INFORMATION SYSTEMS FOR BIOTECHNOLOGY


August 2003
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:



EMERGING TERMINOLOGY – WORDS ARE NEVER ENOUGH
Zac Hanley, Kieran Elborough

In a recent article, Nielsen implies that words fail us. Specifically, he argues that a failure to develop an appropriate, agreed terminology early in the debate over the acceptance and adoption of genetically modified organisms (GMOs) in agriculture has impeded public understanding, to say nothing of approval. Applying advanced biology to agriculture requires `consumer-pull' and the research community has failed to reflect the breadth and diversity of its technical successes with sufficiently clear concepts, metaphors, and explanation. Without this, the debate is too simplified, and as a result polarized. Nielsen believes that the current GMO categorizations are importantly uninformative. What does it mean when something is labeled genetically modified? Are some kinds of genetic modification more preferable than others, but obscured by careless overuse of broad terms like genetically modified and transgenic? Can we draw any useful distinctions if we are prepared to expand our vocabularies?

Roses by other names
To serve his aim, Nielsen differentiates five types of genetic modification and coins four new terms (he is then able to rewrite the definition of transgenic). He builds his argument on a foundation concept that could be called `genetic distance' —how far has the heritable material in question `traveled' to `arrive' in the GMO under consideration? A gene that has been rearranged or rewritten within a genome, perhaps through breeding, mutation, or sexual recombination, has traveled no distance at all; the resulting `non-transgenic GMO' he calls intragenic. Genes introduced to an organism from closely related species via more advanced breeding techniques or genetic engineering are famigenic. Linegenic organisms contain heritable material from species of the same phylogenetic lineage but separated by a species barrier that prevents classical breeding. A transgenic organism contains DNA from an unrelated species. Lastly, xenogenic refers to heritable material that arises from wholly artificial means, for example synthetic promoters.

Nielsen's attempt is not the final word, and he speaks of "further refined taxonomy-based nomenclature." Nor is it the first—there have been several other attempts to introduce a practical vocabulary along such lines. A longstanding concept in breeding is that of levels within the gene pool, where the primary gene pool corresponds to the traditional concept of a biological species and the secondary gene pool includes all species that will cross with that species in question. Beyond these and of relevance to GMO work, the tertiary gene pool represents the maximum reach of high-tech breeding (perhaps via embryo rescue, protoplast fusion, etc.) while the quaternary gene pool contains heritable material from all other species. This last level can only be accessed by genetic engineers with recombinant, in vitro methods. Over the last thirty years these demarcations have shifted and blurred in response to changes in available technology, but in outline they map moderately well onto Nielsen's intra-, fami-, line- and trans-genic definitions. More recently, Tester has proposed the terms tweaking (equivalent to intragenic manipulations), close transfer (equivalent to famigenic transfers within the secondary gene pool), and wide transfer (everything else). All three systems are equivalent in their implicit ranking by `genetic distance'. But are such systems necessary, sufficient, or at all useful?

Nielsen seeks to rescue the current variety of available technologies and products from the ignominy of a single tarnished label—transgenic. Tester is attempting to overcome a similar unhelpful vagueness in genetic modification. But these are rearguard actions: Transgenic for example is formed from `transgenosis', defined as "bacterial gene transfer and subsequent expression in eukaryotic cells." In other words, transgenic was initially defined, within the limited concepts and technology of the time, more or less exactly as Nielsen (re)defines it. In the last thirty years the definition has slipped, or rather spread, to encompass all in vitro manipulations of heritable material. This slippage suggests that resistance is futile: Transgenic has a well-entrenched meaning to the public and attempts to redefine it spread confusion or suspicion rather than enlightenment. Definitions are descriptions of an evolving consensus and are not available for renegotiation under most circumstances. It is clear that the biological R&D community has failed to educate, that is, to explain its concepts in meaningful ways. Debate has indeed been skewed as a result, but the process will not be deflected by legislating new word meanings. Nielsen's offerings seem likely to us to be understood by many as quibbling between `degrees of unnaturalness'. This is far from his intent but is understandable. There are also solid reasons why the biotechnology community will not adopt Nielsen's or Tester's proposals either.

Developments defying description
Our species has been ingenious in accessing the available or theoretical variety in, for example, crop species using breeding, mutation, artificial selection, hybridization, embryo rescue, transgenesis, and more. Our latest techniques do not fit into any of the proposed schemes. Now, early in the biotechnology era, it is merely difficult to shoehorn most of the GMOs on release into categories based on genetic distance. The scientific, commercial, and regulatory environment ensures that the most difficult, expensive, and novel processes are employed only to access the quaternary gene pool to create transgenics. But soon such shoehorning will be impossible.

Consider work by Zhang and coworkers. In unmodified tobacco, their gene of interest (coding region and associated regulatory elements) is located in the nucleus and encodes a prepeptide, requiring processing by chloroplast import machinery for activation. For their experiments they coupled sequence encoding mature peptide with chloroplast regulatory sequences and inserted this construct directly into the chloroplast genome. Active enzyme was manufactured without transport or processing. What genetic distance has been traversed in this instance? The DNA had moved only within the cell—such tweaking represents an intragenic event. However it is widely accepted that the chloroplast is descended from cyanobacteria—have Zhang and coworkers then created a transgenic by wide transfer from the quaternary gene pool? Alternatively, the exact combination of precise transfer, coding region truncation, and appropriate regulation achieved in vitro has probably never existed before and would be unlikely to evolve exactly as seen—is it therefore xenogenic?

This is not hairsplitting; it is a deficit in all of the classification schemes. Antisense and RNAi, gene shuffling in vitro or in vivo, BIBACs, artificial genetic codes, even the routine use of the viral 35S promoter in overexpression studies, all could be called xenogenic by some, transgenic/wide transfer by others, even intragenic/tweaking by a few. Which gene pools are being accessed in each case? Worse, the widespread use of marker genes derived from bacteria apparently renders most GMOs transgenic or xenogenic, containing wide transfers from the quaternary gene pool. In summary, all assignments are subjective. Such categories cannot survive in science any more than in education or in the GMO debate. They may of course serve other purposes, for example as marketing tools intended to differentiate biotechnology producers from processes or products, or as loose jargon in some scientific communication contexts.

Actions speak louder
Nielsen avows that finer degrees of categorization better equip the public to articulate its concerns. The public is thereby empowered and the biotechnology industry is appropriately congratulated for open, honest, transparent communication. But in all of the offered systems, suggested categories are too one-dimensional to capture the true diversity of the technologies, and too subjective to raise the level of the debate.

The answer is not to devise or extend such systems in an effort to anticipate every existing and emerging technology. Instead, we must recognize that the debate over the acceptability of GMOs is not based on determining a publicly agreed maximum genetic distance over which genes may be transferred. Objections are more commonly based on the complex politics of agriculture, competitive friction with other food brands such as `organic', and environmental protection. Terminology is not in short supply, and it is not clear how to bridge the philosophical incompatibilities that one commentator has diagnosed as the fundamental fracture point in the GMO debate. This is, however, irrelevant: Between the extremes is a large public which, as above, wants to be empowered, to understand the what and the why of biotechnology, and needs more information.

More than fifty years of theory and empirical testing have shown that education does not begin with the memorizing of specialist jargon. New knowledge can only be assimilated when it fits with and builds on concepts already understood, and the learner must first be involved and provided with context and an overview. In a 1999 survey approximately a third of Europeans still believed that only genetically engineered tomatoes contain genes; articles offering exotic terminology and unfamiliar concepts such as famigenic and tertiary gene pools to the public are falling on stony ground. All commentators, including the advocates of these new terminologies, recognize the need to educate and inform, but almost all severely underestimate the degree to which the science community has failed in the past to do so. Only when we have done an excellent job of explaining what we are talking about can we enjoy the luxury of encapsulating it all in a pithy word or phrase like intragenic. Such simplicity comes after much hard work on the details. Until that point, emerging technologies will always make emerging terminologies obsolete.

References

1. Nielsen. (2003) Transgenic Organisms—Time For Conceptual Diversification? Nature Biotechnology 21(3): 227-8.

2. Bhullar et al. (2003) Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiology 132: 988-98.

3. Harlan & de Wet. (1971) Toward a rational classification of cultivated plants. Taxonomy 20: 509-17.

4. An example is Abedinia et al. (2000) Accessing genes in the tertiary gene pool of rice by direct introduction of total DNA from Zizania palustris (Wild rice). Plant Molecular Biology Reporter 18(2): 133-8.

5. Tester. (1999) Seeking clarity in the debate over the safety of GM foods. Nature 402: 575.

6. The Oxford English Dictionary 2nd edition reports the first usage as Doy et al. (1973) Biological and molecular evidence for transgenosis of genes from bacteria to plant cells. PNAS 70: 723-6.

7. Zhang et al. (2001) Targeting a nuclear anthranilate synthase a-subunit gene to the tobacco plastid genome results in enhance tryptophan biosynthesis, return of a gene to its pre-endosymbiotic origin. Plant Physiology 127(1): 131-41.

8. Saner. (2001) Real and metaphorical moral limits in the biotech debate. Nature Biotechnology 19(7): 609.

9. Press release available at
http://europa.eu.int/comm/research/press/2000/pr2704en-ann2.html.

Zac Hanley, Kieran Elborough
Consultants in Plant Biotechnology
New Zealand
biotech@greengenz.com



HOW TO IMPROVE PLANT PROTEIN QUALITY: ERR ON THE SIDE OF GOODNESS
William R. Folk

During the past quarter century, improvements in cereal crop yields have significantly lessened, and this is likely to anticipate future food shortages—for greater demands are being made upon cereal crop production by increases in population and livestock and the concentration of animal production systems. Not adequately responding to these needs will prove to be costly and painful. Already we are witnessing the deforestation and degradation of our lands, the pollution of our waterways, and the development and spread of diseases caused by unsafe agricultural production systems. Appropriate applications of biotechnology can help address these problems by improving cereal crop productivity and nutritive value.

We must enrich the proteins of our important crops with lysine, threonine, methionine, and tryptophan, as they limit the growth of humans and many livestock and limit efficient utilization of the more abundant amino acids in our foods and feeds. Ideally, the proteins of the most important crops should be tailored to the specific dietary needs of the humans and livestock for which they are used.

We have developed a biotechnology for improving plant seed protein quality1. The method involves altering the fidelity of protein synthesis so as to cause substitution of the nutritionally limiting amino acids for more prevalent amino acids in seed storage proteins. This approach has advantages over several others that have been attempted, such as expressing high levels of a foreign protein with the requisite amino acids, for it only requires expression in plants of altered plant tRNAs that promote the desired amino acid substitutions. In our recent study, we utilized altered plant tRNAlys species that code lysine at alternative codons to increase the content of lysine in rice grain. The same approach should also work with tRNAs that code threonine, methionine, and tryptophan to increase the content of these amino acids in plant proteins. Importantly, this is not an artificial, totally new process, nor does it require expression of new genes and proteins that might cause concern about safety.

Protein synthesis naturally incurs some errors, and different species use this infidelity to their own particular advantage. For example, viruses utilize suppression of nonsense codons by tRNAs during protein synthesis in plant cells to expand their coding capacity, in order to make additional proteins with properties important for replication2. Errors in protein synthesis and the natural sequence variability of plant seed storage genes cause seed storage proteins to be very heterogeneous. Our approach employs expanding this naturally occurring infidelity and protein sequence heterogeneity to improve nutritional quality of the seeds and grains that form the basis of most human and animal nutrition. By limiting the substitutions in these proteins to relatively conservative replacements, protein expression or structure is not greatly impaired. As no single modified protein is expressed at high level, the allergenicity or other properties of food and feeds that are important for safety and public acceptance should not be affected.

A question that immediately comes to mind is what effect enhancement of the frequency of errors during protein synthesis might have upon the biological fitness of plants. Most proteins are very tolerant of amino acid substitutions, as long as the errors do not alter residues that are involved in folding or catalysis3. Seed storage proteins, because of their natural heterogeneity, may be particularly amenable to modification by errors in protein synthesis. However, too many errors might cause particularly detrimental agronomic properties because other proteins required for growth or reproduction might be affected.

If the frequency of errors is kept below the threshold that reduces agronomic quality, a reduction in fitness can be advantageous, as it will prevent plants altered by the method to escape and to be perpetuated in the wild4. This important principle of using biological control to minimize the risk of spread of bacteria containing recombinant DNAs has been universally accepted and practiced for many years, and can serve equally well to minimize the dissemination of plants that contain recombinant DNAs.

We determined there to be a "window of opportunity" in which sufficient substitution of nutritionally limiting amino acids can occur during protein synthesis in rice plants to affect an improvement of protein quality, without noticeably damaging plant reproduction or growth1. Using microprojectile bombardment, we introduced, into rice callus, DNAs encoding altered tRNAlys species that cause lysine to be introduced in place of amino acids that are particularly abundant in seeds: glutamine, asparagine, and glutamic acid. Also, DNAs encoding altered tRNAlys species that cause lysine to be incorporated at chain termination codons, which normally end protein synthesis or that frequently interrupt protein synthesis of seed storage proteins, were introduced into calli. Rice plants were regenerated from these calli and the substituted lysines in the seed storage proteins were detected by antibodies against the modified seed storage protein glutelins, and by amino acid analyses of the prolamins (the predominant seed storage proteins) and of the intact rice grains. No significant changes in the types of proteins in the grains were detected. This contrasts with the significant effects upon seed storage proteins of the opaque 2 (o2) mutation that is the basis of Quality Protein Maize5.

Substantial amounts of lysine substitution were achieved. In some plants 2-3% of the glutelins were modified, with 40-75% increases in lysine in their prolamins. A 6% overall increase in lysine content was achieved in the grains of these rice plants, which is greater than that achieved by other methods, including random mutagenesis. Errors occurring during protein synthesis in these plants had no obvious effect upon their growth or reproduction in greenhouse cultivation, but field tests are required to fully assess their agronomic yields and to determine their biological fitness.

In principle, introduction of DNAs with the altered tRNA genes is not necessary to achieve an equivalent result. Site-specific mutagenesis of the endogenous tRNA genes of plants most likely will produce similar altered coding; but there are presently no methods to achieve such targeted changes. If such methods become available, then it may not be necessary to introduce additional DNAs into plants to change them.

The increased content of lysine in the rice of these plants is still not sufficient to provide the quantity of lysine required for optimal human and animal nutrition. Further improvements should be possible by combining different types of altered tRNAlys in one plant, by promoting the utilization of the altered tRNAs during seed protein synthesis, and possibly by enhancing the synthesis of lysine. These approaches are currently being explored.

It is likely that the protein quality of other major crops upon which the world relies for food and feed can also be nutritionally improved by application of this method. Glutelins, the major storage proteins of rice, contain higher amounts of lysine than prolamins, the major storage proteins of maize, sorghum, and pearl millet, crops that are particularly important in semi-arid parts of the world where sources of complementary plant proteins are difficult to obtain. If the introduction of altered tRNAs into these crops achieves comparable increases in lysine, as has been observed for rice, the protein nutritive quality of these important crops will also be very significantly improved.

Acknowledgements: I thank members of my laboratory, particularly Dr. Xing Rong Wu, Dr. Zhi Hong Chen and Sarah Scanlon for their assistance and advice. Financial support was provided by the USDA and the University of Missouri.

References

1. Wu XR, Chen ZH, and Folk WR. (2003) Enrichment of cereal protein lysine content by altered tRNAlys during protein synthesis. Plant Biotechnology Journal 1:187-194; http://www.blackwell-synergy.com/links/doi/10.1046/j.1467-7652.2003.00017.x/abs/

2. Beier H and Grimm M. (2001) Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. Nucleic Acids Research 29: 4767-4782.

3. Bowie JU, Reidhaar-Olson JF, Lim WA, Sauer RT. (1990) Deciphering the message in protein sequences: Tolerance to amino acid substitutions. Science 247: 1306-1310.

4. Burke JH, Rieseberg LH. (2003) Fitness effects of transgenic disease resistance in sunflowers. Science 300: 1250.

5. Hunter BG, Beatty MK, Singletary GW, Hamaker BR, Dilkes BP, Larkins BA, Jung R. (2002) Maize opaque endosperm mutations create extensive changes in patterns of gene expression. The Plant Cell 14: 2591-2612.

William R. Folk
Professor of Biochemistry
University of Missouri-Columbia
folkw@missouri.edu


ENGINEERING FOR REDUCED SATURATED FATTY ACIDS IN OILSEED CROPS
Tawanda Zidenga

Oilseed crops have been bred by conventional methods over many years to improve yields and fatty acid composition. The levels of different fatty acids in the crop influence how it is used. By genetically modifying the biochemical pathways leading to the production of fatty acids, scientists hope to produce crops with fatty acid profiles that meet specific uses and purposes.

Plant seed oils are usually evaluated according to the level of saturated fatty acids in them. For example, canola has the lowest level of 7%, peanut oil has 17%, palm oil 49% and coconut oil has saturated fatty acid levels as high as 86%. Saturated fat tends to increase blood cholesterol levels, and high levels of blood cholesterol have been linked to an increase in coronary heart disease risk. One method of breeding for reduced fatty acid in canola is to induce mutations in microspores and select those plants that show lower levels of saturated fatty acids. However, this method involves more chance and less precision. One of the challenges of modern biotechnology is the reduction of saturated fatty acids in oilseed crops through metabolic engineering. This demands an understanding of the complex biochemical pathways that lead to the biosynthesis of fatty acids in plants.

In a recent study published in the May issue of the Plant Biotechnology Journal, Yao and colleagues from Saskatchewan, Canada, have reported that the Arabidopsis ADS1 gene encodes a fatty acid D9 desaturase that could be useful in genetic engineering for modifying the level of fatty acids in oilseed crops1. Prior to their publication, it had been confirmed that the heterologous over-expression of an acyl-CoA D9 desaturase gene in plants can modify the level of fatty acid saturation. A modification of fatty acid composition in tobacco was achieved through over-expression of the yeast D9 acyl-CoA desaturase gene (OLE1) in tobacco plants.2 Similar results were also obtained in transgenic tobacco seeds following the over-expression of a rat liver stearoyl-CoA D9 desaturase gene (RDS)3.

In their study, Yao and colleagues aimed to identify the function of the Arabidopsis ADS1 gene by transgenic expression in Brassica juncea, followed by analysis of the fatty acid composition of the transgenic lines. Their data pointed to the fatty acid desaturase activity of the ADS1 gene, therefore making it a candidate gene in the transgenic modification of saturated fatty acids in oilseed crops. Brassica juncea hypocotyls were transformed with Agrobacterium tumefaciens strain GV3101 bearing plasmid pRB01, which contained one Arabidopsis ADS1 cDNA under the control of the seed-specific napin promoter and also the kanamycin resistance gene, NPTII, as a selectable marker. Of the 49 putative transformants, 33 were determined to be positive by PCR analysis of the leaf genomic DNA using primer pairs NPT1 and NPT2 to amplify the NPTII coding sequence. Lines failing to produce the amplification product were marked as non-transformed control lines. Results of PCR analysis were confirmed by Southern blotting.

Both the negative and positive lines were grown under greenhouse conditions and allowed to self-pollinate. The mature seeds at harvesting (T1 generation) were analyzed for the fatty acid composition. Saturated fatty acids levels were significantly reduced in the positive transgenic lines, ranging from about 1% to 2%. This decrease was mainly attributed to a decrease in palmitic and stearic acids, although other saturated fatty acids (arachidic acid, behenic acid, and lignoceric acid) were also slightly reduced. Furthermore, in the transgenic lines, the decrease in stearic acid was accompanied by a slight increase in oleic acid, a monounsaturated fatty acid that lowers cholesterol in human blood plasma. The results suggested that the ADS1 gene product may preferentially use stearic acid as the substrate for its desaturation. It is therefore advantageous to use the ADS1 gene in genetic engineering for oilseed crop improvement because of its substrate specificity and accompanying positive effect on the levels of oleic acid.

It was important to confirm that the reduction in fatty acid levels was due to transgenesis. To achieve this, the researchers used RT-PCR, a technique that obtains cDNA by reverse transcriptase followed by PCR amplification. The ADS1 gene was amplified from all the selected positive transgenic lines while no RT-PCR product was detectable from the non-transformed control lines. The performance of the ADS1 transgene was further evaluated under field conditions, and it had the same effect of reducing the level of saturated fatty acids.

This study therefore provides an option in genetic engineering for the modification of fatty acids in oilseed crops. More work still needs to be done to understand the complex process of fatty acid and oil biosynthesis in crops. Also, it will be necessary to do comparisons of the available candidate genes, such as OLE1, RDS, and ADS1 to see which is appropriate in a particular crop and situation. In addition, fatty acids play a wide range of roles in plants, including storage and membrane integrity. They also play a role in metabolic regulation. Modifying the fatty acid composition therefore can affect the functions of the whole plant, and detailed studies will be required to prevent negative effects of the technology.

Sources

1. Yao K et al. (2003) Expression of the Arabidopsis ADS1 gene in Brassica juncea results in a decreased level of total saturated fatty acids. Plant Biotechnology Journal 1: 221-229.

2. Polashock JJ, Chin CK, and Martin CE. (1992) Expression of the yeast D9 fatty acid desaturase in Nicotiana tabacum. Plant Physiol. 100: 894-901.

3. Moon H, Hazebroek J, and Hilderbrand DF. (2000) Changes in fatty acid composition in plant tissues expressing a mammalian D9 desaturase. Lipids 35: 471-479.

Tawanda Zidenga
Crop Science Department
University of Zimbabwe
tawandazidenga@justice.com


TRANSGENIC ASPEN TREES WITH ALTERED LIGNIFICATION: GOOD NEWS FOR PULP AND PAPER INDUSTRY
P. Janaki Krishna

A major component in all trees is `lignin', which is a complex compound that resists digestion by animals and impairs preparation of paper from wood. In angiosperm trees, lignin is produced through polymerization of guaiacyl and syringyl monolignols with a syringyl/guaiacyl (S/G) ratio of <2-2.5. Wood pulping kinetics show that increasing the lignin S/G ratio by one unit would approximately double the rate of removal of lignin. This indicates that wood pulp production is based on lignin quantity and reactivity (i.e., S/G constituent ratio).

The desire to remove or reduce lignin has prompted tree biotechnologists to focus research efforts on lowering lignin quantity and increasing lignin reactivity by either knocking out specific genes that produce crucial precursors for lignin or introducing genes that suppress the lignin production. However, neither the genetic reduction of lignin nor genetic transformation through suppression of monolignol pathway has had a significant effect on S/G ratio. Efforts to modify S/G ratio in trees have not succeeded in reducing lignin, thus suggesting that lignin quantity and the S/G ratio are regulated independently in the lignin biosynthetic pathway.

Reports indicate that the syringyl monolignol pathway in angiosperm trees is controlled by three genes, encoding coniferaldehyde 5-hydroxylase (CAld5H), 5-hydroxy-coniferaldehyde O-methyltransferase, and sinapyl alcohol dehydrogenase. Of these, CAld5H plays a key role in monolignol biosynthesis and the lignin S/G ratio. However, multigene modifications through conventional breeding techniques in trees that have long life cycles are very difficult.

A group of scientists from North Carolina State University (Raleigh, NC) and the US Dairy Forage Research Center (Madison, WI) recently reported the modification of multiple lignin traits in aspen (Populus tremuloides) using an Agrobacterium-mediated cotransformation system. The multigene cotransformation system was derived using four pB1101-based binary vectors containing a CaMV 35S- driven monolignol pathway cDNA sequence. About 48 independent transgenic plants were regenerated after cocultivating leaf tissues with a mixture of four independent Agrobacterium strains. Out of 48 transgenics, 35%, 27%, 19%, and 19% contained one, two, three, and four trans-gene constructs, respectively. The team then applied this system in aspen to manipulate lignin content and S/G ratio.

The transformation of aspen with multiple genes was performed using an aspen xylem-specific promoter-GUS binary plasmid DNA. Subsequently the GUS fragment was replaced with either an antisense Pt4CL1 or a sense LsCAld5H cDNA with respect to the xylem-specific promoter (Pt4CL1P). These constructs were transferred to A. tumefaciens C58 strain and used to coinoculate aspen leaf discs to produce transgenic trees. Of the forty phenotypically normal transgenic lines produced, 37%, 40%, and 23% contained antisense Pt4CL, sense LsCAld5H, and antisense Pt4CL + sense LsCAld5H gene constructs, respectively. These plants were grown independently in a greenhouse and 10-month-old trees from each group were selected randomly during the growing season to study various characteristics. Wild type aspen trees derived from in vitro micropropagation were used as the control.

Analyses were performed to determine lignin histochemistry, protein immunolocalization, and lignin and carbohydrate characterization on tree clones expressing low lignin, a high S/G ratio, a high cellulose/lignin ratio, or a combination of these traits. The Cald5H-up-regulated transgenic trees had accelerated maturation (wall thickening) and lignification of secondary xylem cells. The results indicated a reduction of lignin content in transgenics by 45% to 50%, an increase of cellulose by 30%, and higher S/G and cellulose/lignin ratios. The transgenic trees also grew faster, thus demonstrating the first successful dual-gene alteration achieved through genetic transformation.

There is a need for more research to study field performance, real wood properties, resistance to insects and diseases, and the possibility of unforeseen ecological impacts of transgenic trees. However, the multigene cotransformation approach in aspen reported by this team may have a positive impact on the genetic improvement of forest trees for pulp and paper production. The results suggest that this multigene cotransformation system would be broadly useful for tree genetic engineering and functional genomics. High-yield plantations with these desirable traits will enable more efficient wood production.

References

1. Laigeng Li et al. (2003) Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. PNAS 100(8): 4939-4944.

2. Transgenic trees hold promise for pulp and paper. North Carolina State University News Release. http://www.sciencedaily.com/releases/2003/04/030408090203.htm

3. Giller Pilate et al. (2002) Field and pulping performance of transgenic trees with altered lignification. Nature Biotechnology 20(6): 607-612.

P. Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise
Hyderabad, India
jankrisp@yahoo.com


LACCASE TRANSFECTION FOR ANTIFUNGAL PROPERTIES IN CROPS
Brian R. Shmaefsky

Leaf spot disease caused by the fungus Cercospora is most noted for its economic impact on sugar beet production. However, members of this deuteromycete genus destroy other crops including alfalfa, asparagus bean, avocado, banana, cassava, chard, celery, coffee, corn, cucumber, fig, garden bean, garden beet, grape, lettuce, papaya, parsley, peanut, radish, soybean, spinach, strawberry, sugar cane, sweet pepper, tobacco, and taro. Many ornamental plants and turf grass also get leaf spot from Cercospora species. Unfortunately, the fungus resides in various plants that are common agricultural and turf weeds.

Robert Lartey and TheCan Caesar-TonThat of the Agricultural Research Service in Sidney, Montana, found a feasible way of controlling leaf spot on sugar beets without the traditional reliance on fungicides. They restricted the pathogenesis of the fungus by degrading its cercosporin toxin with the enzyme laccase1. Cercosporin toxin damages the plant when, in the presence of oxygen and sunlight, it induces oxygen radical and superoxide production. These compounds then degrade cell membrane fatty acids and consequently disrupt the plant cells. Laccase counteracts the cercosporin toxin by redirecting its normal breakdown, thus preventing it from producing the destructive superoxides and disabling the fungi's ability to produce leaf spotting. Using laccase to prevent cell death inhibits fungal growth by diminishing the fungi's food source. It also reduces secondary infections by pathogenic bacteria that take advantage of the fungal damage.

Cercosporin toxin is a polyaromatic compound related to another fungal secretion, hypericin, which is also known as cyclosan. (Refer to the diagram at http://www.biosite.dk/staabi/cercosporin.htm for cercosporin's chemical structure.) Cercosporin toxin is insoluble in neutral water and many organic solvents. It is usually extracted in pyridine and basic organic solvents. Sunlight and oxygen degrade cercosporin toxin into lipid-soluble components and various superoxides. On the surface of the plants, the reaction causes necrosis due to cell membrane degradation. Fungi producing cercosporin toxin feed on dead leaf epithelium cells and cause further damage to the plant from digestive enzymes that break down cell wall components and proteins.

Laccase belongs to a diverse group of enzymes called oxidoreductases. It is also known as benzenediol:oxygen oxidoreductase, diphenol oxidase, laccase b, phenoloxidase, and urishiol oxidase. These oxygen acceptors, also known as oxygen scavengers, modify ortho- and para-diphenols and related organic molecules such as quinols and aminophenols. They have low substrate specificity and require copper as a cofactor. In addition to Laetisaria arvalis, laccase has been isolated from the fungi Coprinus cinereus, Cryptococcus neoformans, Lentinula edodes, Melanocarpa allomyces, Phanerochaete flavido-alba, and Trametes versicolor. Jeffrey F.D. Dean, University of Georgia, has isolated four gene families of laccase from Arabidopsis and believes other types occur in a variety of plants. T. Bertrand et al.2 have characterized the enzyme and produced a residue map and motifs available at http://www.biochem.ucl.ac.uk/bsm/pdsum/1kya/main.html.

In laboratory studies, Lartey and his team inhibited the action of cercosporin toxin by simply spraying leaves with a laccase solution. The laccase used in their study was extracted from the soil fungus Laetisaria arvalis1. Lartey's team is currently conducting in vivo greenhouse studies and will pursue field trials to evaluate the effectiveness of laccase compared to fungicidal treatments. Lartey hails laccase applications as an environmentally safe strategy to control cercosporin leaf spot.

Lartey's study was not the first attempt at looking for ways to degrade cercosporin toxin. Thomas Mitchell at North Carolina State University investigated bacterial isolates that biodegrade the toxin3. His research team grew various soil bacteria for 48 hours on media containing cercosporin toxin. Xanthomonas campestris pv. zinniae and X. campestris pv. pruni proved to be effective by reducing the cercosporin content of the media by 90% over the 48-hour incubation, as indicated by a color change in the media from brown to green. (Cercosporin and its breakdown products have pH sensitive chromophore groups.) The final breakdown product was the nontoxic xanosporic acid. Mitchell did not characterize the enzyme isolate in this study. However, he did attribute cercosporin degradation to an oxidoreductase in the bacterial cytochrome P450 system.

Margaret Daub, also at North Carolina State University (http://www.cals.ncsu.edu:8050/botany/faculty/mdaub/mdaub.html), is examining other metabolic engineering strategies to impart Cercospora resistance in crops. Her laboratory is working to isolate the fungus genes responsible for resistance to the toxin as well as to isolate and characterize genes from bacteria that encode toxin degradation. They are also investigating the role that pyridoxine (vitamin B6) in Arabidopsis and Nicotiana has in combating the superoxides produced by cercosporin toxin.

Greg Upchurch, USDA, and Jon Duvick, Pioneer Hi-Bred International, initiated a strategy using genetic modification to control leaf spot on corn. They used a gene gun to insert the CFP (cercosporin facilitator protein) gene from Cercospora into corn and tobacco cultured in vitro. Both systems were successful for CFP expression4. The CFP gene protects the fungi from the toxic effects of their own cercosporin toxin. Upchurch and Duvick surmised that for the CFP to work in crops it must be transported to the leaf surface of the transgenic plant. This requirement is not impossible to satisfy because the fungus has membrane mechanisms that readily secrete the protein. The effectiveness of CFP expression in protecting transgenic plants from Cercospora is not yet determined.

Laccase expression in transgenic crop plants would be advantageous for exploiting the enzyme's effectiveness at controlling Cercospora leaf spot disease. First, the initial success of using laccase spray makes it a feasible form of cercosporin biological control. Other important factors are that laccase is already approved by USDA for field trials in transgenic plants and is found naturally in edible mushrooms. The major limitation is that there is currently no expression system that permits the secretion of laccase by leaf epithelial cells. However, the studies on CFP may provide the information needed to express secreted laccase. Expression and cloning methods for laccase have already been studied and provide the background for developing an expression vector system for particular crops5. VTT Biotechnology in Finland already markets laccase and modified oxidoreductases used in commercial applications (http://www.vtt.fi/bel/coe/enzymtec.htm).

Aside from reducing the need for fungicides, one major environmental benefit of using transgenic laccase plants would be that the enzyme appears to impart no harmful effects to plants that may unintentionally receive the gene through purported horizontal gene transfer mechanisms. Also, its placement in wild plants should not create superweeds. It appears that many weed species subject to Cercospora leaf spot disease produce laccase or have other genes that impart some natural resistance.

References

1. Spillman A. (2003) Enzyme may protect sugar beets from leaf spot disease. Agricultural Research 51(5): 14. http://www.ars.usda.gov/is/AR/index.html

2. Bertrand T et al. (2002) Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics. Biochemistry 41(23): 7325-33.

3. Mitchell TK, Chilton WS and Daub ME. (2002) Biodegradation of the polyketide toxin cercosporin. Applied and Environmental Microbiology 68(9): 4173-4181. http://aem.asm.org/cgi/content/full/65/11/4908

4. Lee J. (1998) Gene turns fungus against itself. Agricultural Research 51(8): 10-11. http://www.ars.usda.gov/is/AR/archive/aug98/gene0898.htm

5. Zhoa J and Kwan HS. (1999) Characterization, molecular cloning, and differential expression analysis of laccase genes from edible mushroom Lentinula edodes. Applied and Environmental Microbiology 65(11): 4908-4913.

Brian R. Shmaefsky
Department of Biology and Environmental Sciences
Kingwood College, Kingwood, TX
brian.shmaefsky@nhmccd.edu



US FILES SOS WITH WTO: END EU'S GM MORATORIUM ASAP
Phillip B. C. Jones

On May 13, the United States, Argentina, Canada, and Egypt filed a case with the World Trade Organization against the European Union over its five-year moratorium on the approval of new genetically modified (GM) crops and GM foods. Although they lack a direct commercial interest, the following countries expressed support as third parties: Australia, Chile, Colombia, El Salvador, Honduras, Mexico, New Zealand, Peru, and Uruguay.

The complaining parties asserted that the European Union has failed to apply a scientific, rules-based review and approval process to agricultural biotech product applications. This position is grounded in the WTO Agreement on the Application of Sanitary and Phytosanitary Measures. While the Agreement recognizes that countries are entitled to regulate crops and food products to protect health and the environment, Article 2 requires that members have "sufficient scientific evidence" for such regulations. Moreover, Annex C instructs that procedures to check and ensure the fulfillment of sanitary or phytosanitary measures must be undertaken and completed without undue delay. According to the complaining parties, a five-year delay is not undue.

CropLife America, the National Corn Growers Association, and the U.S. Grains Council welcomed the decision to file the case. According to the NCGA, the moratorium has cost U.S. corn exporters $300 million a year.

The European response was not quite as upbeat. The European Commission expressed its regrets about the suit as legally unwarranted, economically unfounded, and politically unhelpful. European officials, who began to refer to the moratorium as the "so-called `moratorium,'" warned that European consumers might respond with a boycott of U.S. food products and businesses.

The chasm between U.S. and EU perspectives is clear. After Pascal Lamy, EU Trade Commissioner, announced that there is no moratorium, the Office of the U.S. Trade Representative responded with quotes from David Byrne (EU Health Commissioner) and EU Environment Commissioner Margot Wallstrom, who have discussed the ending of the moratorium. As an example of the European Union's willingness to move forward on the processing of applications, the European Commission pointed to two cottonseed oils for food use that were recently placed on the EU market following authorization. According to the USTR, however, these highly processed oils, which lack GM components, did not go through an approval procedure requiring assent of EU member states. Rather, the United Kingdom classified the products as being equivalent to conventional cottonseed oils, and the European Commission acknowledged that conclusion.

Under WTO rules, the two sides had 60 days to settle the case. However, negotiations between American and European officials broke down on June 19. The complaining parties requested the WTO Dispute Settlement Body to form a three-person panel. Within several months, the two sides should present their arguments before the panel, a process that EU officials suggest would proceed at least through November. The losing party can appeal the panel's decision to the WTO Appellate Body, which would provide its report within three months. There is no further appeal. Countries are often given 6 to 15 months to comply with the WTO's final ruling.

Pascal Lamy has dismissed the WTO course of action as pointless, predicting that there will be new GMO authorizations by the time that the two sides get to the real litigation. On July 2, the EU Parliament passed rules for the labeling of genetically altered food products. Twenty days later, the European Agriculture ministers adopted the new regulations, which are expected to pave the way for new biotech foods to be sold in Europe by this fall.

Under the new rules, GM products and byproducts of GM crops that contain more than 0.9 percent genetically altered material will have to carry the label: "This product is produced from Genetically Modified Organisms." The rules also require business operators to retain information on GM products from production through every stage of the commercial chain. In this way, each GM product can be traced from its point of origin to the supermarket. Finally, the new laws allow the 15 EU nations to set their own rules to prevent seeds from fields of GM crops from blowing onto fields of conventional or organically-grown crops.

In mid-July, the EU took another step to end the moratorium by instituting its own litigation. The EU Commission filed lawsuits at the European Court of Justice in Luxembourg against 11 EU governments for failing to implement laws on testing and licensing GMOs.

While the latest EU efforts may end the moratorium, the new rules on labeling and traceability pose difficulties for U.S. agriculture. In fact, Bob Callanan, a spokesman for the American Soybean Association, characterized the rules as "outrageously stupid." The U.S. State Department warned that the new labeling rules could lead to the imposition of a new set of nontariff barriers. The GM food fight continues.

Laser patent case corrects vision of experimental use doctrine
Last fall, the Court of Appeals for the Federal Circuit published its Madey v. Duke University decision on the immunity from patent infringement liability known as the "experimental use doctrine." Although the opinion is in line with precedent, it seems to be an unwelcome eye opener for some. Representatives from universities and non-profit organizations declared that the Madey case is a radical departure from prior law that nullifies the experimental use doctrine. According to the American Council on Education, the decision could chill non-commercial research at many universities.

In a way, the case began in 1988 when Duke University recruited Dr. John M.J. Madey from Stanford University. At Stanford, Madey had created a laser research program and had obtained sole ownership of two patents that covered several pieces of equipment in his free electron laser lab. Madey relocated his research program to Duke until his resignation in 1998.

When Duke researchers continued to use some of the equipment in Madey's old lab, Madey sued the university on various grounds, including patent infringement. A federal district court dismissed the patent infringement claims, holding that Duke benefited from the experimental use defense, because Madey's patented laser technology had been used for experimental or non-profit purposes. Madey appealed the decision, and on October 3, 2002, the Court of Appeals for the Federal Circuit reversed the lower court's holding.

The Federal Circuit found that the lower court had an overly broad concept of experimental use. The appellate court explained that the profit or non-profit status of the infringer is not determinative. Rather, the question is whether the infringing act furthers the infringer's legitimate business and is not solely for amusement, to satisfy idle curiosity, or strictly for philosophical inquiry. Duke's legitimate business objectives, the court decided, include educating students and luring research grants. The infringing acts were in accordance with these objectives. A copy of the Madey opinion can be found on the Federal Circuit's website (http://www.fedcir.gov/dailylog.html).

Duke appealed to the U.S. Supreme Court, and the Association of American Medical Colleges and at least 29 higher education organizations filed an amicus brief, requesting that the Court reverse the Federal Circuit's decision. Before deciding whether to take the appeal, the Supreme Court asked the Solicitor General to file a brief expressing the views of the United States government, an entity that has unsuccessfully asserted the experimental use defense on several occasions.

In May, the Solicitor General announced that the position of the United States is that the Supreme Court should deny the petition for review. For now, the lower courts can clarify the scope of the experimental use defense after additional facts are gathered about the allegedly infringing uses of Madey's patented inventions. On June 27, the Supreme Court denied Duke's appeal. A copy of the Madey brief can be found on the Justice Department's website
( http://www.usdoj.gov/osg/briefs/2002/2pet/6invit/2002-1007.pet.ami.inv.html).

Selected References

1. Ballard, A.M. 2003. Several organizations, universities join Duke in challenge to equipment ownership ruling. Medical Research Law & Policy 2:170, March 5, 2003.

2. European Commission. 2003. GMOs – Keeping you informed. Europa Newsletter, July 8, 2003. Available at: http://europa.eu.int/newsletter/index.htm.

3. Miller, S. and Kilman, S. 2003. A global journal report: Europe is poised to tighten rules on modified crops. Wall Street Journal, A1, July 1, 2003.

4. Rainford, C. 2003. WTO biotech suit earns widespread cheers in US. Agriculture Online, May 13, 2003. Available at: http://www.agriculture.com.

5. Schroeder, M. and Miller, S. 2003. U.S. to file complaint at the WTO. Asian Wall Street Journal, A2, May 14, 2003.

6. USTR. 2003. Procedure and timeline for a WTO case. Available at: http://www.ustr.gov/new/biotech-wtocase.htm.

Phillip B. C. Jones, PhD., J.D.
Spokane, Washington
PhillJones@nasw.org





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