According to the invention, there is provided null-LOX-1 barley and plant products produced thereof, such as malt manufactured by using barley kernels defective in synthesis of the fatty acid-converting enzyme lipoxygenase-1. Said enzyme accounts for the principal activity related to conversion of linoleic acid into 9-hydroperoxy octadecadienoic acid, a lipoxygenase pathway metabolite, which--through further enzymatic or spontaneous reactions--may lead to the appearance of trans-2-nonenal. The invention enables brewers to produce a beer devoid of detectable trans-2-nonenal-specific off-flavors, even after prolonged storage of the beverage.

According to the invention, there is provided null-LOX-1 barley and plant products produced thereof, such as malt manufactured by using barley kernels defective in synthesis of the fatty acid-converting enzyme lipoxygenase-1. Said enzyme accounts for the principal activity related to conversion of linoleic acid into 9-hydroperoxy octadecadienoic acid, a lipoxygenase pathway metabolite, which--through further enzymatic or spontaneous reactions--may lead to the appearance of trans-2-nonenal. The invention enables brewers to produce a beer devoid of detectable trans-2-nonenal-specific off-flavors, even after prolonged storage of the beverage.

Current U.S. Class:	800/320 ; 426/11; 426/64; 435/184; 435/185; 800/278; 800/298
Current International Class:	A01H 5/00 (20060101); C12C 1/00 (20060101); C12C 11/00 (20060101); C12N 15/82 (20060101)
Field of Search:	800/320

Primary Examiner: Ibrahim; Medina A
Attorney, Agent or Firm: Merchant & Gould P.C.

What is claimed is:

1. A barley plant or a portion of said plant, comprising mutated LOX-1 protein lacking all or at least a portion of amino acids 520 to 862 of wild type barley LOX-1 (SEQ ID NO: 3 or 7), wherein said plant exhibits null LOX-1 activity.

2. A barley plant or portion of said plant comprising mutated LOX-1 protein truncated at or between amino acids 378 and 665 of wild-type barley LOX-1 (SEQ ID NO: 3 or 7), wherein said plant exhibits null LOX-1 activity.

3. The barley plant or portion according to claim 2, wherein said mutated LOX-1 protein consists essentially of amino acids 1-378 of wild-type barley LOX-1 (SEQ ID NO: 3 or 7).

4. The barley plant or portion according to claim 3, wherein said mutated LOX-1 protein has the amino acid sequence of SEQ ID NO: 4 or 8.

5. The barley plant or portion according to claim 2, wherein said mutated LOX-1 protein is encoded by a gene that comprises a premature stop codon at or between nucleotides 2311 and 3574 of wild-type genomic barley lox-1 (SEQ ID NO: 1 or 5).

6. The barley plant or portion according to claim 5, wherein said mutated LOX-1 protein is encoded by a gene that comprises a stop codon at nucleotides 3572-3574 of wild-type genomic barley lox-1 (SEQ ID NO: 1 or 5).

7. The barley plant or portion according to claim 5, wherein said mutated LOX-1 protein is encoded by the nucleic acid sequence of SEQ ID NO: 2 or 6.

8. The barley plant or portion according to claim 1, wherein said plant has ATCC accession No. PTA-5847 or PTA-5584.

9. The barley plant or portion according to claim 1, wherein said portion comprises a kernel.

10. The barley plant or portion according to claim 1, wherein said portion comprises an embryo.

11. The barley plant or portion according to claim 1, wherein said LOX-1 activity is determined in homogenized embryo tissue of M3 or M4 plant kernels.

12. A malt composition comprising malted kernels of the plant of claim 1.

13. A malt composition comprising malted kernels of the plant of claim 6.

1. FIELD OF THE INVENTION

The present invention relates to plant biotechnology, disclosing barley and malt defective in synthesis of the lipoxygenase (LOX) enzyme LOX-1, thus providing a new raw material for industrial usage. For example, said raw material can be used for manufacturing a new and distinctive flavor-stable beer having no or negligible quantities of the off-flavor compound trans-2-nonenal (T2N). Said T2N is formed by the sequential action of LOX pathway enzymes, where the LOX-1 represents the primary activity, conferring dioxygenation of linoleic acid to yield 9-hydroperoxy octadecadienoic acid (9-HPODE). Barley and plant products of the invention exhibit no or only negligible quantities of 9-HPODE. In addition, the invention relates to beverages produced using said barley and/or malt.

2. BACKGROUND OF THE INVENTION

One of the research goals related to modern beer production is determining the molecular factors for beer quality and stability. A large fraction of beer is produced on the basis of barley (Hordeum vulgare, L.). It is a monocotyledonous crop plant grown in many parts of the world, not only due to its economic importance as a source of industrial products, such as beer, but also as a source of animal feed. The United States is now one of the leading producers of malting barley, with around 13% of the world crop; Canada, Australia and Europe together account for about 70% of the production (Bios Intern., 2001).

A continuing effort of barley breeders is to develop stable, high-yielding cultivars that are agronomically sound. To accomplish this goal, attempts have included random mutagenesis by chemical treatment or irradiation to modify traits of interest, for example to alter the expression of specific genes that may have deleterious effects on plant growth and crop productivity in general--but also on traits conferring added quality to a product manufactured from the crop. It is well established that sodium azide, NaN.sub.3, is a useful chemical to mutagenize barley. Specifically, NaN.sub.3-derived mutagenesis has been used to induce genetic changes in barley to generate mutants blocked in the synthesis of anthocyanins and proanthocyanidins (von Wettstein et al., 1977; von Wettstein et al., 1985; Jende-Strid, 1991; Jende-Strid, 1993; Olsen et al., 1993). A second example relates to barley kernels mutagenized with NaN.sub.3 to screen for high levels of free phosphate with the aim to identify low-phytate mutants (Rasmussen and Hatzak, 1998); a total of 10 mutants out of 2,000 screened kernels were identified. Although a major drawback in barley genetics has been the inability to specifically study gene function through reverse genetics, forward genetic screens--e.g. following NaN.sub.3-induced mutagenesis--continue regarding improvements that relate to nutritional and product quality parameters of barley and malt.

Except in a gross and general fashion, a breeder cannot predict the outcome of new plant lines under development in a conventional plant breeding process. This unpredictability is mainly caused by the lack of control at the cellular level, more specifically at the level of nuclear DNA--the complexity of which is enormous. A number of other factors influence the outcome of a plant breeding process, for example the climate and soil quality at the geographical location of plant propagation. As a result, different barley breeders that use conventional techniques will never develop plants with identical traits. In the conventional breeding process, a most difficult task is the identification of plants that are genetically superior, not only with respect to the trait of interest, but also with respect to physiological issues of relevance for plant growth. The selection process is particularly difficult when other confounding traits mask the trait of interest. When present-day plant breeding procedures include DNA sequence determination of the mutated gene, it is at a late stage of the breeding program--i.e. after mutant characterization, for example as recently described for screening of chemically induced mutations in Arabidopsis and other plants (Colbert et al., 2001).

Thus far, the creation of gene-indexed loss-of-function mutations on a whole-genome scale has been reported for the yeast Saccharomyces cerevisiae (Giaever et al., 2002). For the plant Arabidopsis, 21,700 of the .about.29,454 predicted genes have been inactivated by the insertion of Agrobacterium T-DNA sequences (Alonso et al., 2003).

Until now, it is not unusual that a conventional breeding process from the first mutagenesis or crossing to marketing of plants or seeds takes >10 years. Specifically, it would be excellent to provide the plant breeder with methods to detect mutations in the gene related to the trait of interest. Such improvements would enhance the level of predictability in breeding programs, especially when the selection of mutants is directed toward those having nonsense mutations in the protein-coding part of the gene of interest. In other cases, it may also be preferred with an early identification of DNA mutations, for example to cancel further breeding with lines characterized by promoter mutations in the gene of intererest or where other DNA mutations influence expression--simply because environmental or physiological factors could confer reversion of the trait induced by the mutagen. Accordingly, there is a demand for finding alternative ways of detecting mutations of interest early in the breeding program. This should make the entire breeding process faster and economically of higher interest, thus maximizing the amount of grain produced on the land.

A major proportion of the barley produced comprises malting varieties, the kernels of which are converted to malt through processes of controlled steeping, germination, and drying of the barley. A small proportion of the malt is used as ingredients in the food industry, whereas the majority of the malt is subsequently used as the main ingredient in the production of malt-derived beverages, including, but not limited to, beer and whisky. In the brewhouse, milled malt is subjected to a mashing process comprising a step-wise increase in temperature of a malt-water suspension which confers partial, enzymatic degradation and extraction of, for example, the kernel polymers starch and .beta.-glucan. Following filtration, the aqueous mash is boiled with hops to yield the wort. Said wort is subsequently fermented with yeast, giving the beer product which--upon maturation--is bottled. The wort can also be used for the production of non-fermented malt beverages.

Palatability and flavor stability of a beverage is an important factor of relevance to the composition of barley and malt. This is because natural flavor molecules derived from said barley and malt--or generated by the action of enzymes extracted from said barley and malt--may confer undesirable taste characteristics to the final product (Drost et al., 1990). In this respect, formation of the volatile compound giving a cardboard-like flavor appears to be of particular biochemical as well as economic interest. In 1970, the molecule responsible for cardboard-like flavor was isolated and identified as T2N, a nine-carbon (C.sub.9) alkenal (Jamieson and Gheluwe, 1970). Since the taste-threshold level for T2N in humans is extremely low, previously determined to be around 0.7 nM or 0.1 ppb (Meilgaard, 1975), products with even minute levels of the aldehyde are regarded as being aged due to the off-flavor taste of the product. Moreover, liberation of T2N from decomposing T2N adducts during beer storage may cause deterioration of the product (Nyborg et al., 1999).

Radioactive labeling studies with plant tissue established that nonenals are derived from the C.sub.18 fatty acid linoleic acid, whereas the hexanals and nonadienals are formed from the C.sub.18 fatty acid linolenic acid (Grosch and Schwartz, 1971; Phillips and Galliard, 1978). These and numerous subsequent observations--for example as summarized by Tijet et al. (2001), Noordermeer et al. (2001), and Matsui et al. (2003)--have been interpreted as evidence that T2N is formed by the sequential action of LOX pathway-specific enzymes, with the action of LOX representing an early enzymatic step. Consistent with this notion, Kurodo et al. (2003) found that malt contains a heat-stable enzymatic factor which is necessary for the transformation of the products made by LOX into T2N. The barley kernel contains three LOX enzymes known as LOX-1, LOX-2 and LOX-3 (van Mechelen et al., 1999). While LOX-1 catalyzes the formation of 9-HPODE--a precursor of T2N--from linoleic acid, LOX-2 catalyzes the conversion of linoleic acid to 13-HPODE which is further metabolized to hexanal (FIG. 1B), a C.sub.6 aldehyde with a taste threshold level of around 0.4 ppm (Meilgaard, supra). Although the product specificity of LOX-3 remains elusive, the very low expression level of the corresponding gene, as shown by van Mechelen et al. (supra), suggests that its contribution to T2N formation is negligible. Research is ongoing to determine if LOX activity is the sole enzymatic source for the generation of linoleic acid hydroperoxide precursors of relevance for the formation of the T2N-specific off-flavors, or whether the process of fatty acid autooxidation contributes as well. It is notable that C.sub.18 hydroperoxides can be further converted by more than seven different families of plant and animal enzymes, with all reactions collectively called the LOX pathway (Feussner and Wasternack, 2002); this pathway is also referred to as the oxylipin pathway. Oxylipins, as their name implies, are oxygenated lipid-derived molecules, which result from the oxygenation of unsaturated fatty acids via the LOX reaction and also include any molecules derived from such oxygenated molecules.

Barley kernels and barley plants having a LOX-1 protein characterized by reduced activity were disclosed in PCT application PCT/IB01/00207 published as WO 02/053721A1 to Douma et al. However, said application does not teach the generation and analysis of barley kernels with inactive LOX-1 enzyme.

Several examples on mutated plants that synthesize low levels of LOX are known. For example, three soybean lines were identified in the early 1980s, each deficient in one of the three LOX enzymes in mature soybean seed: (i) LOX-1. Although the molecular basis of the LOX-1 null mutation remains uncertain, it correlates with the absence of the corresponding mature mRNA (Hildebrandt and Hymowitz, 1982; Start et al., 1986); (ii) LOX-2. Transcripts for the mutated gene were detected, and a single base change was observed which replaces a histidine ligand to the active site iron, leading to enzyme instability (Davies and Nielsen, 1986; Wang et al., 1994); (iii) LOX-3. LOX-3 null mutants exhibited no detectable levels of the corresponding transcript, probably as a consequence of cis-acting elements in the gene promoter (Kitamura et al., 1983; Wang et al., 1995).

In pea seed, a null-LOX-2 line was found to carry a defect leading to the absence of most LOX-2 protein (Forster et al., 1999). Since this line exhibited a great decrease in the amount of mRNA for LOX-2, it was suggested that the mutation caused a dramatic reduction in mRNA stability.

In rice, immunoblot screening of extracts revealed the presence of two natural cultivars, Daw Dam and CI-115, each lacking one of three LOX enzymes (Ramezanzadeh et al., 1999). It was determined that the amount of hexanal, pentanal, and pentanol in normal rice with all three LOXs was markedly induced during storage, while that in Daw Dam and CI-115 was reduced in the range from 66% to 80%. Despite that the results suggest the absence of LOX enzymes in rice grains alleviate oxidative deterioration, the molecular determinants which impart the LOX-less characteristics of Daw Dam and CI-115 remain elusive.

Both antisense-mediated and co-suppression-mediated transgenic depletion of genes for LOX have proved useful to elucidate the function of specific LOX enzymes and their corresponding products in plant defense signaling. In Arabidopsis, for example, depletion of a LOX enzyme led to a reduction in the wound-induced accumulation of jasmonic acid (Bell et al., 1995). And results of antisense-mediated depletion of a gene encoding LOX established the involvement of the corresponding enzyme in the incompatibility trait of a tobacco plant resistant to a fungal pathogen (Rance et al., 1998). A third example where transgenic approaches have been used to elucidate LOX functions relates to the role of a potato LOX, denoted LOX-H1, in growth and development of potato plants (Leon et al., 2002). It was shown that LOX-H1 depletion resulted in a marked reduction of volatile aliphatic C.sub.6 aldehydes, compounds involved in plant defense responses and acting as either signaling molecules for wound-induced gene expression or as antimicrobial substances. A further study showed that transgenic potato plants depleted in the expression of a gene for a LOX enzyme exhibited abnormal tuber development (Kolomiets et al., 2001). However, specific oxylipins that accounted for the tuber phenotype were not identified. In another study, antisense-mediated depletion of potato LOX-H3 suppressed the inducible defense response of the plant, concominant with a higher tuber yield (Royo et al., 1999). Collectively, these data suggest that expression of genes encoding LOX enzymes is important in plant development, possibly with some LOX enzymes playing a defensive role against pathogens, whereas other LOX enzymes generate products that may act to regulate cell development.

It is also of importance to note that tomato fruits with 2-20% reduced levels of two LOX enzymes showed no significant changes in flavor volatiles when compared to wild-type fruits (Griffiths et al., 1999). This finding suggests that either very low levels of LOX are sufficient for the generation of aldehydes and alcohols, or that other LOX enzymes are active in the generation of these compounds.

The role of LOX enzymes is also related to issues outside the field of manufacturing beer, such as LOX-catalyzed generation of hydroperoxy fatty acids that inhibit mycotoxin formation in plants susceptible to fungal contamination, for example as disclosed in U.S. Pat. No. 5,942,661 to Keller. Although the role for LOX enzymes in plant defense and wounding responses remains less clear, the enzymes are induced upon wounding and pathogen challenge (Bell and Mullet, 1991; Bell and Mullet, 1993; Melan et al., 1993; Sarvitz and Siedow, 1996). LOX enzymes' role in wounding and plant defence could be to produce reactive fatty acid hydroperoxides against pathogens (Rogers et al., 1988). Alternatively, LOXs may be induced by stresses to produce signal molecules, such as methyl jasmonate (Bell et al., supra).

Strategies have also been described where 13-HPODE, produced by the action of a LOX enzyme, acts as a substrate for hydroperoxide-converting enzymes to produce flavor-active aldehydes (Noordermeer et al., 2002; Husson and Belin, 2002). Similar processes are disclosed in numerous patents, e.g. U.S. Pat. No. 6,150,145 to Hausler et al. and U.S. Pat. No. 6,274,358 to Holtz et al.

Also, LOX enzymes have been shown to contribute several beneficial effects to bread-making (Casey, 1997). Moreover, U.S. Pat. No. 6,355,862 B1 to Handa and Kausch discloses that fruit quality can be enhanced by inhibiting production of LOX, such as giving a longer shelf life to the product.

3. SUMMARY OF THE INVENTION

There exists thus an unmet need for barley plants with essentially no LOX-1 activity, because beverages prepared from such plants will have very low levels of T2N. In addition, such plants may be useful for other purposes.

Surprisingly, the present invention discloses methods for preparing barley plants with no or very little LOX-1 activity. In particular, the invention discloses null mutations in the gene for LOX-1. The prospective benefits of the invention include a total elimination of T2N from the corresponding branch of the LOX pathway, and the invention thus provides a superior way for controlling T2N levels in the barley kernel; and beer produced from these kernels exhibit exceptional taste stability after prolonged storage, even at elevated temperatures.

Interestingly, the present invention also provides methods for early mutation detection, and hence the disadvantages of late mutant characterization have been solved by the present invention. This makes use of a new attractive procedure for generating improved malting barley cultivars, introducing the sequential use of phenotype characterization and DNA sequence determination of target genes in a mutant population at an early time point in the breeding process. Isolated plants can be further improved using a variety of plant breeding methods.

The present invention solves the current problems, limitations and disadvantages related to the presence of active LOX-1 enzyme in barley. First, this invention provides a novel, efficient screening method that significantly reduces the time and labor for screening chemically mutagenized barley. Second, the present invention includes novel null-LOX-1 barleys, for example useful in the production of flavor-stable beer.

The theoretical background art for plant LOX mutants, as described above, is related to plants having reduced levels of LOX activity. In contrast, the present invention overcomes the limitations and disadvantages related to low or residual LOX activity by providing ways to effectively generate null-LOX-1 barley plants. Specific differences include: (i) In contrast to barley plants disclosed in PCT application PCT/IB01/00207 published as WO 02/053721A1 to Douma et al., plants of the present invention comprise essentially no LOX-1 activity, preferably the plants are true null-LOX-1 plants--i.e. the plants exhibit a total lack-of-function of LOX-1 protein; (ii) The true null-LOX-1 trait described herein could be identified by screening for the presence of a nonsense mutation in the corresponding gene. Accordingly, barley plants homozygous for that trait would be completely blocked in the synthesis of active enzyme--irrespective of growth conditions or environmental effects. This is an ideal property in the art of plant breeding and contrasts the outcome of a possible molecular scenario in the soybean LOX mutants of the background art, where biotic or abiotic conditions could affect changes in the physiological state of cells to confer mRNA stabilization with subsequent translation of LOX; (iii) Where the trait of relevance in LOX mutants of soybean and rice comprised reduced levels of the odor-intense compound hexanal in a staple food, the present invention relates to lower levels of the taste-specific compound T2N in a beverage; (iv) The soybean and rice LOX mutants are affected in molecules of the LOX pathway downstream of 13-HPODE, while the null-LOX-1 trait relates to that branch of the LOX pathway which comprises molecules downstream of 9-HPODE; (v) While the soybean mutants comprise irradiation-induced mutations in genes for LOX, and Daw Dam and CI-115 represent selected naturally occurring cultivars of rice breeding lines, mutations in barley plants having the null-LOX-1 trait were induced by the chemical NaN.sub.3.

Hence, it is an objective of the present invention to provide barley plants, parts or fragments thereof comprising less than 5%, preferably less than 1% of the LOX-1 activity of a wild-type barley plant.

It is a second objective of the invention to provide kernels from a barley plant comprising less than 5%, preferably less than 1% of the LOX-1 activity of a wild-type plant.

A third objective of the present invention is to provide compositions comprising a barley plant, or parts or fragments thereof comprising less than 5%, preferably less than 1% of the LOX-1 activity of a wild-type barley plant.

It is a further objective of the present invention to provide malt compositions comprising a processed barley plant comprising less than 5%, preferably less than 1% of the LOX-1 activity of a wild-type barley plant. Malt compositions may preferably be pure malt compositions. However, malt compositions may also be for example blends of barley and malt.

It is also an objective of the present invention to provide beverages having stable organoleptic qualities, wherein said beverages are manufactured using the barley plant of the invention or part thereof. In particular, it is preferred that said beverages are manufactured using the malt composition, such as a pure malt composition or a blend of barley and malt, described herein above. In a preferred embodiment of the invention said beverages consist of beer.

It is an additional objective of the invention to provide a beverage having stable organoleptic qualities, wherein said beverage is manufactured using a barley plant and wherein the ratio of 9,12,13-trihydroxyoctadecenoic acid to 9,10,13-trihydroxyoctadecenoic acid within said beverage is at the most 1.8. Preferably, said beverage is beer.

Moreover, an objective of the invention is to provide compositions, such as food compositions, feed compositions, or fragrance raw material compositions that comprise the barley plant according to the invention or parts thereof.

In addition, it is an objective of the present invention to provide methods for expressing a recombinant protein in a barley plant according to the invention, wherein said method comprises transforming said plant with a nucleic acid sequence comprising, as operably linked components a promoter expressable in barley plants or parts thereof, a DNA sequence encoding said recombinant protein, and a transcriptional termination region, thereby expressing said recombinant protein in said barley plant.

Further, an objective of the present invention is to provide methods for reducing the levels of a protein in a barley plant of the invention, wherein said method includes transforming said plant with a nucleic acid sequence comprising, as operable linked components, a promoter expressable in barley plants or parts thereof, a DNA sequence, and a transcriptional termination region, wherein expression of said DNA sequence reduces the expression of a gene encoding said protein by antisense, or co-suppression or RNA interference.

An additional objective of the present invention is to provide methods of preparing a barley plant comprising less than 5%, preferably less than 1% of the LOX-1 activity of a wild-type barley plant comprising the steps of: (i) Determining the LOX-1 activity in wild-type barley kernels or parts thereof; and (ii) Mutagenizing barley plants and/or barley kernels and/or barley embryos, thereby obtaining generation MO barley; and (iii) Breeding said mutagenized barley plants, kernels and/or embryos for at least 2 generations, thereby obtaining generation Mx barley plants, wherein x is an integer .gtoreq.2; and (iv) Obtaining kernels or parts thereof from said Mx barley plants; and (v) Determining the LOX-1 activity in said kernels or parts thereof; and (vi) Selecting plants wherein the LOX-1 activity of the mutagenized kernels or parts thereof is less than 5% than the LOX-1 activity of the wild-type kernels or part thereof; thereby obtaining a barley plant comprising less than 5% of the LOX-1 activity of a wild-type barley plant.

Still further, it is an objective of the present invention to provide methods of producing a beverage having stable organoleptic qualities comprising the steps of: (i) Providing a malt composition according to the invention; (ii) Processing said malt composition into a beverage; thereby obtaining a beverage with stable organoleptic qualities.

It is an additional objective of the present invention to provide methods of producing a malt composition with low LOX-1 activity, comprising the steps of (i) Providing kernels according to the invention; (ii) Steeping said kernels; (iii) Germinating the steeped kernels under predetermined conditions; (iv) Treating germinated kernels with heat; thereby producing a malt composition with low or no LOX-1 activity.

In one preferred embodiment, the present invention is based on the unpredicted outcome of functional studies of barley mutant D112, which revealed a total loss-of-function with respect to the major 9-HPODE-forming enzyme LOX-1. It was a surprising discovery to detect a 10%:90% distribution of 9-HPODE:13-HPODE in biochemical assays designed to determine the product profile following LOX-catalyzed conversion of linoleic acid. Given the extremely low taste-threshold of T2N, it was even more surprising that degradation of residual 9-HPODE and the like in kernels of mutant D112 only caused very low liberation of T2N--well below the taste threshold level--during aging of beer products manufactured from malt of said kernels.

Examination of the results from analyses using wild-type and null-LOX-1 kernels provide clear evidence that high LOX-1 activity can intensify the stale cardboard flavor of T2N, thus confirming an important role of the LOX pathway in controlling formation of the alkenal. This conclusion contrasts the notion of Liegeois et al. (supra), who suggested that LOX activity only contributes with a small fraction of the T2N precursor molecules.

The null-LOX-1 trait can be introduced into established malting barley varieties, thus allowing production of flavor-stable beverages with prolonged shelf lives. This approach will not only be independent of the geographical region where mutant D112-derived barley is grown, but also independent of the location where mutant D112-derived beer is produced and sold to customers. Barley plants of mutant D112, or plants derived thereof, are potentially an important economical factor for farmers that grow the crop, and for breweries that use it as a raw material for beer production. Other applications that depend on raw materials without 9-HPODE/9-HPOTE-forming activities are also anticipated to benefit from the properties of barley mutant D112.

In accordance with one embodiment of the invention, there is provided several novel malting barley mutants, for example the mutants designated D112 or A618. The present invention is therefore related to the kernels of barley mutants D112 or A618, to the plants of barley D112 or A618 and to methods for producing a barley plant derived from crossing barley mutants D112 or A618 with itself or another barley line. Moreover, the present invention comprises null-LOX-1 variants generated by mutagenesis or transformation of barley mutant D112 or A618. Thus, all plants produced using barley mutants D112 or A618--or a derivative thereof--as a parent are within the scope of this invention.

In another aspect, the invention provides regenerable cells for use in tissue culture of barley mutant plant D112 or A618. The tissue culture will preferably be used for regeneration of plants having the characteristics of the foregoing barley plants, including morphological and genetic characteristics. The regenerated cells in such tissue cultures will be embryos, protoplasts, meristematic cells, callus, pollen, anthers, etc. It is understood that the present invention provides barley plants regenerated form the tissue cultures of the invention.

In a preferred embodiment, the present invention comprises malt derived from null-LOX-1 barley kernels.

The present invention also relates to wort compositions prepared from null-LOX-1 barley plants or parts thereof or from malt compositions prepared from such barley plants.

The invention further comprises beverages, such as beer manufactured using either null-LOX-1 barley kernels of the present invention or malt derived from said kernels.

In addition, the invention relates to a plant product produced from a null-LOX-1 barley plant or parts thereof. Said plant product may be any product resulting from processing of said barley plant or part thereof. Preferably, said plant product is selected from the group consisting of malt, wort, fermented beverages such as beer, non-fermented beverages, food products such as barley meal and feed products.

It is also an object of the present invention to provide null-LOX-1 barley kernels exhibiting such levels of disease resistance that are indistinguishable from wild-type barley plants or even have improved disease resistance.

Still further, the invention comprises null-LOX-1 barley kernels and malt derived from said kernels, where both kernels and malt exhibit reduced levels of mycotoxins.

Also, the present invention comprises null-LOX-1 barley varieties with enhanced disease resistance relative to wild-type plants. Further, null-LOX-1 barley having reduced disease resistance relative to wild-type plants are disclosed, provided that other characteristics of said plants provide benefits that are more important than the property of reduced disease resistance.

In addition, the present invention provides null-LOX-1 barley kernels useful for the production of LOX pathway-derived fragrances, including green note compounds.

Moreover, the present invention provides for transgenic plants of null-LOX-1 barley mutants D112 or A618, or plants derived thereof, where the introduced gene(s) confer such traits as herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral diseases, enhanced nutritional quality, and industrial usage. The gene may be an endogenous barley gene or, alternatively, a transgene introduced through genetic engineering techniques.

These and other features, aspects, and advantages of the present invention will be better understood when related to the following definitions, descriptions, examples, appended claims as well as accompanying sequence listings and drawings.

3.1 Definitions

In the description, figures, and tables which follow, a number of terms are used. In order to provide the specifications and claims, including the scope to be given such terms, the following definitions are provided:

As used herein, "a" can mean one or more, depending on the context in which it is used.

The term "agronomic trait" describes a phenotypic trait of a plant that contributes to the performance or economic value of said plant. Such traits include disease resistance, insect resistance, virus resistance, nemtatode resistance, drought tolerance, high salinity tolerance, yield, plant height, days to maturity, kernel nitrogen content and the like.

By "antisense nucleotide sequence" is intended a sequence that is in inverse orientation to the normal coding 5'-to-3' orientation of that nucleotide sequence. When present in a plant cell, the antisense DNA sequence preferably prevents normal expression of the nucleotide sequence for the endogenous gene, and may disrupt production of the corresponding, native protein.

The term "barley" in reference to the process of making beer, particularly when used to describe the malting process, means barley kernels. In all other cases, unless otherwise specified, "barley" means the barley plant (Hordeum vulgare, L.) including any varieties.

By "disease resistance" is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. In this way, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms. Alternatively, the disease symptoms caused by the pathogen are minimized or reduced.

A "cereal" plant as defined in this publication is a member of the Graminae plant family cultivated primarily for their starch-containing seeds. Cereal plants include barley (Hordeum), wheat (Triticum), rice (Oryza), maize (Zea), rye (Secale), oat (Avena), sorghum (Sorghum), and Triticale, a rye-wheat hybrid.

By "encoding" or "encoded," in the context of a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g. introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g. in cDNA). The information by which a protein is encoded is specified by the use of codons.

As used herein, "expression" in the context of nucleic acids is to be understood as the transcription and accumulation of sense mRNA or antisense RNA derived from a nucleic acid fragment. "Expression" used in the context of proteins refers to translation of mRNA into a polypeptide.

By "flavor molecules" is intended aldehydes and/or alcohols that are produced and are constituents of odor and/or taste in plants. In particular, flavor molecules include certain volatile alcohols and aldehydes. Examples of flavor molecules which are volatile include but are not limited to hexanal, (3Z)-hexenal, (2E)-hexenal, (2E)-hexenol, (3Z)-nonenal, (2E)-nonenal. The invention can be used to modulate levels of flavor molecules in plants.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (promoter and terminator). Eukaryotic genes are discontinuous with proteins encoded by them, consisting of "exons" interrupted by "introns.". After transcription into RNA, the introns are removed by splicing to generate a mature messenger RNA (mRNA). The "splice sites" between exons are typically determined by consensus sequences acting as splice signals for the splicing process, consisting of a deletion of the intron from the primary RNA transcript and a joining or fusion of the ends of the remaining RNA on either side of the excised intron. In some cases alternate or different patterns of splicing can generate different proteins from the same single stretch of DNA. A native gene may be referred to as an endogenous gene.

"Gene-silencing" is a method to alter gene expression. It refers to RNA silencing, which is a post-transcriptional gene-silencing mechanism conserved among various organisms. The method includes post-transcriptional gene silencing (PTGS) and RNA interference (RNAi). PTGS is a gene-silencing phenomenon of endogenous and exogenous homologous genes. Although most examples on PTGS are on the effects caused by co-suppression constructs or expression of transgenes in antisense orientation, it has also been observed in plants of conventional breeding programs, e.g. the Lgc1 mutation in rice (Kusaba et al., 2003). This mutation was found to suppress glutelin expression via RNA silencing, possibly due to a 3.5-kbp deletion between two highly similar genes for glutelin that forms a tail-to-tail inverted repeat that might produce a double-stranded RNA molecule--and thus a potent inducer of RNA silencing. A second form of RNA silencing is known as RNA interference (RNAi), where the basic premise is the ability of double-stranded RNA to specifically block expression of its homologous gene when injected or ingested into cells (Goenczy et al., 2000).

As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

The term "germination" as used herein means the beginning or resumption of growth by a barley kernel in various compositions, such as normal soil as found in nature. Germination can also take place in the soil of pots placed in growth chambers an the like, or for example take place on wet filter paper placed in standard laboratory Petri dishes. Germination is generally understood to include hydration of the kernels, swelling of the kernels and inducing growth of the embryo. Environmental factors affecting germination include moisture, temperature and oxygen level. Root and shoot development are observed.

"Green notes" is a term describing volatile flavor and fragrance molecules present in numerous plants, and characterized in organoleptic terms as fresh green and grassy. These molecules are produced by the plant from the degradation of lipids and free fatty acids, such as linoleic acid and linolenic acid.

As used herein, the term "isolated" means that the material is removed from its original environment. For example, a naturally-occuring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynycleotides or polypeptides could be part of a composition, and still be isolated because such vector or composition is not part of its natural environment.

The term "kernel" is defined to comprise the cereal caryopsis, also denoted internal seed, the lemma and palea. In most barley varieties the lemma and palea adhere to the caryopsis and are a part of the kernel following threshing. However, naked barley varieties also occur. In these, the caryopsis is free of the lemma and palea and threshes out free as in wheat. The terms "kernel" and "grain" are used interchangeably herein.

"Kernel maturation" or "grain development" refers to the period starting with fertilization in which metabolizable reserves, e.g. sugars, oligosaccharides, starch, phenolics, amino acids, and proteins are deposited, with and without vacuole targeting, to various tissues in the kernel (grain), e.g. endosperm, testa, aleurone, and scutellum, leading to kernel (grain) enlargement, kernel (grain) filling, and ending with kernel (grain) desiccation.

The term "LOX-1 activity" refers to the enzymatic activity of the barley LOX-1 enzyme. In particular, in the context of the present invention "LOX-1 activity" is the enzyme catalyzed dioxygenation of linoleic acid to 9-HPODE. Even though the LOX-1 enzyme is capable of catalyzing other reactions, for the purpose of determining the activity of LOX-1 according to the present invention only the 9-HPODE forming activity should be considered. FIG. 1B outlines the biochemical pathway wherein linoleic acid is converted to T2N.

The term "low-LOX" refers to the presence of one or several mutations in one or several endogenous genes, causing a partial loss-of-function of a specified LOX enzyme, preferably with respect to--but not restricted to--enzymic activity. For example, the barley plants disclosed in PCT application PCT/IB01/00207 published as WO 02/053721 A1 to Douma et al. produce a mutated LOX-1 enzyme having 10% residual activity compared with the corresponding wild-type enzyme. "Low-LOX" with reference to a plant refers to a plant having partial loss-of-function of the specified LOX enzyme.

"Malting" is a special form of germination of barley kernels taking place under controlled environmental conditions, including, but not limited to maltery steep tanks and germination boxes. In accordance with the process of the present invention, malting begins to occur during and/or after the barley kernels have been steeped. The malting process may be stopped by drying of the barley kernels. A malt composition prepared from null-LOX-1 barley is understood to comprise null-LOX-1 malt, such as pure null-LOX-1 malt or any blend of malt comprising null-LOX-1 malt.

"Mashing" is the incubation of milled malt in water. Mashing is preferably performed at a specific temperature and in a specific volume of water. The temperature and volume of water is important as this affects the rate of decrease of enzyme activity derived from the malt, and hence the amount of especially starch hydrolysis that can occur. Mashing can occur in the presence of adjuncts, which is understood to comprise any carbohydrate source other than malt, e.g. barley (including null-LOX-1 barley), maize or rice adjunct, used principally as an additional source of extract. The requirements for processing of the adjunct in the brewery depend on the state and type of adjunct used, and in particular the starch gelatinization or liquefaction temperatures. If the gelatinization temperature is above the normal malt saccharification temperature, then the starch is gelatinized and liquefied before adding to the mash.

"Mutations" include deletions, insertions, transversions and point mutations in the coding and noncoding regions of a gene. Deletions may be of the entire gene or of only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certains cells or tissues of the plant and are not inherited to the next generation. Germline mutations can be found in any cell of the plant and are inherited.

The term "null-LOX" refers to the presence of a mutation in a LOX-encoding gene, causing a total loss-of-function of the encoded LOX enzyme. Mutations that generate premature termination (nonsense) codons in a gene encoding LOX represent only one mechanism by which total loss-of-function can be obtained. Molecular approaches to obtain total loss-of-funtion of a LOX enzyme comprise the generation of mutations that cause a total absence of transcripts for said enzyme, or mutations that totally inactivate the encoded enzyme. "null-LOX" with reference to a plant refers to a plant having a total loss-of-function of the specified LOX enzyme.

"Operably linked" is a term used to refer to the association of two or more nucleic acid fragments on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence, i.e. that the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

"PCR" or "polymerase chain reaction" is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159 to Mullis et al.).

"Plant" or "plant material" includes plant cells, plant protoplasts, plant cell tissue cultures from which barley plants can be regenerated, plant calli, and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, flowers, kernels, leaves, roots, root tips, anthers, or any part or product of a plant.

By the term "plant product" is meant a product resulting from the processing of a plant or plant portion. Said plant product may thus for example be malt, wort, a fermented or non-fermented beverage, a food or a feed product.

As used herein, "recombinant" in reference to a protein is a protein that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition by deliberate human intervention.

"RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript. When an RNA sequence is derived from post-translational processing of the primary transcript, it is referred to as the mature RNA. "Messenger RNA" or "mRNA" refers to the RNA that is without introns and that can be translated into proteins by the cell. "cDNA" refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to a double-stranded form using, for example, the Klenow fragment of DNA polymerase I. "Sense RNA" refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene. The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e. at the 5' non-coding sequence, 3' non-coding sequence, introns, or the protein coding sequence. "Functional RNA" refers to sense RNA, antisense RNA or other RNA that may not be translated into a protein but yet has an effect on cellular processes.

Unless otherwise noted, "T2N" means the free form of T2N. By the term "T2N potential" is described the chemical substances which have the capacity to release T2N, or be converted into T2N, in one or more reactions. The T2N potential can be measured as the concentration of T2N in a solution, e.g. in wort or beer, following an incubation (e.g. for 2 h) at an elevated temperature (e.g. 100.degree. C.) and low acidity (e.g. pH 4.0). This sample treatment causes liberation of T2N from the T2N potential, e.g. from "T2N adducts," a term used to describe T2N conjugated to one or more substances, including, but not limited to, protein(s), sulfite, cellular debris, cell walls, or the like. In general, T2N adducts per se are not sensed by humans as off-flavors. However, T2N released from said T2N adducts, for example by heat or acid, may give rise to an off-flavor.

"Tissue culture" indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant, for example protoplasts, calli, embryos, pollen, anthers, and the like.

"Transformation" means introducing DNA into an organism so that the DNA is maintained, either as an extrachromosomal element (without integration and stable inheritance) or chromosomal integrant (genetically stable inheritance). Unless otherwise stated, the method used herein for transformation of E. coli was the CaCl.sub.2-method (Sambrook and Russel, supra), and Agrobacterium-mediated transformation of the barley cultivar Golden Promise was basically as described by Tingay et al. (1997) and Wang et al. (2001), except that only the cultivar Golden Promise was used as host.

A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

As used herein, "transgenic" includes reference to a cell that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, transgenic cells express genes that are not found in an identical form within the native form of the cell, or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all as a result of deliberate human intervention. The term "transgenic" in reference to plants, particularly barley plants, as used herein does not encompass the alteration of the cell by methods of traditional plant breeding, e.g. NaN.sub.3-derived mutagenesis, and by naturally occurring events such as those occuring without deliberate human invention.

The term "wild-type barley plant" refers to a conventional barley plant, preferably the term refers to the barley plant, from which the barley plants of the invention have been derived, i.e. the parent plants. In one preferred embodiment of the invention, the "wild-type barley plant" is selected from the group consisting of cv.s Celeste, Lux, Prestige, Saloon, and Neruda. More preferably, the "wild-type barley plant" is cultivar Barke.

4. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing (summarized in Table 9), which forms a part of this application. Said table lists the nucleic acids and polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier [SEQ ID NO:]. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications.

The Sequence Listing contains the one letter code for nucleotide and amino acid sequence characters as defined in conformity with the standardized recommendations (Cornish-Bowden, 1985; IUPAC-IUB Joint Commission on Biochemical Nomenclature, 1984), which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules governing sequence disclosures in patent applications.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is divided into two flow diagrams A and B. FIG. 1A shows how NaN.sub.3-mutagenized barley kernels may be propagated. Kernels of generation M0 grow into plants that develop kernels of generation M1. These may be sown and develop into M1 plants which produce new kernels of generation M2. Next, M2 plants grow and set kernels of generation M3, which may be harvested and used for screening analyses. M3 seeds may also be sown, and flowers of the corresponding plants used for crossings to obtain plants of generation M4. FIG. 1B is a simplified representation how the biochemical LOX pathway operates to degrade linoleic acid, eventually yielding T2N.

FIG. 2 is a graphic comparison of total LOX activities measured in embryo extracts of cv. Barke, mutant D112, and in a control sample comprising heat-inactivated extract of cv. Barke embryos.

FIG. 3 displays a graphic comparison of total LOX activities measured in embryo extracts of mutant A618, cv. Neruda, and in a control sample comprising heat-inactivated extract of cv. Barke embryos.

FIG. 4 shows a comparison of total LOX activities measured in kernels of 12 individual M4 progeny lines of mutant D112. The activities of control samples consisting of kernel extracts of cv. Barke, and heat-inactivated kernel extracts of cv. Barke are included in the comparison.

FIG. 5 summarizes the results of analyses for total LOX activity in 90 individual kernel extracts of M5 progeny lines of mutant D112. The activities of control kernel extracts of cv. Barke, and heat-inactivated kernel extracts of cv. Barke are included in the comparison.

FIG. 6 gives a summary of a comparison of total LOX activities measured in 40 individual kernel extracts of M4 progeny lines of mutant A618. The activities of control samples with kernel extracts of cv. Barke and heat-inactivated kernel extracts of cv. Barke are included in the comparison.

FIG. 7 consists of two separate immunoblots, showing that immunoreactive LOX-1 protein is not detectable in kernel extracts of mutant D112, generation M3. Each immunoblot was probed with an antibody to barley LOX-1, and the samples consisted of extracts of E. coli cells expressing recombinant LOX-1 (lane 1), kernel extracts of cv. Vintage (lane 2), mutant line G (lane 3 and lane 7), cv. Barke (lane 6 and lane 8), and separate lines of mutant D112, generation M3 (lanes 4-5 and 9-16). The position of an immunoreactive LOX-1 protein is indicated.

FIG. 8 shows two separate immunoblots, detailing the absence of LOX-1 in kernel extracts of mutant A618, generations M3 and M4. Each immunoblot was probed with an antibody to barley LOX-1, and the samples consisted of kernel extracts of mutant line G (lane 1), cv. Neruda (lane 6 and lane 16). Extracts of randomly chosen M3 and M4 kernels that have not been through the LOX selection procedure were separated in lanes 2-5 and 8-12, respectively; all of these extracts contained a LOX-1 immunoreactive protein. The null-LOX-1 phenotype of a kernel extract of mutant A618, generation M3 (lane 7), was inherited in separate M4-progeny lines of mutant A618-82 (lanes 8-12). The position of an immunoreactive LOX-1 protein is indicated.

FIG. 9 schematically illustrates the genetics of the backcrossing program for mutant D112 to cv. Prestige. The wild-type LOX-1 trait is assigned NN, while the null-LOX-1 mutant trait is nn.

FIG. 10 provides an illustration of seven separate immunoblots, each probed with an antibody to barley LOX-1. The immunoblots show the presence or absence of the immunoreactive LOX-1 protein in kernels of separate plants of the first backcross generation of mutant D112 to cv. Prestige (lanes 1-6 and lanes 9-14), and the presence or absence of the immunoreactive LOX-1 protein in kernels of the second backcross generation of mutant D112 to cv. Prestige (lanes 17-22, lanes 25-30, lanes 33-38, lanes 41-45, and lanes 48-52). Control kernel extracts of mutant D112, lacking immunoreactive LOX-1 (lanes 7, 15, 23, 31, 39, 46, 53), and cv. Prestige, containing immunoreactive LOX-1 (lanes 8, 16, 24, 32, 40, 47, 54), were used as controls. The position of an immunoreactive LOX-1 protein is indicated.

FIG. 11 is a simplified, schematic overview of the beer production process without the use of adjuncts, but including steeping of the barley grain (1), malting (2), kiln drying (3), milling of the dried malt (4), mashing (5), filtration (6), wort boiling in the presence of added hops (7), fermentation in the presence of yeast (8), beer maturation (9), beer filtration (10), bottling (11), and labeling (12). The individual processes can be grouped into sections comprising malt production (1-3), wort production (4-7), fermentation (8-9), and preparation of the finished beer (10-12).

FIG. 12 focuses on graphs showing the accumulation of free T2N during forced aging at 37.degree. C. The aldehyde was measured in beer produced from malt of null-LOX-1 mutant D112 (.DELTA.), and control malt of cv. Barke (.smallcircle.). The taste threshold level for T2N in beer is approximately 0.05 ppb.

FIG. 13 displays the chromatograms of HPLC analyses used to assay for the formation of 9- and 13-HPODEs in barley tissues. The levels of HPODEs were analyzed by measuring the absorbance at 234 nm, with the results given in milli absorbance units (mAU). Peaks of the elution profiles that correspond to 9-HPODE and 13-HPODE are indicated by arrows. FIG. 13A shows the chromatogram of 9-HPODE and 13-HPODE standards. FIG. 13B is a chromatogram of HPODEs formed in extracts prepared from mature embryos of cv. Barke. FIG. 13C is a chromatogram of HPODEs formed in extracts prepared from mature embryos of low-LOX kernels. FIG. 13D is a chromatogram of HPODEs formed in extracts of mature embryos of the null-LOX-1 mutant D112.

FIG. 14 depicts the chromatograms of HPLC analyses used to assay for the formation of 9- and 13-HPODEs in malt. The levels of said HPODEs were analyzed by measuring the absorbance at 234 nm, with the results given in milli absorbance units (mAU). Peaks of the elution profiles that correspond to 9-HPODE and 13-HPODE are indicated by arrows. FIG. 14A shows the chromatogram of 9-HPODE and 13-HPODE standards. Chromatogram peaks corresponding to 9-HPODE and 13-HPODE are indicated by arrows. FIG. 14B is a chromatogram of HPODEs formed in extracts of malt from cv. Barke. FIG. 14C is a chromatogram of HPODEs formed in extracts of malt from low-LOX barley. FIG. 14D is a chromatogram of HPODEs formed in extracts of malt from the null-LOX-1 mutant D112.

FIG. 15 is a map showing the organization of the gene for barley LOX-1, spanning the start codon (ATG) and stop codon (TM). The schematic drawing of the 4,165-bp-long sequence shows 7 exons (filled boxes) and 6 introns (lines). The position of the mutations identified in the gene for LOX-1--i.e. specific for mutant line G (low-LOX), mutant A618 and mutant D112--are indicated by arrows.

FIG. 16 summarizes the predicted molecular differences related to the gene for LOX-1 of wild-type, mutant A618 and mutant D112 barley plants. The information listed in the columns marked "Result," "Length in amino acids," and "Mass in kDa" is predicted from the DNA sequence.

FIG. 17 provides ways used to perform RT-PCR mutant analysis and transcript verification related to the barley gene encoding LOX-1. In A is shematically shown the principle for RT-PCR detection of a specific transcript for the gene encoding LOX-1 in developing embryos of cv. Vintage and low-LOX-1 mutant line G. Primers consisted of FL821 [SEQ ID NO: 11] and FL852 [SEQ ID NO: 12], which anneal in the exons flanking the 83-bp-long intron 5; PCR product differences using either genomic DNA or mRNA templates are indicated. In B is shown the result of a RT-PCR agarose gel analysis, where focus was on the detection of a specific transcript related to the gene encoding LOX-1 in developing embryos of barley, cv. Vintage and mutant line G. Lanes 1 and 5 contained marker fragments, and lanes 2, 3, and 4 contained the PCR products derived from embryo tissues of cv. Vintage after 20, 40, and 60 days after flowering (DAF), respectively. Lanes 6, 7 and 8 contain the products derived from embryo tissues of mutant line G after 20, 40, and 60 DAF, respectively. In C, lanes 1-5 show the result of an experiment similar to that detailed for lanes 1-5 in B, while lanes 6, 7 and 8 contained the products derived from RT-PCR detection of a mutant D112 embryo-specific transcript of the gene for LOX-1 after 20, 40, and 60 DAF, respectively. In D is shown an electropherogram that resulted from a sequencing reaction of a RT-PCR fragment specific for the gene for LOX-1. Sequence analysis revealed that the RT-PCR target RNA was free of DNA. The black triangle points to the splice point, indicating correct splicing of the transcript.

FIG. 18 details the results of a SNP-assisted detection of barley mutant D112. The analysis was based on the generation of a specifc PCR fragment pattern using two sets of PCR reactions per sample, as schematically illustrated in A (primer set 1 consists of FL820 [SEQ ID NO: 13] and primer FL823 [SEQ ID NO: 15], and primer set 2 consists of FL820 [SEQ ID NO: 13] and FL825 [SEQ ID NO: 14]). In B is shown the result of a PCR pattern analysis of elite breeding material. Genomic DNA of plants were subjected to PCR analyses. Results shown in lanes 2-3 (plant 1), 4-5 (plant 2), 6-7, (plant 3), 8-9 (plant 4), 10-11 (plant 5), 12-13 (plant 6), 14-15 (plant 7), 16-17 (plant 8), and 18-19 (plant 9) utilized primer combination 1 (even numbered lanes) or primer combination 2 (odd numbered lanes). Comparison of the banding pattern with that shown in A revealed that plants 1, 2, 4, 5, 7, and 8 were homozygous mutants, while the genotype of plants 3, 6, and 9 could be classified as homozygous wild-type. Marker DNA was separated in lanes 1 and 20.

FIG. 19 demonstrates the principle of multiplex SNP analysis of barley samples containing material of mutant G or mutant D112. The analysis utilized multiplex PCR reactions, such that the length of the fragment amplified could be related to the genotype of the material added. Amplification of a 370-bp fragment would indicate that a malt sample contained material derived from mutant line G, while the amplification of a 166-bp fragment would point to the presence of material derived from mutant D112. Panel A is a schematic illustration detailing how specific primer pairs, each with one primer that contains a sequence which is specific for the mutant of interest (asterisk; for mutant line G nucleotide number 2279 in the genomic clone for LOX-1, and for mutant D112 position 3574). The primer combination FL918 [SEQ ID NO: 16] and FL920 [SEQ ID NO: 17] was used for detection of the mutant line G-specific mutation, while FL820 [SEQ ID NO: 13] and FL823 [SEQ ID NO: 15] were utilized for detection of the mutant D112-specific base change. In B is shown how the relative quantities of mutant-specific material (lanes 2-7: mutant line G; lanes 8-13: mutant D112) in samples may enhance the synthesis of a specific PCR fragment (lanes 2 and 8: no mutant material added; lanes 3 and 9: 20% mutant material added; lanes 4 and 10: 40% mutant material added; lanes 5 and 11: 60% mutant material added; lanes 6 and 12: 80% mutant material added; lanes 7 and 13: 100% mutant material). Lane 1 consisted of marker fragments.

FIG. 20 presents the result of SDS-PAGE of affinity-purified, His-tagged LOX-1 from E. coli cells transformed with the vector plasmid pET19b (lanes 2-5), expression plasmid pETL1 (lanes 6-10), and expression plasmid pETL2 (lanes 11-15). Proteins from fractions comprising unbound proteins (lanes 2, 6, 11); first wash (lanes 3, 7, 12); second wash (lanes 4, 8, 13); first eluate (lanes 5, 9, 14); and second eluate (lanes 10 and 15) were analyzed. The upper arrow indicates the position of recombinant LOX-1 (corresponding to wild-type LOX-1), while the lower arrow indicates the postion of truncated, recombinant LOX-1 (corresponding to LOX-1 in barley mutant D112). Lane 1 comprised separated marker proteins.

FIG. 21 illustrates plasmid inserts for transformation of barley. In A is illustrated an expression cassette consisting of the maize ubiquitin-1 promoter and intron 1 (collectively denoted the UBI promoter), directing constitutive expression of the bar gene (BAR), which encodes the selectable marker phosphinothricin acetyl transferase. Transcription termination is provided by the NOS terminator sequence (N). In B is illustrated an expression cassette consisting of the aforementioned UBI promoter, here directing constitutive expression of the barley cDNA sequence for LOX-1 in sense or antisense oritentation. In C is illustrated an expression cassette consisting of the UBI promoter directing constitutive expression of a intron-containing hairpin construct, where the sequence of intron 1 of the Arabidopsis gene for fatty acid desaturase FAD2 intron 1 (Int), flanked by the sense arm (.fwdarw.) and antisense arm (.rarw.) of an approximately 200-bp-long fragment of the gene for LOX-1. Transcription termination is provided by the NOS terminator sequence (N). For the generation of barley plants exhibiting co-suppression of the gene for LOX-1, plasmid mixtures are used that comprise equal amounts of expression plasmids comprising the inserts detailed in A and B. For the generation of barley plants exhibiting total silencing of the gene for LOX-1, mixtures are used that comprise equal amounts of expression plasmids comprising the inserts in A and C.

6. DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity of description, and not by way of limitation, the detailed description of the invention is divided into the following subsections: (i) Barley plant; (ii) Preparing null-LOX-1 barley; (iii) Composition; (iv) Chemical mutagenesis; (v) Selection of barley mutants; (vi) Plant breeding; (vii) Barley crossings; (viii) LOX enzymes; (ix) LOX pathway products; (x) T2N potential; (xi) Disease resistance; (xii) Mycotoxins; (xiii) Fragrances; (xiv) Heterologous expression of genes encoding LOX. 6.1 Barley plant

The present invention relates to barley plants and parts thereof comprising less than 1% of the LOX-1 activity of a wild-type barley plant. The barley plants of the invention comprising less than 1% LOX-1 activity are herein also referred to as "null-LOX-1 barley plants."

The barley plant may be in any suitable form. For example, the barley plant according to the invention may be a viable barley plant, a dried plant, a homogenized plant, such as milled barley kernels. The plant may be a mature plant, an embryo, a germinated kernel, a malted kernel or the like.

Parts of barley plants may be any suitable part of the plant, such as kernels, embryos, leaves, stems, roots, flowers or fractions thereof. Fractions may for example be a section of a kernel, embryo, leaves, stem, root or flower. Parts of barley plants may also be a fraction of a homogenate or milled barley plant or kernel.

In one embodiment of the invention, parts of barley plants may be cells of said barley plant, preferably viable cells, that may be propagated in tissue cultures in vitro.

In a preferred embodiment of the invention, null-LOX-1 barley plants comprise less than 5%, preferably less than 3%, more preferably less than 1%, preferably less than 0.5%, even more preferably less than 0.1% of the activity of a wild-type barley plant. The activity may be determined by any suitable method, preferably however, the activity is determined using the method in Example 1 herein below. In a very preferred embodiment of the invention, the null-LOX-1 barley plants have essentially no LOX-1 activity, more preferably no LOX-1 activity at all. "Essentially no LOX-1 activity" means no detectable LOX-1 activity using an assay for LOX-1 activity as described herein below.

The almost absent LOX-1 activity of the null-LOX-1 barley may for example be the result of that said barley comprises a malfunctioning LOX-1 protein, such as a mutant LOX-1 protein. However, the null-LOX-1 barley comprises only very little or, more preferably, no LOX-1 protein, such as less than 5%, preferably less than 3%, more preferably less than 1%, preferably less than 0.5%, more preferably less than 0.1% LOX-1 protein compared to a wild-type barley plant. More preferably, the null-LOX-1 barley comprises essentially no LOX-1 protein, most preferably no LOX-1 protein at all. "Essentially no LOX-1 protein" is meant to cover no detectable LOX-1 protein. The LOX-1 protein may be detected by any suitable means known to the person skilled in the art, however, preferably the protein is detected by techniques wherein LOX-1 protein is detected by specific antibodies to LOX-1. Said techniques may for example be Western blotting or ELISA. Said specific antibodies may be monoclonal or polyclonal, preferably however, said antibodies are polyclonal recognizing several different epitopes within the LOX-1 protein. LOX-1 protein may also be detected indirectly, for example by methods determining LOX-1 activity, by methods detecting mutations in the gene encoding LOX-1 or by methods detecting expression of the LOX-1 gene. Mutations in the gene encoding LOX-1 may for example be detected by sequencing said gene. Expression of the gene for LOX-1 may for example be detected by Nothern blotting or RT-PCR. In one preferred embodiment of the invention, LOX-1 protein is detected using the methods outlined in Example 4 of the instant publication.

The term LOX-1 protein is meant to cover the full length LOX-1 protein of barley as set forth in [SEQ ID NO: 3] or [SEQ ID NO: 7] or a functional homologue thereof. The active site of LOX-1 is situated in the C-terminal part of LOX-1. In particular is the region spanning amino acid residues 520-862 or parts thereof relevant for LOX-1 activity. Accordingly, in one embodiment null-LOX-1 barley preferably comprises a gene encoding a mutant form of LOX-1 lacking some or all of amino acids 520-862 of LOX-1. Said mutant LOX-1 may also lack other amino acid residues which are present in wild-type LOX-1.

Accordingly, null-LOX-1 barley may comprise a truncated form of LOX-1, which is not functional, such as an N-terminal or a C-terminal truncated form. Preferably, said truncated form comprises no more than 800, more preferably no more than 750, even more preferably no more than 700, yet more preferably no more than 690, even more preferably no more than 680, yet more preferably no more than 670 consecutive amino acids of LOX-1, such as no more than 665, for example no more than 650, such as no more than 600, for example no more than 550, such as no more than 500, for example no more than 450, such as no more than 425, for example no more than 399 consecutive amino acids of LOX-1 of [SEQ ID NO: 3]. Preferably, said truncated form comprises only an N-terminal fragment of LOX-1. Hence, preferably said truncated form comprises at the most the 800, more preferably at the most the 750, even more preferably at the most the 700, yet more preferably at the most the 690, even more preferably at the most the 680, yet more preferably at the most the 670, even more preferably at the most the 665 N-terminal amino acids of [SEQ ID NO: 3], such as no more than 665, for example no more than 650, such as no more than 600, for example at the most the 550, such as at the most the 500, for example at the most the 450, such as at the most the 425, for example at the most the 399 N-terminal amino acids of [SEQ ID NO: 3].

In one very preferred embodiment, the truncated form may consist of amino acid 1-665 of [SEQ ID NO: 3].

In a preferred embodiment of the invention, the barley plant comprises a gene transcribed into mRNA encoding LOX-1, wherein said mRNA comprises a nonsense codon or a stop codon upstream of the stop codon of wild-type LOX-1 mRNA. Such a nonsense codon is herein designated a premature nonsense codon. Preferably all genes transcribed into mRNA encoding LOX-1 of said plant comprise a premature non-sense codon or a stop codon. The non-sense codon or stop codon is preferably situated at the most 800, more preferably at the most the 750, even more preferably at the most the 700, yet more preferably at the most the 690, even more preferably at the most the 680, yet more preferably at the most the 670, even more preferably at the most the 665 codons down-stream of the start codon. The sequence of wild-type genomic DNA encoding LOX-1 is given in [SEQ ID NO: 1] or [SEQ ID NO: 5].

In one preferred embodiment the barley plant comprises a gene encoding LOX-1, wherein pre-mRNA transcribed from said gene comprises the ribonucleic acid sequence corresponding to [SEQ ID NO: 2].

In another preferred embodiment of the invention, the barley plant comprises a gene encoding mutant LOX-1 wherein said gene comprises at least one, such as 1, for example 2, such as 3, for example 4, such as 5 mutations in at least one, such as 1, for example 2, such as 3 splice sites. Preferably, said mutation(s) results in that said at least one splice site is non-functional. mRNA transcribed from such a gene will thus be abnormal due to aberrant splicing. Accordingly, it is preferred that mRNA transcribed from the LOX-1 gene of the null-LOX-1 barley plant according to the invention encodes no protein or a protein comprising only the N-terminus of LOX-1. Said protein may comprise other sequences encoded by the abnormal mRNA, which are not derived from the gene for LOX-1. In this context, the N-terminus of LOX-1 comprises amino acid 1 to amino acid N, wherein N is an integer in the range of 2 to 800, more preferably in the range of 2 to 750, yet more preferably in the range of 2 to 700, even more preferably in the range of 2 to 650, yet more preferably in the range of 2 to 600, even more preferably in the range of 2 to 550, yet more preferably in the range of 2 to 500, yet more preferably in the range of 2 to 450, even more preferably in the range of 2 to 400, yet more preferably in the range of 2 to 378.

In one embodiment of the invention the barley plant comprises a gene encoding a mutant LOX-1, wherein said gene has a mutation in a splice site leading to mRNA encoding a protein consisting of amino acids 1 to 378 of [SEQ ID NO: 3] as well as an additional amino acid sequence not derived from LOX-1. Preferably, said mutant LOX-1 consists of the sequence as outlined in [SEQ ID NO: 8].

In a very preferred embodiment of the invention the gene encoding mutant LOX-1 of the null-LOX-1 barley plant comprises a nonsense mutation, said mutation corresponding to a G.fwdarw.A substition at position 3574 of [SEQ ID NO: 1]. More preferably the null-LOX-1 barley plant is a plant designated D112 having American Type Culture Collection (ATCC) deposit accession No. PTA-5487.

In another very preferred embodiment of the invention the gene encoding LOX-1 of the null-LOX-1 barley plant comprises a non-functional intron 3 donor splice site. LOX-1 mRNA of said plant thus encodes a protein containing amino acids 1-378 of LOX-1 and additional amino acids from intron 3, comprised in [SEQ ID NO: 8]. More preferably, the null-LOX-1 barley plant is a plant designated A618 having American Type Culture Collection (ATCC) deposit accession No PTA-5584.

The barley plants according to the invention may also be the progeny of a null-LOX barley plant. Hence, the barley plant may be the progeny of the plant designated D112 having ATCC deposit accession No. PTA-5487 or the plant designated A618 having ATCC deposit accession No. PTA-5584.

The barley plant according to the invention may be prepared by any suitable method known to the person skilled in the art, preferably by one of the methods outlined herein below (see for example Section 6.2 "Preparing null-LOX-1 barley").

In one embodiment of the invention it is preferred that the null-LOX-1 barley plant according to the present invention has plant growth physiology and grain development comparable to wild-type barley. It is hence preferred that the null-LOX-1 barley plant is similar to wild-type barley in respect of plant height, number of tillers per plant, onset of flowering and/or number of grains per spike.

It is also an aspect of the invention to provide a null-LOX-1 barley plant, wherein said plant is characterized by: (i) having enhanced disease resistance; or (ii) having reduced potential for the production of mycotoxins; or (iii) comprising regenerable cells for use in tissue culture; or (iv) any combination of the traits of (i) to (iii). 6.2 Preparing Null-LOX-1 Barley

The barley plant according to the invention may be prepared by any suitable method known to the person skilled in the art. Preferably, the barley plant of the invention is prepared by a method comprising the steps of mutagenizing barley plants or parts thereof, for example barley kernels, followed by screening and selecting barley plants for plants with less than 5% LOX-1 activity. Interestingly, the present invention in one aspect relates to a new and very efficient screening method, significantly superior to the screening method described in for example WO 02/053721 to Douma et al. The new screening method allows reproducibly to identify barley plants with no or very little LOX-1 activity. This new screening method includes obtaining kernels or parts thereof, such as embryos, from mutagenized barley and determining the LOX-1 activity in said kernels or parts thereof.

Accordingly, it is an objective of the present invention to provide methods of preparing a barley plant comprising less than 5% of the LOX-1 activity of a wild-type barley plant comprising the steps of: (i) Determining the LOX-1 activity in wild-type barley kernels or parts thereof; and (ii) Mutagenizing barley plants and/or barley cells and/or barley tissue and/or barley kernels and/or barley embryos thereby obtaining generation M0 barley; and (iii) Breeding said mutagenized barley plants, kernels and/or embryos for at least 2 generations, thereby obtaining generation Mx barley plants, wherein x is an integer .gtoreq.2; and (iv) Obtaining kernels or parts thereof from said Mx barley plants; and (v) Determining the LOX-1 activity in said kernels or parts thereof; and (vi) Selecting plants wherein the LOX-1 activity of the mutagenized kernels or parts thereof is less than 5% than the LOX-1 activity of the wild-type kernels or part thereof; thereby obtaining a barley plant comprising less than 5% of the LOX-1 activity of a wild-type barley plant.

Step (ii) in the above list may involve mutagenizing living material selected from the group consisting of barley plants, barley cells, barley tissue, barley kernels and barley embryos, preferably selected from the group consisting of barley plants, barley kernels and barley embryos, more preferably barley kernels. It is preferred that the LOX-1 activity of mutagenized kernels is determined using the same kind of material as that used for the determination of the LOX-1 activity of wild-type barley kernels, i.e it is preferred that the barley kernel or parts thereof of step (i) is the same kind of barley kernel or parts thereof as that mentioned in step (iv). By way of example, if the LOX-1 activity of wild-type barley is determined in embryos of wild-type barley, it is preferred that step (iv) comprises determining LOX-1 activity in embryos of mutagenized barley plants.

Mutagenesis may be performed by any suitable method. In one embodiment, mutagenesis is performed by incubating a barley plant or a part thereof, for example barley kernels or individual cells from barley with a mutagenizing agent. Said agent is known to the person skilled in the art, for example, but not limited to sodium azide (NaN.sub.3), ethyl methanesulfonate (EMS), azidoglycerol (AG, 3-azido-1,2-propanediol), methyl nitrosourea (MNU), and maleic hydrazide (MH).

In another embodiment, mutagenesis is performed by irradiating, for example by UV, a barley plant or a part thereof, such as the kernel. In preferred embodiments of the invention the mutagenesis is performed according to any of the methods outlined herein below in Section 6.4 "Chemical mutagenesis." A non-limiting example of a suitable mutagenesis protocol is given in Example 1.

It is preferred that the mutagenesis is performed in a manner such that the expected frequency of desired mutants is at least 0.5, such as in the range of 0.5 to 5, for example in the range of 0.9 to 2.3 per 10,000 grains, when screening M3 barley.

In a preferred embodiment, mutagenesis is performed on barley kernels. The mutagenized kernels are designated generation MO (see also FIG. 1A).

Subsequent to mutagenesis, barley plants, or parts thereof, that comprise less than 5%, preferably less than 1% LOX-1 activity are selected. Selection may be performed according to any suitable method known to the person skilled in the art. Preferably, selection comprises obtaining a sample from a barley plant, such as from a barley kernel, determining the activity of LOX-1 in said sample and selecting plants, wherein said sample has less than 5%, or preferably less than 1% of the LOX-1 activity of a wild-type barley plants.

The sample may be taken from any suitable part of said plant. Preferably, however, the sample is taken from the kernel, more preferably the sample is taken from the embryo tissue of a kernel, yet more preferably the sample consists of embryo tissue of a kernel. In general, the sample must be homogenized using any suitable method prior to determining the LOX-1 activity.

In a preferred embodiment, the sample is taken from generation Mx kernels, wherein x is an integer .gtoreq.2, preferably x is an integer in the range of 2 to 10, more preferably in the range of 3 to 8. In a very preferred embodiment LOX-1 activity is determined on M3 kernels or a sample derived from kernels. In that embodiment, it is preferred that mutagenised barley kernels (generation M0) are grown to obtain barley plants which are crossed to obtain kernels M1. The procedure is repeated until M3 kernels are available (see also FIG. 1A).

Determination of LOX-1 activity may be carried out using any suitable assay, preferably by one of the methods outlined herein below. In particular, it is preferred that the assay monitors the dioxygenation of linoleic acid to 9-HPODE by LOX-1. In general, assaying will therefore involve the steps of: (i) Providing a sample prepared from a barley kernel or part thereof; and (ii) Providing linoleic acid; and (iii) Incubating said sample with said linoleic acid; and (iv) Detecting dioxygenation of linoleic acid to 9-HPODE.

Detection may be performed directly or indirectly. Any suitable detection method may be used with the present invention. In one embodiment of the invention, linoleic acid hydroperoxides are detected. Linoleic acid hydroperoxides may for example be detected by coupling degradation of said linoleic acid hydroperoxides with an oxidative reaction, which develops a detectable compound. For example, this may be done as described in Example 1. In another embodiment 9-HPODE is detected directly, for example by spectrophotometric methods, such as HPLC as described in Example 9. In one embodiment of the invention, LOX-1 activity is determined simply by determining the amount of 9-HPODE in a sample from a barley kernel. This may be done by any suitable method known to the person skilled in the art, for example as outlined in Example 9.

It is important at what pH the determination of LOX-1 activity is performed. Preferably, said determination is performed at a pH which allows high activity of LOX-1, but only low activity of LOX-2. Hence, determination of LOX activity is preferably done at a pH in the range of 3 to 6.5, for example in the range of 3 to 4, such as in the range of 4 to 5, for example in the range of 5 to 6, such as in the range of 6 to 6.5. Preferably, the pH is around 3, such as around 3.5, for example around 4, such as around 4.5, for example around 5, such as around 5.5, for example around 6, such as around 6.5, for example around 7. Preferred methods for selecting barley plants according to the invention are described herein below in the Section 6.5 "Selecting barley mutants."

A preferred example of a method for determination of LOX-1 activity is given in Example 1.

The selection procedure may be adapted for microtitre plate assay procedures, or other known repetitive, high-throughput assay formats that allow the rapid screening of many samples. It is preferred that at least 5,000, such as at least 7,500, for example at least 10,000, such as at least 15,000 mutagenized barley plants are analyzed for LOX-1 activity.

Subsequent to the selection of useful barley plants with less than 5% LOX-1 activity, one or more additional screenings may optionally be performed. For example, selected mutants may be further propagated, and subsequent generations may be screened again for LOX-1 activity.

Subsequent to selection of useful barley plants, these may be subjected to breeding, such as conventional breeding. Methods of breeding are described herein below (Section 6.6 "Plant breeding" and Section 6.7 "Barley crossings").

The barley plant according to the invention may, however,