RubisCo). This type of primary carbon dioxide fixation and enrichment is normally used by CAM and C ₄ plants. However, there is a temporal separation between PEPCase (night) and RubisCo reaction (day) in CAM plants, while C ₄ plants spatially separate PEPCase (mesophyll cells) and RubisCo (bundle of vaginal cells), because both enzymes are of course also in competition Carbon dioxide (Dennis and Turpin 1990; Mohr and Schopfer 1978; Strasburger et al. 1991; Taiz and Zeiger 1991; Ting 1985).

Latzko and Kelly (1983) suggest ten more biochemical reasons for the presence of PEPCase. However, these further possible uses do not result in any immediate energetic advantages. A substrate of the PEPCase is phosphoenol pyruvate. Among other things, this can be generated from pyruvate by the PPDK, so that the PEPCase may also be limited by the replenishment of phosphoenolpyruvate. However, the enzymatic characteristics and metabolic functions of PPDK from non-photosynthetically active tissues or C ₃ plants are not yet fully understood (Fißlthaler et al. 1995; Rosche et al. 1994).

Previous patent applications have shown that constitutively increased levels of PPDK increase lead to very different effects (Sheriff 1995; Sheriff 1995). Tobacco plants with Larger amounts of plastid PPDK in all tissues produced more seeds than that Wild-type and grew faster, but showed no increased amounts of starch. Tobacco plants with larger amounts of a cytosolic PPDK in all tissues, however, produced in In extreme cases, no more seeds and then grew more slowly. In no case were increased amounts of starch were detected in the fruit.

My idea now is to express additional plastid or cytosolic PPDK in defined tissues such as roots or potato tubers. This Expression leads to unexpected but economically interesting results. So grow Tobacco plants with additional PPDK in the roots faster than the wild type and Potato tubers with increased amounts of PPDK produce more starch than the wild type.

A possible lack of phosphoenolpyruvate has probably been remedied. My The main assumption is that the PPDK is the limiting factor for this. You bring it Plants to produce larger quantities of PPDK would have to face such a bottleneck get fixed. In order to prove this thesis, the PPDK cDNA was chosen from the optional  CAM plant Mesembryanthemum crystallinum in Nicotiana tabacum (Samsun SNN) or Solanum tuberosum (Desirée) introduced and overexpressed in roots or tubers. The Test system tobacco serves only as a model here and was due to the easy handling chosen. The PPDK from M. crystallinum serves as a representative of this enzyme class and was chosen based on its availability.

solutions

The cDNA of the M. crystallinum PPDK codes for a plastid enzyme of 103 kDa with and 94 kDa without a plastid recognition sequence (Fißlthaler 1993; Fißlthaler et al. 1995). This cDNA was cloned in a sense orientation into a binary expression vector (Bevan 1984) (see Fig. 3). In a further approach, the cDNA was manipulated so that this originally plastid enzyme remains in the cytosol. For this purpose, 183 bp were removed from the 5 'end of the cDNA (see FIG. 2), which led to the deletion of 120 bp of the plastid signal sequence. A second ATG (at +258 bp) of the reading frame was used as the translation start, so that the plastid signal sequence was deleted down to nine amino acids. The PPDK then no longer gets into the plastids and remains in the cytosol. Such constructs are called APPDK in the following.

In both cases the ppdk cDNA was under the control of the B33 patatin promoter specific for potato tubers (Rocha-Sosa et al. 1989). Tobacco received additional genetic information from these binary vectors (see Fig. 3) with the help of Agrobacterium tumefaciens. The T-DNA of the binary plasmid is integrated into plant genomes by A. tumefaciens. The integration of the T-DNA into the plant genome takes place at an undetermined location of the genome, so that variations in the expression of the genes have to be expected with different transformants, since this is influenced by the chromosomal localization (Schell 1987).

Fig. 4 shows that more PPDK was produced in the B33-PPDK plants than in the wild type. This difference, as was to be expected, is more pronounced in the potato tubers than in the tobacco roots, because the B33 promoter is only weakly induced in roots, while it is expressed very strongly in potato tubers.

The growth of the PPDK-overexpressing tobacco lines was then determined. The PPDK overexpressing tobacco plants grew faster than the wild type ( Fig. 5). Their fresh weight 3 months after sowing was about 2 times as large (B33-PPDK) or 2.3 times as large (B33-ΔPPDK) as that of the wild type. At the same time, these plants were about 1.3 times larger than the wild type, and they grew about 1.3 times faster in the last two months ( Fig. 5; relative growth).

PPDK-overexpressing potatoes produced up to 3.5 times greater amounts of starch per gram fresh weight in the tubers than the most productive wild type plant ( Fig. 6; starch; diluted 1:10). When the amounts of other metabolism intermediate were determined from the same samples, a different picture emerged: On average, the wild-type tubers contained more fructose, glucose, malate and sucrose than the transgenic tubers ( Fig. 6). Since the tubers were only prepared a week after harvesting, the amounts of sucrose in all plants are unusually low.

Conclusions

The following conclusions can be drawn from the data: With the help of additional cytosolic or plastid PPDK, which is produced in roots, plants can accelerate Growth. Potatoes with additional cytosolic or plastid PPDK, which is produced in tubers, produce more starch than the wild type. Former Patent applications showed that constitutively increased amounts of PPDK differed greatly Effects (Sheriff 1995; Sheriff 1995). Tobacco plants with larger quantities plastid PPDK in all tissues produced and grew more seeds than the wild type faster, but showed no increased amounts of starch. Tobacco plants with larger quantities a cytosolic PPDK in all tissues, however, did not produce any in extreme cases Seeds more and then grew more slowly. In no case were increased amounts of starch detected. The expression of additional PPDK in starch-storing tissues appears to Increase this storage capacity. The reason for these effects could not be clarified.

credentials

Aoyagi K. and Bassham JA (1986) Appearance and accumulation of C ₄ carbon pathway enzymes in developing wheat leaves. Plant Physiol. 80, 334-340.

Bevan M. (1984) Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12, 8711-8721.

Boehringer (1995) Methods of enzymatic bioanalysis and food analysis.

Boehringer Mannheim GmbH Biochemicals, eds: Sandhofer Str. 116, D-68298 Mannheim.

Borlaug N.E. and Dowswell C.R. (1988) World revolution in agriculture. In Book of the Year 1988, eds) Chicago: Encyclopedia Britannica, pp. 514.

Dennis D.T. and Turpin D.H. (1990) Plant Physiology, Biochemistry and Molecular Biology. Longman Group UK Limited, Essex

Fißlthaler B. (1993) The pyruvate, phosphate dikinase from the optional Crassulaceae Acid metabolism plant Mesembryanthemum crystallinum: sequencing and Characterization of the gene. Free University of Berlin, dissertation

Fißlthaler B., Meyer G., Bohnert H.J. and Schmitt J.M. (1995) Age-dependent induction of pyruvate, othophosphate dikinase in Mesembryanthemum crystallinum L. Planta 196, 492-500.

Horsch R.B., Fry J.E., Hoffmann N.L., Eichholtz D., Rogers S.G. and Fraley R.T. (1985) A simple general method for transferring genes into plants. Science 227, 1229-1231.

Lambers H. (1985) Respiration in intact plants and tissues. Its regulation and dependence on environmental factors, metabolism and invaded organisms. In Higher Plant Cell Respiration, (Douce R. and Day D.A., eds) Berlin: Springer-Verlag, pp. 418-473.

Latzko E. and Kelly GJ (1983) The many-faceted function of phosphoenolpyruvate carboxylase in C ₃ plants. Physiol. Veg. 2, 805-815.

Meyer A.O., Kelly G.J. and Latzko E. (1978) Pyruvate orthophosphate dikinase of immature wheat grains. Plant Sci. Lett. 12, 35-40.

Mohr H. and Schopfer P. (1978) Textbook of Plant Physiology. Springer publishing house, Berlin.

Rocha-Sosa M., Sonnewald U., Frommer W., Stratmann M., Schell J. and Willmitzer L. (1989) Both developmental and metabolic signals activate the promoter of a class I patatin genes. EMBO 8, 23-29.

Rosche E., Streubel M. and Westhoff P. (1994) Primary structure of the photosynthetic pyruvate orthophosphate dikinase of the C ₃ plants Flaveria pringlei and expression analysis of pyruvate orthophosphate dikinase sequences in C ₃ , C ₃ -C ₄ and C ₄ Flaveria species. Plant Mol. Biol. 26, 763-769.

Schell J. (1987) Transgenic Plants as Tools to Study the Molecular Organization of Plant Genes. Science 237, 1176-1183.

Sheriff A. (1995) Method of obstructing or preventing the formation of breathing vegetable tissue, in particular for the production of seedless plants by additional cytosolic pyruvate, phosphate dikinase. File number of the patent application at German Patent Office: 195 31 678.9.

Sheriff A. (1995) Process for increasing plant seed production and for Accelerated plant growth through additional plastid pyruvate, phosphate Dikinase. File number of the patent application at the German Patent Office: 195 31 783.1.

Strasburger E., Noll F., Schenck H. and Schimper A.F.W. (1991) Textbook of botany. Gustav Fischer Verlag, Stuttgart.

Taiz L. and Zeiger E. (1991) Plant Physiology. The Benjamin / Cummings Publishing Company, Inc., Redwood City.

Ting I.P. (1985) Crassulacean acid metabolism. Ann. Rev. Plant. Physiol. 36, 595-622. Voet D. and Voet J.G. (1992) Biochemistry. VCH, Weinheim.

Wilson D. and Jones J.G. (1982) Effect of selection for dark respiration rate of mature leaves on crop yields of Lolium perenne cv. S23. Ann. Bot. 49, 313-320.  

Legends

Fig. 1: Metabolism scheme according to Latzko and Kelly (1983) shows one way of refixing respiratory carbon dioxide.

Fig. 2: Metabolism scheme according to Aoyagi and Bassham (1986) shows a possibility for carbon dioxide enrichment.

Fig. 3: Schematic drawing of the constructed binary vectors with M. crystallinum-PPDK insertion (B). At the very top (A), the PPDK cDNA is shown with some restriction sites (Fißlthaler 1993). The PPDK cDNA sequence from M. crystallinum is 3173 bp long, of which 9 bp at the 5 'end is the linker sequence from the cDNA synthesis and the 3' terminal 12 bp is the polyA end which is attached after the transcription. The start codon is at position 63, the termination codon of the longest open reading frame at position 2910. The protein derived from the cDNA sequence of the PPDK from M. crystallinum consists of 949 amino acids, which corresponds to a molecular weight of 103244 daltons. The pre-sequence of the PPDK from M. crystallinum has a length of 74 amino acids, so that the mature protein processed in the chloroplast has a size of approximately 94 kDa. The amino acid composition of the mature, processed protein gives a pI of 7.6. 5 'and 3' untranslated areas of the cDNA are shown as terminal hatched boxes. In the sense PPDK, the PPDK cDNA is shown in the correct orientation (left 5 ', right 3' untranslated area; the dotted box represents the plastid signal sequence with the three ATGs in the reading frame; the first ATG is normally used as the translation start ). A indicates a deletion of 183 bp at the original 5 'end. Restriction mapping of Bin19 can be seen in (C).

BR, BL: right and left border region of T-DNA from Agrobacterium tumefaciens; Km: Kanamycin resistance with bacterial regulatory elements; nptll: neomycin phosphotransferase (Kanamycin resistance) with herbal regulatory elements; B33: promoter of a class I Patatinens; ocs polyA: polyadenylation signal of the octopine synthase gene from Agrobacterium tumefaciens.

The transformation of the tobacco plants using the A. tumefaciens-mediated gene transfer was carried out according to Horsch et al. (1985).

Fig. 4: PPDK protein levels of PPDK-overexpressing potato tubers (see also Fig. 3) were examined by Western blots. An antiserum against PPDK was used for the detection. 6 µg crude extract of a salt-pressed M. crystallinum served as positive controls. Untransformed potatoes (Wt) served as controls. 6 µg of the soluble tuber proteins were used. The transgenic potato plants analyzed were the F ₀ generation of independent transformants. 6 µg of the soluble tobacco root proteins were used. The analyzed transgenic tobacco plants were the F ₁ generation of independent transformants.

Fig. 5: Differences in the growth rate of wild type (Wt) and the PPDK overexpressing tobacco lines (B33-PPpK and B33-ΔPPDK) 3 months after sowing. The transgenic plants each consist of six different lines (independent transformants). 8 wt 26 B33-ΔPPDK and 21 B33-PPDK plants were analyzed to determine the fresh weight. 15 Wt, 43 B33-ΔPPDK and 59 B33-PPDK plants were analyzed to determine the size and relative growth. The plants were grown in uniform earth.

Fig. 6. Quantitative determination of metabolic products (starch, sucrose glucose, fructose and malate, Boehringer 1995) of the PPDK overexpressing potato lines. For the analysis, 4 wild-type potato plants and 13 potato tubers overexpressing the PPDK and 14 of the APPDK were analyzed. The individual transgenic plants were independent transformants (F ₀ generation). The graphic shows individual analyzes, since only single plants but not several plants in a line could be used for the investigation. This makes it easy to see the difference in the effect of independent transformants. Each sample was measured twice. The mean of both measurements is shown in each case. In addition, the starch measurement was carried out with two different sample quantities. Both measurements are listed (undiluted and diluted 1:10).

Claims (12)

1. A process for the production of plants with an increased growth rate and increased yield, characterized in that an additional pyruvate, phosphate dikinase is expressed in these plants in the cytosol or in the plastids and additional phosphoenol pyruvate is thereby produced. 2. Process for the production of plants with increased growth rate and increased yield, characterized in that in these plants an additional pyruvate in the root, Phosphate dikinase is expressed and thereby additional phosphoenol pyruvate is produced. 3. Process for the production of plants with increased growth rate and increased yield, characterized in that in these plants in the tuber or in the fruit a additional pyruvate, phosphate dikinase (PPDK) is expressed and thereby additional Phosphoenolpyruvat is produced. 4. Process for the production of plants with increased growth rate and increased yield, characterized in that the chimeric DNA construct B33-PPDK is included. 5. Process for the production of plants with increased growth rate and increased yield, characterized in that in these plants the chimeric DNA construct B33-ΔPPDK is included. 6. Process for the production of transgenic plant cells, characterized in that they can be obtained according to any one of claims 1-5. 7. A process for the production of transgenic plants, characterized in that after one of claims 1-5 can be obtained. 8. A method for producing transgenic plants according to claim 7, characterized characterized that they are useful plants. 9. A process for the production of transgenic potato plants, characterized in that they are obtained according to any one of claims 1-5. 10. A process for the production of transgenic tobacco plants, characterized in that they are obtained according to any one of claims 1-5. 11. Chimeric gene B33-PPDK, characterized in that plants with increased Growth rate and increased yield can be generated. 12. Chimeric gene B33-ΔPPDK, characterized in that plants with increased Growth rate and increased yield can be generated.