WO1999067392A2 - Modified plant metabolism using pyrophosphate-dependent phosphofructokinase - Google Patents

Abstract

This invention is directed to the modification of metabolism in photosynthetic organisms. Photosynthetic organisms have been successfully transformed with nucleic acid encoding an unregulated pyrophosphate:fructose 6-phosphate 1 phosphotransferase (PFP) protein capable of expressing a PFP protein which modifies metabolism without being regulated by the host organism. Transgenic plants and seeds have been produced which yield modified carbohydrate, oil, fiber, sugar content as a result of alteration of glycolysis.

Description

MODIFIED PLANT METABOLISM USING PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKTNASE

RELATED APPLICATION(S)

This application claims benefit of application 60/089,927, filed June 19, 1998, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Carbohydrate metabolism is a complex process in plant cells. Through the process of photosynthesis, sugars are synthesized and are used for a variety of purposes. Starch and fatty acids are synthesized in the plastids. A variety of processes result in a multitude of carbohydrate, protein, fiber and oil products.

However, attempts to manipulate the allocation of carbon resources within plant cells by changing the ratios of enzymes involved in primary carbon metabolic pathways is usually unsuccessful, or results in insignificant changes, because of compensatory pathways in plants. Thus, most attempts to manipulate plant metabolism through genetic engineering concentrate on manipulating enzymes at the end of a pathway.

Pyrophosphate:fructose 6-phosphate 1 -phosphotransferase (PFP) catalyzes the phosphorylation of fructose-6-phosphate, thus supplying the plant with fructose- 1, 6-bisphosphate during glycolysis. Little is known regarding the role of this important enzyme in photosynthetic organisms. Attempts have been made to study PFP in plants but, due to compensatory pathways and feedback regulation of the endogenous plant gene, it has been difficult to determine how PFP affects end product synthesis in plants and seeds.

The ability to regulate the partitioning between starch and sugar is of great commercial value in many plants and seeds. In tuberous plants, the ability to prevent starch breakdown and the subsequent accumulation of reducing sugars, such as glucose and fructose, during storage is important to the food and horticultural industries. SUMMARY OF THE INVENTION

This invention relates to constructs and methods for the modification of carbohydrates in photosynthetic organisms. In particular, this invention relates to nucleic acid constructs comprising a gene which is not endogenous in photosynthetic organisms and which encodes a PFP protein, preferably a Giardia PFP enzyme.

Another aspect of this invention involves vectors comprising expression constructs containing nucleic acid sequence(s) encoding a non- plant or unregulated PFP enzyme operably linked to a promoter sequence, either tissue-specific, constitutive or inducible, for transfer of these sequences and integration into the genome of the cell of a photosynthetic organism. For example, plant transformation vectors may include Agrobacterium T-DNA border region(s) to provide for transfer of the sequences to the plant cell.

A method for modifying the level of one or more metabolic products of a plant, plant part or photosynthetic cell, wherein said method comprises: a. transforming a plant, plant part or photosynthetic cell with a recombinant DNA construct containing an unregulated PFP; b. optionally regenerating the plant part or photosynthetic cell to generate a whole plant; and c. subjecting the transgenic plant, plant part, or photosynthetic cell to conditions wherein the unregulated PFP is expressed; wherein the level of the one or more metabolic products of the plant, plant part or photosynthetic cell are modified relative to an untransformed plant, plant part or photosynthetic cell. The methods of the invention are particularly useful for modifying the plant, tissue or cellular levels of fiber, oil and protein. The invention further describes stably transformed transgenic plants, plant cells or plant tissues or parts or cell cultures thereof made according to the methods. Preferred plants include Brassica sp. (e.g., canola), alfalfa, corn (maize), sorghum, soybean, sunflower, wheat, rice, rye, cotton, barley, turfgrass and potato.

Also considered part of this invention are photosynthetic organisms, especially plants, plant cells and tissues, containing nucleic acid sequences encoding the non-plant or unregulated PFP enzyme. Seeds and descendants of these transformed organisms are included as well.

In yet another aspect of this invention, transformed plant cells having reduced or increased soluble sugars or starch content are considered. Of particular interest are plant cells in starch storage organs, such as roots, tubers or seeds.

Further it is recognized that the synthesis or degradation of sugars and starch through the use of the PFP enzyme in photosynthetic organisms also affects the quantity of other metabolic products such as lipids, fiber, and protein. Thus, this invention relates to constructs and methods for manipulating the amount and ratio of many different metabolic products in these organisms, particularly plants. Specifically, reduction or enhancement in the levels of the soluble sugars, proteins, fiber and oil through manipulation of either plant or non plant pyrophosphate dependent phosphofructokinase is part of this invention. It is therefore recognized that modulation of glycolysis using the PFP enzyme or through inhibition of endogenous PFP has many commercial applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a diagram of the relationship of the glycolytic pathway between the cytosol and the plastids of plants.

Fig. 2 is a schematic representation of the 95.820 construct, which was used to clone the Giardia PFP gene.

Fig. 3 is a schematic representation of the 95.1160 construct, which was used to transform Agrobacterium. Fig. 4 is the polynucleotide sequence (SEQ ID NO: 1) and encoded amino acid sequence (SEQ ID NO:2) of the Giardia lamblia PFP gene.

Fig. 5 is a histogram depicting the level of enzyme activity in leaves. The enzyme activity in Units per gram of fresh weight tissue (y-axis) in the four T₃ lines PFP-1, PFP- 16, PFP-20, and PFP-23, and transgenic control pRD400, is shown for the four enzymes Giardia PFP, tobacco PFP, PFK, and FBPase. Fig. 6 is a bar graph showing the level of sucrose in μmoles per gram of fresh weight leaf tissue (y-axis) for transgenic plants expressing Giardia PFP (black bars) compared to control plants (white bars). Samples were taken at 7 hours (initiation of light period) and 19 hours. Light intensity was 500 μmole per meter² per second, and temperatures were set at 22 °C (day) and 18°C (night). The data was pooled from four replicates each of lines PFP-1, PFP-16, PFP-20, and PFP-23, or Null-1, Null-16, Null-20, and Null-23.

Fig. 7 is a bar graph showing the level of starch in μmoles glucose equivalents per gram of fresh weight leaf tissue (y-axis) for transgenic plants expressing Giardia PFP (black bars) compared to control plants (white bars). Samples were taken at 7 hours (initiation of light period) and 19 hours. Light intensity was 500 μmole per meter² per second, and temperatures were set at 22 °C (day) and 18°C (night).

Fig. 8 is a bar chart depicting the level of photosynthesis in transgenic plants (black bars) and null segregants (white bars) under different levels of light intensity. Carbon dioxide consumption in μmoles CO₂ per meter² leaf area per second is shown on the y-axis, and irradiance (0, 300 and 1200 μmoles quanta per meter² surface area per second) is shown on the x-axis. Fig 9 is a plot showing the correlation between the level of PFP activity (in μmol per minute per gram fresh weight tissue) and percent fiber in seeds nine days after anthesis. Each transgenic (•) and null segregant (O) data point represents a single measurement of a single plant.

Fig. 10 is a plot showing the correlation between the level of PFP activity (in μmol per minute per gram fresh weight tissue) and protein in milligrams per gram of fresh weight tissue in seeds nine days after anthesis. Each transgenic (•) and null segregant (O) data point represents a single measurement of a single plant.

Fig. 11 is a plot showing the correlation between the level of PFP activity (in μmol per minute per gram fresh weight tissue) and percent lipid in seeds nine days after anthesis. Each transgenic (•) and null segregant (O) data point represents a single measurement of a single plant. Fig. 12 is a plot showing the correlation between the level of PFP activity (in μmol per minute per gram fresh weight tissue) and starch, in μmoles hexose equivalents per gram fresh weight tissue in seeds nine days after anthesis. Each transgenic (•) and null segregant (O) data point 5 represents a single measurement of a single plant.

Fig. 13 is a graph showing reduced biomass accumulation in transgenic plants (•) relative to non-transgenic plants (■) as total grams of shoot and root dry weight at various days after transplanting.

Figs. 14A and 14B are histograms comparing the levels of fiber (Fig. 10 14A) and lipid (Fig. 14B) in mature seeds of transgenic plants and seeds of nulls.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a nucleic acid construct which encodes an unregulated pyrophosphate-dependent phosphofructokinase in plants or other

15 photosynthetic organisms. In particular, this invention relates to a nucleic acid construct comprising a gene from the parasitic protist, Giardia lamblia, which encodes an enzyme which catalyses the interconversion of fructose-6- phosphate and fructose- 1,6-bisphosphate, and to functional segments thereof which encode a protein or polypeptide which also catalyses the

20 interconversion of these two compounds, or which similarly alters the levels of starch, carbohydrate, protein, lipid, and/or fiber in plants or other photosynthetic organisms.

By "lipid" is meant the oils and fats naturally present in vegetative materials and especially the oil in oleaginous seeds, including saturated and

25 unsaturated fats. The term "carbohydrate" is used herein in its usual context to mean a water-soluble saccharidic compound of carbon, hydrogen, and oxygen that contains the saccharose grouping. Monosaccharides, disaccharides, and low molecular weight polysaccharides are included in the term "carbohydrates." Monosaccharides useful herein include simple sugars such

30 as fructose and glucose. Disaccharides include such compounds as sucrose, maltose, and lactose. By "protein" and "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). By "fiber" is meant complex carbohydrates resistant to digestion by mammalian enzymes, such as the carbohydrates found in plant cell walls and seaweed, and those produced by microbial fermentation. Examples of these complex carbohydrates are lignin, brans, celluloses, hemicelluloses, pectins, gums and mucilages, seaweed extract, and biosynthetic gums. Sources of the cellulosic fiber include whole plants, vegetables, fruits, seeds, cereals.

Pyrophosphate:fructose-l-6-phosphotransferase, also called pyrophosphate-dependent phosphofructokinase, or PFP, catalyzes a reversible phosphorylation of fructose-6-phosphate (F6P) using inorganic pyrophosphate (PPi) as a phosphate donor. Absent in plastids, but with a near-ubiquitous distribution in the cytosol of plants, PFP is often more active than the ATP- dependent phosphofructokinase (PFK) and is activated by the regulator metabolite, fructose-2,6-bisphosphate. PFP obviously plays an important role in plant metabolism, but its actual contribution has been difficult to determine. There have been several unsuccessful attempts to modify PFP activity in plants. For example, transformation of plants using DNA constructs encoding the subunit of PFP in an antisense orientation does not result in significant changes in carbon partitioning or in growth rate compared to controls (Paul et al. (1995) Planta 196:277-283).

The expression of a heterologous bacterial pyrophosphatase in plant tissues has been shown to elevate the level of sucrose, glucose and fructose in these plants (Jelitto et al. (1992) Planta 188:238-244). It is believed that utilization of pyrophosphate would upset the equilibrium of enzymes involved in sucrose synthesis in the direction of product formation. In another study, expression of maize sucrose phosphate synthase in transgenic tomatoes also led to an increase in the level of soluble sugars (Worrell et al. (1991) Plant Cell 3:1121-1130). The use of pyrophosphatase and sucrose phosphate synthase for the manipulation of sugar levels (as well as cold and drought tolerance and as a remedy for cold sweetening in potatoes) have also been studied. However, the manipulation of PFP to alter the synthesis or degradation of starch and hexose sugars is a novel feature of this invention. Removal of PFP activity through anti-sense constructs led to a slight increase in the level of soluble sugars (Hajirezaei et al. (1994) Planta 192:16- 30). PFP activity was not, however reduced to zero and it has yet to be shown whether complete elimination of the activity of this enzyme leads to a significant increase in the levels of soluble sugars. PFP catalyzes a freely reversible reaction, the interconversion of fructose-6-phosphate and fructose- 1,6-bisphosphate while either using or generating pyrophosphate. Manipulation of enzymes involved in pyrophosphate metabolism also have a significant effect on the levels of sucrose and other soluble sugars.

In the present invention, PFP levels have been manipulated in plants and are shown to have a significant effect on starch. Further, it is demonstrated that pyrophosphate-dependent phosphofructokinase (PFP) can also play a role in the control of carbohydrate levels in plants. These alterations in carbohydrate levels also result in changes to the amounts of ultimate products that are formed from the intermediates of glycolysis, e.g., oil, fiber, lignin, and protein.

In most plants, the carbon resource used for oil biosynthesis is sucrose, which is synthesized in the leaves and transported to the developing seed. Within the cells of the seed, either invertase or the sucrose synthase pathway first converts the sucrose to hexose and the glycolytic pathway then oxidizes the hexose to pyruvate. The pyruvate dehydrogenase complex can further oxidize this to acetyl-CoA, the immediate carbon precursor for fatty acid biosynthesis.

Fatty acid synthesis in plants occurs in plastids, especially leucoplasts which are specialized for fatty acid biosynthesis in seeds. Leucoplasts synthesize saturated and unsaturated fatty acids from acetyl-CoA. The fatty acids are exported into the cytosol of cells and stored in lipid bodies as triacylglycerol.

By manipulation of a novel non-plant, unregulated PFP, starch levels can be dramatically reduced in leaves. Previous attempts to reduce sugar formation from starch, using various genes (U.S. Patent No. 5,648,249), antisense (U.S. Patent No. 5,646,023) or ribozymes have not been very successful.

There are several potential applications for this technology and indication that more complete reduction in the level of the native plant enzyme than has yet been achieved may also lead to an increase in the levels of soluble sugars such as sucrose, fructose and glucose.

The parasitic protist, Giardia lamblia, contains a gene that, by homology to other genes, clearly encodes for the enzyme PFP. The gene has also been engineered to express in bacteria (e.g., E. coli), and is confirmed to have PFP activity. The amino acid homologies with the and β subunits of the potato enzyme are approximately 35% and 50% respectively. The enzyme from Giardia is, however, significantly different from the PFP of plants; in particular, the activity of PFP is not dependent on the activator fructose-2,6- bisphosphate, that is, when transformed into a plant, it behaves as an unregulated PFP. By "unregulated PFP" is meant a pyrophosphate-dependent phosphofructokinase enzyme that interconverts fructose-6-phosphate and fructose- 1,6-bisphosphate without being regulated by the level of fructose-2,6- bisphosphate, which is a regulatory mechanism in plants. The enzyme is preferably highly active in the plant or plant cell. By "highly active", it is meant the PFP has an activity level substantially the same as the endogenous PFP in the presence of fructose -2,6-bisphosphate. That is, a PFP that, when isolated from one organism and placed in a different (photosynthetic) organism, catalyzes the interconversion of fructose-6-phosphate and fructose- 1 ,6-bisphosphate without being regulated by the levels of fructose-2,6- bisphosphate. The PFP enzyme is made up of two subunits: one catalytic and one regulatory. When a plant is transformed with the PFP from another plant, the regulatory subunit responds to the level of fructose-2,6-bisphosphate. Applicants have found however, that when a plant is transformed with the PFP from Giardia sp., the single subunit of the Giardia PFP does not respond to levels of fructose-2,6-bisphosphate, and so is unregulated. It is intended, therefore, that the term "unregulated PFP" include not only those PFP enzymes that, when placed within a host plant, fail to respond to regulation (e.g., the Giardia PFP), but also those PFP enzymes in which the catalytic and/or regulatory subunits have been mutated such that the enzyme is active and non-responsive to fructose-2,6-bisphosphate regulation, e.g., a plant PFP wherein the subunits have been mutated or otherwise altered so as to render it non-responsive to regulation by the plant into which it has been transformed.

For instance, one could use site-directed mutagenesis to alter the binding site of either the the activator fructose-2,6-bisphosphate or the binding site of the substrates. Crystal structures are available for nonplant PFK and FBPase enzymes, and these two enzymes use the same carbon substrates (fructose-6-phosphate and fructose- 1,6-bisphosphate) as does PFP, and PFP and FBPase are allosterically regulated by frustose-2,6-bisphosphate, e.g. , FBPase is inhibited while PFP is activated. Based on what is already known about these related enzymes, one can deduce the analogous residues in the PFP sequences by conserved sequence homology and computer protein modeling. By using such information, meaningful protocols can be formulated to accomplish site-directed mutagenesis of a plant PFP to transform the PFP from a regulated PFP to an unregulated PFP useful in the present invention. Plant PFP, unlike Giardia PFP, is composed of two subunits, designated and β, which are believed to exist as heterotetramers consisting of two α and two β subunits. The β subunit is reported to contain the substrate binding sites (fructose-6-phosphate, pyrophosphate, fructose- 1,6- bisphosphate, phosphate), giving catalytic activity (Carlisle et al. (1990) J. Biol. Chem. 265:18366-18371). Fructose-2,6-bisphosphate activates PFP by eliciting a conformational change. This site can be mutagenized so as to make this conformational change permanent, thereby producing a mutant PFP that is constantly active, as is Giardia PFP. Substantial information is available on the essential amino acid residues of the catalytic site of PFP, including the crystal structure of E. coli PFK, sequence analysis, and computer modeling. Two aspartate residues have been identified as having a catalytic role after site-directed mutagenesis of the Propionibacterium freudenreichii PFP sequence (Green et al. (1993) J. Biol. Chem. 265:5085-5088). Similarly, mutagenesis of specific arginine residues of the protozoan Naegleria fowler i PFP sequence was reported to result in substantial reductions of the reaction rate (Hinds et al. (1998) Arch. Biochem. Biophys. 349:41-52). The potato PFP enzyme is also reported to contain an unspecific essential arginine (Montavon et al. (1993) Plant Physiol. 101:765-771). A number of PFP sequences are available for plants, including potato, castor bean, grapefruit, and Arabidopsis, allowing deduction of conserved essential amino acid residues for other plant PFP enzymes. Another strategy would be to mutagenize the fructose-2,6- bisphosphate binding site. Sites for mutagenesis would be chosen based on computer modeling and amino acid residues that are conserved between plant PFP enzymes and residues known to be essential for binding in PFP enzymes from other organisms, as deduced from crystal structures of fructose-2,6- bisphosphate complexed with FBPase (Liang et al. (1992) Proc. Natl. Acad. Sci. USA 59:2404-2408), or crystal structures of ADP (which has a similar phosphate grouping with fructose-2,6-bisphosphate) complexed with PFK (Van Praag (1997) Intl. J. Biol. Macromolecules 2i:307-317).

The present invention therefore is intended to encompass transformed plants containing such PFP enzymes which have been manipulated so as to be active and insentitive to fructose-2,6-bisphosphate, as is the Giardia PFP enzyme.

"Mutated or otherwise altered" is intended to include selective mutation, spontaneous mutation, chemical mutagenesis, mutagenesis by genetic engineering, and mutation resulting from mating or other forms of exchange of genetic information, including, e.g., base changes, deletions, insertions, inversions, translocations, duplications, or frame-shifts. By "unregulated," it is not intended that the activity of the enzyme is never to be controlled, but rather, that the expression of the enzyme may be regulated (e.g., turned "on" or "off in certain tissues or under certain conditions) in ways other than by the level of fructose-2,6-bisphosphate. In the present invention, a plant is challenged with an enzyme of primary carbohydrate metabolism, the activity of which it is unable to control. To do this, the coding sequence of the Giardia PFP gene was connected in a sense orientation to a tandem 35S CaMV promoter (Figures 2 and 3 and Example 1). The resulting construct was transformed into tobacco, Brassica sp., and alfalfa. In this manner, plants have been produced which demonstrate a variety of unregulated PFP activities. Since PFP catalyzes a freely reversible reaction that either utilizes or generates pyrophosphate, a metabolite that is involved in reactions controlling the synthesis and breakdown of sucrose, the presence of the Giardia PFP affects the level of starch, protein, oil and fiber in these plants.

By "plant" is meant a photosynthetic organism, and parts and cells of such organisms. The term is therefore meant to include whole plants, plant parts (e.g., tissues, seeds, roots, shoots, flowers, cutting, fruits, etc.), and cells, e.g., including tissue culture of such plants and plant parts. Descendants of such plants, plant parts and cells, e.g., tissue cultures of such plants, plant parts and cells, and descendants of such tissue cultures, are also intended to be included by the term "plant."

By "photosynthetic organism" is meant green plants, algae, fungi, and other organisms capable of photosynthesis when found in a wild type state, whether such organisms are multicellular or unicellular.

Data collected from these plants indicates that (1) plants have been produced in which expression of PFP is elevated from 2-fold to 80-fold compared to the levels of enzyme measured in nulls; (2) plants expressing greater than 20-fold the PFP activity found in null plants contain lower levels of sucrose, glucose and fructose; (3) early-developing seeds from plants expressing 80-fold the normal level of PFP have increased production of oil bodies and protein, and are retarded in their production of lignin (although they reach normal levels at maturity) compared to seeds of nulls, and are inhibited in embryo development. These data also suggest that a complete removal of the endogenous PFP enzyme may lead to an increase in the soluble sugar levels. The use of an unregulated PFP indicates, contrary to what the prior art teaches, that plants can be modified by changing PFP activity. Previous attempts to reduce the level of PFP in transgenic plants have involved manipulation, either sense or anti-sense, of the plant enzyme itself. Previous published attempts were successful in reducing PFP activity to low levels affecting sugars, however, there were no obvious phenotypic or growth differences. This invention indicates that complete removal or inactivation of the gene encoding PFP can have varying results.

The invention described herein demonstrates that the glycolytic and gluconeogenic pathways can be manipulated by expressing and thus increasing PFP expression over and above the endogenous levels in a photosynthetic organism. The increased PFP activity has an effect on other enzymes in the glycolytic pathway, pushing glycolysis in one direction depending on the plant tissue and the stage of development. In seeds, for example, oil formation is accelerated early in seed formation and the synthesis of fiber (e.g., lignin) is delayed, leading to a higher ratio of oil to fiber in the seeds of transgenic plants.

By "stage of development" is meant a particular point in the development and life cycle of the plant, e.g., germination, onset of flowering, seed set, seed production, development of tubers (in tuberous plants), senescence, etc.

Over 300 transgenic tobacco plants have been produced which incorporate the Giardia gene encoding PFP. Replicates of four different T3 homozygous lines (PFP-1, PFP- 16, PFP-20, and PFP -23) were compared to their homozygous nulls segregants or the pRD400 transgenic control (pRD400, wild-type plants).

Transgenic seeds of the four transformed lines were found to have a higher percent germination rate than did seeds from the null segregants.

Transgenic plants also differed from null plants in carbohydrate levels. Expression of Giardia PFP in transgenic plants produced a significant change in sucrose levels (Figure 7). Sucrose levels are reduced in source leaves at 7 hours (initiation of light period) and increased at 19 hours. Starch levels, as shown in Figure 7, were markedly reduced at 7 and at 19 hours indicating that carbohydrates could be utilized more rapidly in the dark in the transgenic plants. Thus, the soluble sugars are probably transported rapidly through the glycolytic pathway, especially during the night in transgenic plants, to sink tissues where these intermediates are used to produce oil, protein, fiber, and other end products.

The protein levels in leaves were also lower than those of the null plants. These measurements were taken on a fresh weight basis. Fresh weights per square meter of leaf area did not differ between transgenic and null plants.

Glycolytic enzyme activity levels were also measured in seeds of transgenic plants and compared to activity levels in nulls. In addition to elevated levels of PFP activity, other enzyme activity levels were also increased as follows: 1 ) PFK activity increased 2.0-fold;

2) FBPase activity increased 1.3-fold;

3) Pyruvate kinase activity increased 2.0-fold; and

4) PEP carboxylase activity increased 1.5 fold.

The effects of increased PFP activity does not result in morphological changes in the trangenic plants and seeds. The shoot to root ratio is unchanged between transgenic plants and nulls. The color of the plants also does not vary.

Shoot and root biomass accumulation between the time of seedling establishment and onset of flowering is lower in the transgenics than in the null segregants (Fig. 13). This effect is amplified under conditions where soil phosphate is limiting. These smaller plants do not yield less seed, however, and smaller plants would allow planting at higher density in the field. Seed production per unit planted area would therefore be increased, which is especially important in oilseed crops such as Brassica. A reduced growth habit would also be advantageous in plants such as turf grasses and some ornamentals. Protein levels in leaves are also reduced, as shown in Table 1, below. The reduction is especially striking in Experiment 3.

Table 1. Content of protein in leaves, in milligrams of protein per gram fresh weight leaf tissue. "Experiment" refers to samplings from three different plantings. Within each planting there were replicates of each transgenic line and null. "High nutrient" refers to fertilizer levels.

The lipid levels of mature transgenic seeds are increased as well. Early in seed development (8-12 days after anthesis) there is an increase in the deposition of lipid (oil bodies) in the seed. The fiber levels are reduced (Figure 9) and the oil levels increased (Figure 11). As shown in Fig. 10, protein levels are also increased. Higher levels of oil and protein, and decreased levels of fiber are highly desirable in seed crops, because the oil and protein are the desirable economic products, while fiber adversely affects digestibility of the seed, thereby limiting its utility as an animal feed. Even in non-animal feed crops, fiber reduction is desirable because such reduction increases the percentage of valuable products (e.g., protein, oil) proportionally.

The normal levels of fiber in mature seeds was 35.9% of fresh weight (Fig. 14A) whereas the lipid content was measured at 32.0% (Fig. 14B). In all transgenics measured, the ratio of fiber to oil was considerably lower. One explanation for this change, without being limited by theory, is that the sucrose transported to the seed is more efficiently and rapidly utilized. Fiber is produced for the seed coat before embryo storage oil. An early onset of storage oil synthesis in the transgenics could divert imported sucrose away from fiber synthesis. Thus, reserves of the substrate PEP could be converted into lipid before fiber synthesis is initiated or is in full progress in the cytosol. This invention provides a method for selectively modifying the sugar, starch, fiber, and oil levels of photosynthetic organisms and seeds. The method involves incorporating an isolated nucleic acid encoding a non-plant or unregulated PFP protein, such as Giardia pyrophosphate-dependent phosphofructokinase (SEQ ID NO:l) or its functional equivalent into a cell or organism and maintaining the cell or organism under conditions appropriate for expression of the encoded PFP enzyme or its functional equivalent. The methods of the present invention can be used to enhance or reduce sugar and oil levels of plant parts, seeds, and plants, as well as in other types of photosynthetic organisms, such as unicellular algae. These methods of modulating carbohydrate and lipid levels are especially useful under conditions of phosphate starvation when other pathways, which depend on ATP are reduced in activity or completely inhibited. The PFP proteins of the invention are incorporated, and are also very useful, for example, to sequester particular compounds in seeds.

It is also understood that the constructs of the present invention can function to modulate carbohydrate levels of all types of cells, preferably plant cells, and in cell lines of organisms, preferably plants. The cells can comprise single-celled prokaryotic organisms; i.e., the cyanobacteria (blue-green algae). Alternatively, the cells can be, plant- like or fungal-like protists (single-celled eukaryotic organisms). Multicellular organisms, including all members of the fungi and plant kingdoms, are also suitable for application of the methods of this invention. Cell lines incorporating the constructs produced through the methods of this invention can be derived from any of these eukaryotic organisms. Tissue cultures and protoplasts can also incorporate constructs capable of expressing non-plant or unregulated PFP and can be useful for production of products and for research purposes. In addition to modulation of sugar levels in transformed cells, the methods of the present invention can be used in conjunction with other enzymes, such as phosphatases; i.e., incorporating phosphate-starvation promoters and enzymes, as well as other molecules into cells and photosynthetic organisms to further modify the accumulation of products. Like many proteins that catalyze part of a major metabolic pathway, these proteins are useful to modify the carbohydrate and lipid accumulation in every part of a plant.

The invention described herein demonstrates for the first time the effects of overproduction of PFP in photosynthetic organisms and demonstrates that this enzyme is important not only under normal physiological environmental conditions, but also under conditions of stress, especially nutrient stress.

A novel aspect of this invention is the finding that the Giardia pyrophosphate-dependent phosphofructokinase is unregulated in a photosynthetic organism and thus unaffected by the regulatory systems in these organisms. Even more surprising is the discovery that the expressed protein affects not only sugar accumulation but can change the quantity and ratio of lipids, fiber and protein in various plant organs. That Giardia pyrophosphate-dependent phosphofructokinase is effectively expressed in a construct in a transformed plant, without requiring additional introduction of a multicomponent enzyme system, is novel, unusual and advantageous. In one embodiment, therefore, levels of soluble sugars in photosynthetic organisms are reduced by elevating the efficiency of PFP activity which reduces sucrose, glucose and fructose levels. These results suggest that the natural role of PFP in plants is in carbon flow, primarily the modification of glycolysis and sucrose synthesis. Expression of PFP stimulates a higher, more efficient level of sugar flux and utilization into products. While not to be limited by theory, the lower starch and soluble sugar levels probably result from acceleration of the sugar flux process, with carbon intermediates probably being subjected to less regulation. Further, the enhancement is stable because the newly introduced gene and encoded protein is not susceptible to regulatory control, thereby reducing the possibility of down-regulation. This novel technology results in major modification of product synthesis in all parts of the plant. The enhancement of PFP activity is caused by a genetic component which can be used to manipulate a major metabolic pathway to result in many useful products. Thus, the discovery that PFP activity in plants can be controlled to produce a novel and significant impact on the ultimate levels and kinds of carbohydrates, oils, lignins and protein found in various plant organs. The enhancement of foreign PFP protein production and its effect on sugar transport can be achieved by stably introducing a DNA construct comprising a nucleic acid encoding a functional PFP polypeptide operably linked to a promoter into a cell of a photosynthetic organism, preferably a plant, and putting the cell under conditions for expression of the protein. This method can enhance PFP activity in cells, tissues and organs of plants at levels which are not found in the naturally- occurring plant.

The ability of the leaves of the transgenic plants expressing Giardia PFP to load sucrose into the phloem (and from there to seeds, roots and emerging leaves) is likely to be related to the phosphate status of the leaf and the inorganic phosphate/inorganic pyrophosphate (Pi/PPi) ratio. Since the reaction catalyzed by PFP utilizes PPi and generates Pi (or vice versa depending on the direction in which the enzyme is working), the reduction in sugar and starch levels could be the result of an effect on the loading of sucrose into the phloem.

Activity of PFP, unlike PFK, does not require ATP to function. Thus, PFK is ATP dependent and PFK modulation of the sugar content of plants will vary under conditions of phosphate sufficiency and phosphate deficiency. PFP, as shown in Figure 1, is not ATP dependent and, under conditions where phosphate is limited, will still continue to drive pathways leading to sugar modulation. Thus, under anaerobic conditions or other conditions of phosphate starvation, PFP will be a controlling factor in varying the sugar levels in various plant tissues, especially in the root where it is expressed. The many- faceted nature of sugar transport mechanisms in plants, suggests that there are a significant number of combinations of promoters and PFP encoding genes that can affect the amounts and ratios of the products of carbon metabolism. The constructs of this invention can include any promoter, tissue- specific, constitutive or inducible, which can drive the expression of a nucleic acid encoding a PFP polypeptide. Those of skill in the art recognize that any of many suitable methods of introduction of vectors can be used to introduce constructs of this invention and the methods are a matter of choice for the particular organism, cell or tissue used, and are not critical to success in expressing the proteins of this invention.

For example, isolated DNA is introduced into plant cells of a target plant by well-known methods, such as Agrobacterium-mediated transformation, microprojectile bombardment, microinjection, electroporation and in plαntα transformation. Cells carrying the introduced isolated and/or recombinant DNA can be used to regenerate transgenic plants which have altered phenotypes, therefore becoming sources of additional plants either through seed production or non-seed asexual reproductive means. The methods of this invention can be used to provide plants, seeds, plant tissue culture, plant parts, cells, and protoplasts containing one or more nucleic acids which comprise a modified or isolated introduced gene encoding a non-plant or unregulated PFP or its functional equivalent which alters glycolysis and the resulting products, such as starch, sugars, fiber and lipids. Plants parts can include roots, leaves, stems, flowers, fruits, meristems, epicotyls, hypocotyls, cotyledons, pollen and embryos.

The present invention also relates to transgenic plants, or cells or tissues derived from such plants, in which glycolysis is altered directly or product synthesis is altered indirectly through application of the methods of this invention. The term "transgenic plants" includes plants or photosynthetic protists which contain introduced DNA which, if transcribed and translated, changes the amount or type of one or more plant products compared to a wildtype (naturally-occurring) plant of the same species or variety grown under the same conditions. Transgenic plants include those into which isolated and/or recombinant nucleic acids have been stably inserted and their descendants, produced from seed, vegetative propagation, cell, tissue or protoplast culture, or the like wherein such alteration is maintained or PFP expression is detectable. The introduced DNA which is originally inserted into the plants or plant cells or protoplasts can include additional copies of other genes found in the naturally-occurring organism.

Those of skill in the art will recognize the methods of this invention for enhancing synthesis of naturally-occurring plant products such as proteins, oils, fiber and/or carbohydrates, and combinations thereof in cells has the possibility of an almost infinite number of applications. Besides enhancing accumulation of naturally-occurring products, the transgenic plants can also contain introduced genes which encode useful products whose accumulation or harvest is facilitated by manipulation of sugar levels. In particular, an increase in oils, reduction of fiber or reducing sugars, and the like.

Enhanced product accumulation as described in the present invention can be used to increase the solubilization and accumulation of such plant- derived products in the edible portions of plants or in the portions of plants intended to be harvested for extraction of these compounds, or even to change the size of plant parts. Transport and products can be altered, for example, in any plant organ; e.g., stems, roots, leaves, flowers and fruits.

Those of skill in the art can understand that the variety of transgenic products which can be produced or whose production can be altered in both prokaryotic and eukaryotic photosynthetic organisms by the methods described herein is broad and encompasses many important naturally- occurring and foreign substances which are regulatory or are products themselves. These include, for example, storage products such as sugars, starches, pigments, lignin, lipids, glycolipids, phospholipids, proteins and the like. If naturally-occurring in the photosynthetic organism, the product may be produced at higher levels, compartmentalized in a different part of the cell, such as the plastid, mitochondrion, or vacuole, or even in a different organ, such as the flower, seed, root or leaf. Thus, lipids can be synthesized and/or accumulated at higher levels to enhance the oil content of the plant or plant part normally producing the lipid by methods provided herein. Further, through the same methods, other product levels can be altered in a plant or a plant organ by incorporating the gene encoding the PFP and other genes encoding further enzymes in the constructs described herein, so that the glycolytic pathway and subsequent metabolic processes are directed to enhancement of a particular product or result in the reduction of a naturally- occurring product in the transformed organism. Alternatively, this invention provides methods for varying the phenotype of seeds and other storage organs of plants. These novel products or combination of products can be provided by enhancing and/or reducing the accumulation of molecules to be stored or by modifying the glycolytic pathway to alter the accumulation of particular products. Thus, in addition to increasing the overall amount of stored substances, thus increasing the nutritive value of the seed, alterations can include modifying the fatty acid and fiber composition in seeds by changing the ratio and/or amounts of the various fatty acids as they are produced. Alternatively, improvements in the amino acid composition of storage proteins can be generated. Of particular interest as target substances are the storage proteins of seeds, such as napin, cruciferin, β-conglycinin, phaseolin, brazil nut protein, other 2S or 7S proteins, or the like, as well as proteins involved in fatty acid biosynthesis, such as acyl carrier protein.

To produce transgenic plants of this invention, a construct comprising the gene encoding an unregulated or non-plant PFP, such as Giardia PFP, or nucleic acid encoding its functional equivalent and a promoter are incorporated into a vector as described in Example 1 or through other methods known and used by those of skill in the art. The construct can also include any other necessary regulators such as terminators or the like, operably linked to the coding sequence. It can also be beneficial to include a 5' leader sequence, such as the untranslated leader from the coat protein mRNA of alfalfa mosaic virus (Jobling, S.A. and Gehrke, L. (1987) Nature 325:622- 625) or the maize chlorotic mottle virus (MCMV) leader (Lommel, S.A. et al. (1991) Virology 57:382-385). Those of skill in the art will recognize the applicability of other leader sequences for various purposes.

Targeting sequences are also useful and can be incorporated into the constructs of this invention. A targeting sequence is used to direct peptides from the cytosol where translation occurs to a different cellular compartment (e.g., organelles, nucleus, vacuole, plasma membrane). Examples of targeting sequences useful in this invention include, but are not limited to, the yeast mitochondrial presequence (Schmitz et al. (1989) Plant Cell 7:783-791), the targeting sequence from the pathogenesis-related gene (PR-1) of tobacco (Cornellisen et al. (1986) EMBO J. 5:37-40), vacuole targeting signals (Chrispeels, M.J. and Raikhel, N.V. (1992) Cell 65:613-616), secretory pathway sequences such as those of the ER or Golgi (Chrispeels, M.J. (1991) Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:21-53). Intraorganellar sequences may also be useful for internal sites, e.g., thylakoids in chlorop lasts. Theg, S.M. and Scott, S.V. (1993) Trends in Cell Biol. 3:186-190.

In addition to 5' leader sequences, terminator sequences are usually incorporated into the construct. In plant constructs, a 3' untranslated region (3' UTR) is generally part of the expression plasmid and contains a polyA termination sequence. The termination region which is employed will generally be one of convenience, since termination regions appear to be relatively interchangeable. The octopine synthase and nopaline synthase termination regions, derived from the Ti-plasmid of A. tumefaciens, are suitable for such use in the constructs of this invention. Any suitable technique can be used to introduce the nucleic acids and constructs of this invention to produce transgenic plants with an altered genome. For grasses such as maize, microprojectile bombardment (see for example, Sanford, J.C. et al., U.S. Patent No. 5,100,792 (1992)) can be used. In this embodiment, a nucleotide construct or a vector containing the construct is coated onto small particles which are then introduced into the targeted tissue (cells) via high velocity ballistic penetration. The vector can be any vector which permits the expression of the exogenous DNA in plant cells into which the vector is introduced. The transformed cells are then cultivated under conditions appropriate for the regeneration of plants, resulting in production of transgenic plants.

Transgenic plants carrying the construct are examined for the desired phenotype using a variety of methods including but not limited to an appropriate phenotypic marker, such as antibiotic resistance or herbicide resistance, or visual observation of the time of floral induction compared to naturally-occurring plants.

Other known methods of inserting nucleic acid constructs into plants 5 include Agrobacterium-mediated transformation (see for example Smith, R.H. et al., U.S. Patent No. 5,164,310 (1992)), electroporation (see for example, Calvin, N., U.S. Patent No. 5,098,843 (1992)), introduction using laser beams (see for example, Kasuya, T. et al., U.S. Patent No. 5,013,660 (1991)) or introduction using agents such as polyethylene glycol (see for example, Golds,

10 T. et al. (1993) Biotechnology, 11 :95-97), and the like. In general, plant cells may be transformed with a variety of vectors, such as viral, episomal vectors, Ti plasmid vectors and the like, in accordance with well known procedures. As indicated above, the method of introduction of the nucleic acid into the plant cell is not critical to this invention.

15 The methods of this invention can be used with in planta or seed transformation techniques which do not require culture or regeneration. Examples of these techniques are described in Bechtold, N. et al. (1993) CR Acad. Sci. Paris/Life Sciences 376:118-93; Chang, S.S. et al. (1990) Abstracts of the Fourth International Conference on Arabidopsis Research, Vienna, p.

20 28; Feldmann, K.A. and Marks, D.M (1987) Mol. Gen. Genet. 208:1-9; Ledoux, L. et al. (1985) Arabidopsis Inf. Serv. 22:1-11; Feldmann, K.A. (1992) In: Methods in Arabidopsis Research (Eds. Koncz, C, Chua, N-H, Schell, J.) pp. 274-289; Chee et al., U.S. patent, Serial No. 5,376,543.

The transcriptional initiation region may provide for constitutive

25 expression or regulated expression. Many promoters are available which are functional in plants. The term "promoter" refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of

30 transcription.

Constitutive promoters for plant gene expression include, but are not limited to, the octopine synthase, nopaline synthase, or mannopine synthase promoters from Agrobacterium, the cauliflower mosaic virus (35S) promoter, the figwort mosaic virus (FMV) promoter, and the tobacco mosaic virus (TMV) promoter. Tissue-specific gene expression in plants can also be provided by the seed-specific promoter napin (Baszczynski et al. (1990) PI Mol. Biol. 74:633-635), the glutamine synthase promoter (Edwards et al. (1990) PNAS 57:3459-3463), the maize sucrose synthetase 1 promoter (Yang et al. (1990) PNAS 57:4144-4148), the promoter from the Rol-C gene of the TLDNA of Ri plasmid (Sagaya et al. (1989) Plant Cell Physiol. 30:649-654), and the phloem-specific region of the pRVC-S-3A promoter (Aoyagi et al. (1988) Mol. Gen. Genet. 273:179-185).

Heat-shock promoters, the ribulose- 1,6-bisphosphate (RUBP) carboxylase small subunit (ssu) promoter, tissue specific promoters, and the like can be used for regulated expression of plant genes. Developmentally- regulated, stress-induced, wound-induced or pathogen-induced promoters are also useful.

The regulatory region may be responsive to a physical stimulus, such as light, as with the RUBP carboxylase ssu promoter, differentiation signals, or metabolites. The time and level of expression of the sense or antisense orientation can have a definite effect on the phenotype produced. Therefore, the promoters chosen, coupled with the orientation of the exogenous DNA, and site of integration of a vector in the genome, will determine the effect of the introduced gene.

Specific examples of regulated promoters also include, but are not limited to, the low temperature Kinl and cor6.6 promoters (Wang et al. (1995) Plant Mol. Biol. 25:605; Wang et al. (1995) Plant Mol. Biol 25:619-634), the ABA inducible promoter (Marcotte Jr. et al. (1989) Plant Cell 1:969-976), heat shock promoters, such as the inducible hsp70 heat shock promoter of Drosphilia melanogaster (Freeling, M. et al. (1985) Ann. Rev. of Genetics 19: 297-323), the cold inducible promoter from B. napus (White, T.C. et al. (1994) Plant Physiol. 106:9X7), the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R.T. et al, Miflin, B.J., Ed. Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3, p 384-438, Oxford University Press, Oxford 1986), the phloem-specific sucrose synthase ASUS1 promoter from Arabidopsis (Martin et al. (1993) Plant J. 4:367-377), the ACS1 promoter (Rodrigues-Pousada et al. (1993) Plant Cell 5:897-911), the 22 kDa zein protein promoter from maize (Unger et al. (1993) Plant Cell 5:831-841), 5 the psl lectin promoter of pea (de Pater et al. (1993) Plant Cell 5:877-886), the phas promoter from Phaseolus vulgaris (Frisch et al. (1995) Plant J. 7:503-512), the lea promoter (Thomas, T.L. (1993) Plant Cell 5:1401-1410), the E8 gene promoter from tomato (Cordes et al. (1989) Plant Cell 7:1025- 1034), the PCNA promoter (Kosugi et al. (1995) Plant J. 7:877-886), the

10 NTP303 promoter (Weterings et al (1995) Plant J. 8:55-63), the OSEM promoter (Hattori et al (1995) Plant J. 7:913-925), the ADP GP promoter from potato (Muller-Rober et al. (1994) Plant Cell 6:601-604), the Myb promoter from barley (Wissenbach et al. (1993) Plant J. 4:411-422), and the plastocyanin promoter from Arabidopsis (Vorst et al. (1993) Plant J. 4:933-

15 945).

Transgenic plants of this invention can contain isolated or recombinant nucleic acids which preferentially modify glycolytic pathways which are present in green tissues, or which are present in actively growing tissues or in storage tissues or organs such as seeds. In this manner, different products can

20 be accumulated, exported or imported to modify the capability of the plant to express and localize one or more products compared to the expression and accumulation of the same product(s) in a plant of the same variety without said introduced isolated or recombinant nucleic acids when grown under identical conditions.

25 Further, this invention includes a method of producing a transgenic plant containing, in addition to isolated nucleic acids which encode a non- plant or unregulated PFP or its functional equivalent so that glycolysis is altered, at least one nucleic acid which encodes a polypeptide for production of a useful foreign product. Coupled with the altered glycolysis in the cells of

30 the plant, it is possible to design a plant wherein, when all of the inserted nucleic acids are expressed, the result is the large scale and inexpensive production of valuable carbohydrates, lipids, or other products in a particular plant tissue or at a particular stage of development.

The methods described herein can be applied to all types of plants and other photosynthetic organisms, including: angiosperms (monocots and dicots), gymnosperms, spore-bearing or vegetatively-reproducing plants and the algae (including the blue-green algae). Further, the methods of this invention are suited to enhance translocation of substances in all prokaryotes. It is understood that prokaryotic organisms lack plastids and other organelles which compartmentalize products of photosynthesis and respiration, but the alteration of glycolysis can be accomplished which alters the production and accumulation of various products through expression of the PFP or its functional equivalent. Further, the methods described herein can be applied, without undue experimentation, to enhance transport of substances in nonphotosynthetic eukaryotes such as the fungi. Transgenic plants containing the constructs described herein can be regenerated from transformed or transfected cells, tissues or portions of plants by methods known to those of skill in the art. A portion of a plant is meant to include any part capable of producing a regenerated plant. Thus, this invention encompasses a cell or cells, tissue (especially meristematic and/or embryonic tissue), tissue cultures, protoplasts, epicotyls, hypocotyls, cotyledons, cotyledonary nodes, pollen, ovules, stems, roots, leaves, and the like. Plants may also be regenerated from explants. Methods will vary according to the plant species.

Seed can be obtained from the regenerated plant or from a cross between the regenerated plant and a suitable plant of the same species. Alternatively, the plant may be vegetatively propagated by culturing plant parts under conditions suitable for the regeneration of such plant parts. For example, plants can be regenerated from cultured pollen, protoplasts, meristems, hypotcotyls, epicotyls, stems, leaves, tubers, tissue cultures, and the like.

The present invention has numerous commercial applications in agriculture, horticulture and processing of plant products. The examples provided below are not intended to be limiting as skilled artisans can find numerous possibilities for an enzyme which can be used to modulation the process of glycolysis.

The ability to regulate the partitioning between of starch and sugars is of great importance in seeds and tubers. For example, the accumulation of hexose sugars in potato tubers during cold storage (cold sweetening) results darkening of the tissues when these tubers are cooked as chips or fries, making them less desirable to consumers. This accumulation results from slow reconversion of starch into sucrose and free hexoses. This process also occurs during the sprouting of tubers.

Increased expression of PFP in the tubers during cold storage can result in a decrease in sucrose synthesis by removal of hexoses through glycolysis and further metabolism. Thus, the use of non-plant or unregulated PFP provides a method of maintaining tuber quality and reducing cold sweetening during prolonged cold storage of potatoes and other tubers. Further, it can provide a method to inhibit the sprouting of tubers in storage without the use of chemical applications of inhibitors.

In another embodiment, the invention can be used to increase the overwintering capacity of plants such as alfalfa, turfgrasses, and some ornamentals. To overwinter successfully, such plants must have sufficient carbohydrate reserves stored in the root systems by autumn. Further manipulation of carbohydrate metabolism is also useful to decrease biomass in situations where desirable, e.g., to produce slow-growing turgrasses, "miniature" ornamentals, etc. Other embodiments of these novel constructs can be used to create and alter the pattern and rate of lignin deposition in the seed coat of plants such as canola and soybean. Elevation of Giardia PFP level in transgenic plants reduces lignin formation in seeds. Thus, extraction of digestible materials and desirable products of seeds becomes less time-consuming and expensive because the seeds are easier to crush. Increased oil to fiber ratio increases agricultural yields of desirable products per acre. The constructs encompassed by this invention provide a means for increasing oil synthesis in plants harvested for their oils, such as Brassica. For example, DNA can be constructed wherein the nucleic acid encoding a non-plant or unregulated PFP is attached to the napin promoter of Brassica so that oil synthesis in Brassica or Canola seeds is increased. Thus, this invention includes methods of increasing oil yields of crop plants and in other photosynthetic organisms.

The manipulation of glycolysis using the constructs of this invention also provides transgenic plants and seeds wherein the storage oil and protein levels can be altered for specific purposes. For example, crop plants such as canola can be produced with high oil to protein ratios, providing higher oil yields per acre. Soybeans can be altered to yield high protein or high oil content, depending on the use intended for the crop. Elevation of PFP levels can result in high oil levels. In other embodiments, the elimination of endogenous PFP activity in transgenic plants can be accomplished by inactivating the endogenous PFP gene. Until now, the actual role of PFP in photosynthetic organisms was not known.

Elimination of endogenous PFP activity in transgenic plants will result in an increase in soluble sugar biosynthesis. There are many applications in which farmers and horticulturalists would benefit from having improved cold and drought tolerant plants due to the increased sugar levels in the above- ground parts. For example, cold and drought tolerant canola means that canola could be planted earlier in the Spring in northern climates and would resist summer droughts. Further, the drought tolerant plants could be grown in more arid areas. Increased sugar production in beet and sugar cane would improve yields of these crops. The same principles can be applied to manipulate sweetness in fruit, e.g., grapes, cherries, apples, tomatoes, nectarines, melons, and the like. Thus, increased sweetness in many fruit and vegetable crops can also be achieved. EXAMPLES

Example 1 . Construction of vectors

In order to examine the effects on plants of an uncontrollable PFP gene, the gene for the PFP enzyme from Giardia lamblia was selected for transformation because unlike plant PFP, it is not dependent on the presence of fructose-2,6-bisphosphate for activation. The gene for Giardia PFP was obtained from Dr. Miklos Muller at Rockefeller University, NY 10021, USA. The sequence of this gene has been published in Rozario, C, Smith, M.W., Muller, M. Biochimica et Biophysica Ada (1995) 260:218-222 (Figure 4; nucleic acid sequence (SEQ ID NO:l) and amino acid sequence (SEQ ID NO:2)). This gene contained no introns and could therefore be used directly for vector construction. PCR primers were designed which contained restriction sites to facilitate the construction of vectors for 1) overexpression of Giardia PFP in E.coli and to check for enzyme activity, and 2) transformation of plants. The primers were as follows:

Original sequence :

5 ' -TGGCTCAATTTGAAATGTCTGCTT - - >

New primer (SEQ ID NO: 3) :

5 ' -TGGCTGAATTCGACATGTCTGCTT - - >

EcoRI Afllll Xha J

<-- 5 ' -ACAATCTTCTAGAATTCTAAATTT-3' Original sequence <-- 3' -TGTTAGGAGATCTTAAGATTTAAA-5' New primer (SEQ ID NO: 4)

The ATG (start) codon is in bold. The two new primers (SEQ ID NO:3 and SEQ ID NO:4) were used to generate a PCR fragment of 1749 bp containing the entire coding region for Giardia PFP. The EcoRI sites were used to clone the fragment into the bacterial expression vector pGΕX-4T-l (Pharmacia Biotech, Uppsala, Sweden). Several plasmids containing inserts of the predicted size were obtained. One of these, designated 95.624, was used to overexpress the insert following the manufacturers instructions provided with the pGEX-4T-l vector. A fusion polypeptide of the correct size was obtained and activity of the overexpressed protein was confirmed by assaying for PFP activity. E.coli does not have this enzyme and any activity can, therefore, be attributed to expression of the Giardia gene. The second vector, that was used for plant transformation, was then constructed. Plasmid 95.624 was digested with ^ HII and Xbal releasing the Giardia PFP fragment with the ATG start codon. This was ligated to pBI525 digested with Ncol and Xbal. AfRll and Ncol are compatible restriction sites although neither site is reconstituted upon ligation and the resulting plasmid, designated 95.820 (Figure 2), contained the tandem 35S-35S promoter and alfalfa mosaic virus transcriptional enhancer (from pBI525) linked to the PFP sequence and followed by the nos terminator, again derived from pBI525.

Plasmid p95.820 was first sequenced to determine that the ATG start codon was in frame, and the plasmid was then digested with EcoRI and Hz^'ttdlll to release two fragments, one an EcoRI-H dIII fragment of about 0.95 kb and the other a H dIII fragment of about 1.7 kb. These were eluted from a 1.0% low melting agarose gel and purified using standard techniques.

These two fragments were then ligated sequentially into the plant transformation vector pRD400 (Datla et al. (1992) Gene 277:383-384). First, pRD400 was digested with EcoRI and H dIII and ligated to the 0.95 kb fragment from p95.820. The resulting plasmid, designated 95.1086, was digested with Hz^'wdlll and ligated to the eluted 1.7 kb H dIII fragment. The orientation of the H dIII fragment was then determined by restriction digestion and sequenced to confirm that the internal Htwdlll fragment within the Giardia PFP coding sequence remained in frame. This final plasmid used for plant transformation was designated 95.1160 (Figure 3).

Example 2. Transformation procedure

Agrobacterium-mediated transformation ofNicotiana tabacum cv. 'Petit Havana SRI' was achieved by the leaf disc method (Horsch et al. (1985) Science 227:1229-1331) and regenerated as described in Gottlob-McHugh et al. ((1992) Plant Physiology 100:820-825). Homozygous lines were selected by Southern blot analysis and the activity of Giardia PFP determined by conducting enzyme assays on various tissue extracts in the absence of fructose-2,6-bisphosphate. The latter is obligately required for activity of the native plant enzyme and, therefore, any activity in the absence of this enzyme can be attributed to the transgene. Western blot analysis was also used to confirm the presence of Giardia PFP polypeptides in transgenic plants using a polyclonal an -Giardia PFP serum raised in a mouse against overexpressed PFP using p95.820 described above. Control plants (nulls) for each selected transgenic line were generated by self-pollination and selection of plants in which the transgene had been lost. Brassica sp. was transformed by the in planta procedure described in patent application PCT/CA98/00859. Alfalfa was transformed by the procedure of McKersie et al. ((1999) Plant Phys. 77P:839-847).

Over 300 transgenic tobacco plants have been produced which incorporate the Giardia gene encoding PFP. Replicates of four different T3 homozygous lines (PFP-1, PFP-16, PFP-20, and PFP-23) were compared to their homozygous nulls segregants or the pRD400 transgenic control (pRD400, wild-type plants).

Example 3. Sampling procedures Plants or plant part samples were immediately frozen in liquid nitrogen after harvest, and stored at -80°C until analysis. Fresh weights were taken on frozen tissues. Before assay, tissues were ground to a fine powder in liquid nitrogen. All biochemicals and enzymes were supplied by Sigma (St. Louis, Missouri, USA) or Boehringer Mannheim (Indianapolis, Indiana, USA).

Example 4. Carhohydrate Extraction

For carbohydrate extraction from plant tissue or seeds, frozen ground powder was extracted in boiling 80% (v/v) ethanol for 10 minutes. After a 5 minute centrifugation at 12,000xg, the supernatant was transferred to a fresh tube and the pellet was re-extracted with 50% (v/v) ethanol. After centrifugation, the two supernatants were combined. This combined supernatant was evaporated using a vacuum desiccator and the residue was resuspended in distilled water before adding O.lx volume of chloroform. Phase separation was hastened by 5 minutes of centrifugation at 12,000xg. The aqueous phase was transferred to a fresh tube and stored at -20°C until assayed.

The aqueous-ethanol-insoluble residue was resuspended in 0.2 M KOH and boiled for 30 minutes to gelatinize the starch. The samples were then cooled and neutralized with 1M acetic acid. Starch was hydro lyzed overnight at 50°C with 10 U/ml α-amylase (EC 3.2.1.1) and 6 U/ml amyloglucosidase (EC 3.2.1.3) in 50 mM sodium citrate-acetic acid (pH 4.6).

Example 5. Assay for Sugars

Glucose, fructose and sucrose were measured spectrophotometrically at 37°C in a 200 μL assay mixture containing 100 mM Hepes-KOH, 3 mM MgCl₂, pH 7.4, 1.1 mM ATP, 0.5 mM NADP, and 0.4 U glucose-6-P dehydrogenase (EC 1.1.1.49) by the successive addition of either 0.4 U hexokinase (EC 2.7.1.1), 0.75 U phosphoglucoisomerase (EC 5.3.1.9), or 10.0 U invertase (EC 3.2.1.26), respectively. Hydrolyzed starch samples were assayed in the above mixture by the addition of 0.4 U hexokinase.

Example 6. Enzyme Assay After grinding tissue or seed samples to a frozen powder, extraction buffer was added which contained 50 mM Hepes-KOH, pH 7.5, 5 mM MgCl₂, ImM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 1 mM benzamidine, 5 mM €-aminocaproic acid, 2 μM leupeptin, 1 mM PMSF, 2% (w/v) insoluble PVP, and 20% (v/v) glycerol. For seed samples, 10 mM thiourea was also included. Tissues were further homogenized in the extraction buffer until thawed. Samples were centrifuged for 2 minutes at 12,000xg and the supernatant was assayed for enzyme activity.

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) activity was measured in a continuous spectrophotometic assay using a SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, CA). The assay mixture contained 50 mM Hepes-KOH, pH 7.5, 5 mM MgCl₂, 10 mM fructose-6-P, 0.3 mM NADH, 0.3 U aldolase (EC 4.1.2.13), 0.3 U glycerol-3- P dehydrogenase (EC 1.2.1.12), 0.87 U triose-P isomerase (EC 5.3.1.1) and 5 μl extract in a 250 μl total volume. Fructose-6-P was pre-treated at pH 2 for 1 hour to destroy any contaminating fructose-2,6-bisphosphate, and was then neutralized with potassium hydroxide. After establishment of a background rate (A₃₄₀-A₄₀₅), 2.5 mM Na₄-pyrophosphate was added to measure the activity of the introduced Giardia PFP enzyme. Tobacco PFP activity was calculated from the difference between the Giardia PFP rate and the rate after the subsequent addition of 5 μM fructose-2,6-bisphosphate.

ATP-dependent phosphofructokinase (PFK) activity was measured in a similar way to PFP using a continuous spectrophotometic assay. The assay mixture contained 50 mM Bicine, pH 8.0, 5 mM MgCl₂, 10 mM fructose-6-P, 0.2 mM NADH, 0.3 U aldolase (EC 4.1.2.13), 0.3 U glycerol-3-P dehydrogenase (EC 1.2.1.12), 0.87 U triose-P isomerase (EC 5.3.1.1) and 10 μl extract in a 250 μl total volume. After establishment of a background rate (A₃₄₀-A₄₀₅), 0.25 mM ATP was added to start the reaction.

The assay mixture for fructose- 1,6-bisphosphatase (FBPase) contained 50 mM PIPES, pH 7.0, 5 mM MgCl₂, 0.2 mM NADP, 1.0 U phosphoglucoisomerase (EC 5.3.1.9), 0.5 U glucose-6-P dehydrogenase (EC 1.1.1.49) and 10 μl extract in a 250 μl total volume. After establishment of a background rate (A₃₄₀-A₄₀₅), 50 μM fructose- 1,6-biphosphate was added to start the reaction.

The assay mixture for pyruvate kinase contained 50 mM Bicine, pH 8.0, 10 mM MgCl₂, 20 mM KCl, 2 mM PEP, 2 mM DTT, 0.2 mM NADH, 0.5 U lactate dehydrogenase and 10 μl extract in a 250 μl total volume. After establishment of a background rate (A₃₄₀-A₄₀₅), 2.0 mM ADP was added to start the reaction.

The assay mixture for PEP carboxylase (EC 4.1.1.31) contained 50 mM Bicine, pH 8.0, 5 mM MgCl₂, 5 mM glucose-6-P, 10 mM NaHCO₃, 2 mM DTT, 0.2 mM NADH, 0.5 U malate dehydrogenase and 10 μl extract in a 250 μl total volume. After extablishment of a background rate (A₃₄₀-A₄₀₅), 2.5 mM PEP was added to start the reaction.

Expression of PFP in mature, expanded and non-senescing leaves of transgenic tobacco plants is shown in Figure 5. Figure 5 is a bar graph showing units of activity (y-axis) of Giardia PFP, plant PFP, PFK and FBPase for the transgenic lines PFP-1, PFP- 16, PFP-20, PFP-23, and null segregant pRD400. Giardia PFP was highly expressed in transgenic plants and its activity was over 50-fold higher than PFP activity in nulls. In comparison, the endogenous PFP activity was also slightly higher in the transgenic plants. PFK activity did not change.

Example 7. Production of mouse-anti- rør<irø-PFP antiserum

Antibodies were raised in CD1 female mice (Charles River Inc., St. Constant, Quebec, Canada) weighing approximately 25 grams each. Pre- imrnune serum was collected from the orbital sinus prior to the first injection. Each injection consisted of 2 μg purified Giardia PFP protein, diluted to 100 μl with sterile PBS (0.15 M NaCl, 0.01M NaPO₄ buffer pH7.4), and mixed with an equal volume (100 μl) of Ribi adjuvant. Adjuvant was obtained from RIBI ImmunoChem Research Inc., (Hamilton, Montana, USA), product R-700 MPL+TDMEMulsion, and reconstituted in 1 ml sterile PBS. Mice were injected subcutaneously on days 0, 21 and 27. On day 34, blood was collected by cardiac puncture (approximately 1 ml). The collected blood was chilled overnight at 4°C, spun at 5000 φm in a bench top microcentrifuge for 5 minutes and the serum recovered (approximately 400 μl). Serum was stored in small aliquots at -20 °C.

Example 8. Photosynthesis and respiration of leaves

Rates of photosynthesis were measured on the youngest fully developed leaves of tobacco plants during vegetative stage of growth (before flowering, 6 weeks from planting) using an open flow gas exchange system. The methods used are described in Long and Hallgreen ("Measurements of CO₂ assimilation by plants in the fields and the laboratory", in: Hall DO, Scurlock JMO, Bolhar-Nordenkampf HR, Leegood RC, Long SP (eds) Photosynthesis and production in a changing environment, Chapman and Hall, London, 1993).

Rates of photosynthesis were measured at light intensities of 300 μmol/meter /second (average light intensity during day growth period) and at 1200 μmol/meter /second (saturating light intensity) by measuπng the rate of CO₂ uptake using an infra red gas analyzer (model S151, Qubit Systems Inc., Kingston, Ontario, Canada). The leaf was placed in a flow through leaf chamber (model Gl 12, Qubit Systems Inc.). The light was provided by a cold, red LED light source (model Al 13, Qubit Systems Inc., Kingston, Ontario, Canada) fitted on top of the chamber. Temperature of the leaf was monitored using a thermister (model SI 71, Qubit Systems Inc.) placed in the bottom of the leaf chamber.

Rates of leaf respiration in the dark were measured as CO₂ evolution rates using the set up described above for photosynthesis measurements except that the leaf cuvette was maintained in the dark by covering it with foil and a dark cloth.

As measured by gas exchange, the photosynthetic and respiration rates were unchanged in transgenic plants compared to nulls. Figure 8 compares pooled data for transgenic plant lines compared to null plant lines for ambient

(300 μmol/meter 2 /second) and high (1200 μmol/meter 2 /second) photosynthetic light conditions. Figure 8 also shows the pooled data for these lines under respiration conditions (0 μmol/meter /second). There are no significant differences in the photosynthetic rates of transgenic plants compared to controls, indicating that the effects of the increased PFP activity affect only the modification of the carbon products of photosynthesis and that there are no direct effects on photosynthesis itself. It is also apparent that mitochondrial activities are not greatly affected. Example 9. Growth Analysis

Tobacco plants were grown in the greenhouse conditions in silica sand and were watered with a hydroponic nutrient solution (Plant Products Co., Brampton, Ontario, Canada). Plants were harvested at weekly intervals over a period of 5 weeks. Growth analysis was performed by measuring accumulation of dry weight of roots and shoots at different stages of plant development. Relative growth rates were calculated as described by Beadle ("The Growth Analysis", in: Hall DO, Scurlock JMO, Bolhar-Nordenkampf HR, Leegood RC, Long SP (eds) Photosynthesis and production in a changing environment, Chapman and Hall, London, 1993). From root and shoot dry weight, total dry weight and shoot to root ratios were calculated.

Example 10. Seedling vigor

Four seeds of each line were placed in a row on a plexiglass slant covered with wet filter paper (5 slants were used, placed on an angle in a tray containing water and were covered with plastic wrap). Seeds were allowed to germinate and the length of the seedling was measured daily for a period of 2 weeks. Relative growth rates of the seedlings were determined from the total length of the seedling. These methods are described in Pollock BM, and Roos EE ("Seed and seedling vigor," in Seed Biology, Kozlowski TT (ed), Academic Press, New York, 1972); and Kuang A, Crispi M, Musgrave ME (1998, "Control of seed development in Arabidopsis thaliana by atmospheric oxygen," Plant Cell Envir. 21: 71-78).

Example 1 1. Seed Composition

Frozen ground powder from seed samples was extracted three times with a monophasic solution of chloroform: methanol: water (1 :2:0.8) as per Bligh and Dyer (1959, Can. J. Biochem. Physiol. 37:911-917). After a 5 minute centrifugation at 12,000 xg, the solvent was transferred to a fresh tube and the remaining insoluble residue containing fiber, protein and starch was dried to constant weight and weighed. Chloroform was added to the solvent to produce a biphasic solution. The aqueous-methanol phase, containing soluble carbohydrates and amino acids, and the chloroform phase, containing lipids, were separately transferred to fresh tubes. Each tube was evaporated to dryness using a vacuum desiccator and were weighed. All component weights are expressed as a percentage of the fresh weight of the original seed sample.

All patents, patent applications, and references cited above are hereby incoφorated by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A method for modifying the level of one or more metabolic products of a plant, plant part or photosynthetic cell, wherein said method comprises: a. transforming a plant, plant part or photosynthetic cell with a recombinant DNA construct containing an unregulated PFP; b. optionally regenerating the plant part or photosynthetic cell to generate a whole plant; and c. subjecting the transgenic plant, plant part, or photosynthetic cell to conditions wherein the unregulated PFP is expressed; wherein the level of the one or more metabolic products of the plant, plant part or photosynthetic cell are modified relative to an untransformed plant, plant part or photosynthetic cell.

2. The method of Claim 1 , wherein the one or more metabolic products are selected from the group consisting of carbohydrate, fiber, oil and protein.

3. The method of Claim 1 , wherein the expression construct comprises a regulatory sequence operably linked to a nucleotide sequence encoding the unregulated PFP, which regulatory sequence directs expression of the nucleotides sequence at a selected stage of development or maturity of the transgenic plant or plant organ.

4. The method of Claim 3, wherein the stage of development is seed or tuber development.

5. The method of Claim 1, wherein the expression construct contains a regulatory sequence operably linked to a nucleotide sequence encoding the unregulated PFP, which regulatory sequence directs tissue-specific expression of the nucleotide sequence in the transgenic plant, plant part or photosynthetic cell.

6. The method of Claim 1 , wherein the transgenic plant, plant part or photosynthetic cell contains at least one expression cassette which contains a nucleotide sequence encoding a second enzyme.

7. The method of Claim 1 , wherein the transgenic plant, plant part or photosynthetic cell contains at least one expression cassette which contains a nucleotide sequence encoding a metabolic product, wherein the metabolic product is selected from the group consisting of carbohydrate, fiber, oil and protein.

8. The method of Claim 1 , wherein the plant part or photosynthetic cell is selected from the group consisting of roots, leaves, stems, flowers, seeds, tubers, rhizomes, sports, embryos, hypocotyls, cotyledons, explants, tissues, cells and protoplasts.

9. The method of Claim 1 , wherein the unregulated PFP is a protist PFP.

10. The method of Claim 9, wherein the unregulated PFP is a Giardia sp. PFP.

11. A stably transformed transgenic plant, plant part, or photosynthetic cell containing modified levels of one or more metabolic products, the plant, plant part, or photosynthetic cell made by the following method: a. transforming a plant, plant part or photosynthetic cell with a recombinant DNA construct containing an unregulated PFP; b. optionally regenerating the plant part or photosynthetic cell to generate a whole plant; and c. subjecting the transgenic plant, plant part, or photosynthetic cell to conditions wherein the unregulated PFP is expressed; thereby producing a stably transformed transgenic plant, plant part, or photosynthetic cell containing modified levels of one or more metabolic products, relative to an untransformed plant, plant part, or photosynthetic cell.

12. A tissue culture of the plant, plant part or photosynthetic cell of Claim 11.

13. The transformed transgenic plant, plant part or photosynthetic cell of Claim 11 , wherein the one or more metabolic products are selected from the group consisting of carbohydrate, fiber, oil and protein.

14. The transformed transgenic plant, plant part or photosynthetic cell of Claim 11, wherein the transgenic plant, plant part or photosynthetic cell contains at least one expression cassette which contains a nucleotide sequence encoding a second enzyme.

15. The transformed transgenic plant, plant part or photosynthetic cell of Claim 11 , wherein the transgenic plant, plant part or photosynthetic cell contains at least one expression cassette which contains a nucleotide sequence encoding a metabolic product, wherein the metabolic product is selected form the group consisting of carbohydrate, fiber, oil and protein.

16. The transformed transgenic plant, plant part or photosynthetic cell of Claim 11 , wherein the plant, plant part or photosynthetic cell is selected from the group consisting of roots, leaves, stems, flowers, seeds, tubers, rhizomes, sports, embryos, hypocotyls, cotyledons, explants, tissues, cells and protoplasts.

17 The transformed transgenic plant, plant part or photosynthetic cell of Claim 11, wherein the unregulated PFP is a protist PFP.

18. The transformed transgenic plant, plant part or photosynthetic cell of Claim 17, wherein the unregulated PFP is a Giardia sp. PFP.

19. The transformed transgenic plant, plant part or photosynthetic cell of Claim 11, wherein the plant is selected from the group consisting of Brassica sp., alfalfa, corn, sorghum, soybean, sunflower, wheat, rice, rye, cotton, canola, barley and potato.

20. A method for modifying the carbohydrate level of a plant, plant part or photosynthetic cell, wherein said method comprises: a. transforming a plant, plant part or photosynthetic cell with a recombinant DNA construct containing a Giardia sp. PFP; b. optionally regenerating the plant part or photosynthetic cell to generate a whole plant; and c. subjecting the transgenic plant, plant part, or photosynthetic cell to conditions wherein the Giardia sp. PFP is expressed; thereby modifying the carbohydrate levels of the plant, plant part or photosynthetic cell.

21. The method of Claim 20, wherein the DNA construct comprises a regulatory sequence operably linked to a nucleotide sequence encoding the Giardia sp. PFP, which regulatory sequence directs expression of said nucleotide sequence at a selected stage of development of the transgenic plant, plant part or photosynthetic cell.

22. The method of Claim 21, wherein the stage of development is seed or tuber development.

23. The stably transformed transgenic plant, plant part or photosynthetic cell of Claim 20 wherein the DNA construct contains a regulatory sequence operably linked to a nucleotide sequence encoding the Giardia sp. PFP, which regulatory sequence directs tissue-specific

5 expression of the nucleotide sequence in the transgenic plant, plant part or photosynthetic cell.

24. The method of Claim 23, wherein the tissue-specific expression occurs in a tissue selected from the group comprising tuber tissue, leaf tissue, or seed tissue.

10 25. The method of Claim 23, wherein the transgenic plant, plant part or photosynthetic cell contains at least one expression cassette which contains a nucleotide sequence encoding a metabolic product, wherein the metabolic product is selected from the group consisting of carbohydrate, fiber, oil and protein.

15 26. The method of Claim 23, wherein the plant part or photosynthetic cell is selected from the group consisting of roots, leaves, stems, flowers, seeds, tubers, rhizomes, sports, embryos, hypocotyls, cotyledons, explants, tissues, cells and protoplasts.

27. A stably transformed transgenic plant, plant part or photosynthetic cell 20 containing modified carbohydrate levels, the plant, plant part or photosynthetic cell made by the following method: a. transforming a plant, plant part or photosynthetic cell with a recombinant DNA construct containing a Giardia sp. PFP; b. optionally regenerating the plant part or photosynthetic cell to 25 generate a whole plant; and c. subjecting the transgenic plant, plant part, or photosynthetic cell to conditions wherein the Giardia sp. PFP is expressed; thereby producing a stably transformed transgenic plant, plant part or photosynthetic cell containing modified carbohydrate levels.

28. A tissue culture of the plant, plant part or photosynthetic cell of Claim 27.

29. The transformed transgenic plant, plant part or photosynthetic cell of Claim 27, wherein the one or more metabolic products are selected from the group consisting of carbohydrate, fiber, oil and protein.

30. The transformed transgenic plant, plant part or photosynthetic cell of Claim 27, wherein the transgenic plant, plant part or photosynthetic cell contains at least one expression cassette which contains a nucleotide sequence encoding a second enzyme.

31. The transformed transgenic plant, plant part or photosynthetic cell of Claim 27, wherein the transgenic plant, plant part or photosynthetic cell contains at least one expression cassette which contains a nucleotides sequence encoding a metabolic product, wherein the metabolic product is selected form the group consisting of carbohydrate, fiber, oil and protein.

32. The transformed transgenic plant part or photosynthetic cell of Claim 27, wherein the plant part or photosynthetic cell is selected from the group consisting of roots, leaves, stems, flowers, seeds, tubers, rhizomes, sports, embryos, hypocotyls, cotyledons, explants, tissues, cells and protoplasts.

33. The transformed transgenic plant, plant part or photosynthetic cell of Claim 27, wherein the plant is selected from the group consisting of Brassica sp., alfalfa, corn, sorghum, soybean, sunflower, wheat, rice, rye, cotton, canola, barley and potato.