Keynoting BIO 2006 held recently in Chicago, former president Bill Clinton discussed the role of biotechnology in addressing food security in the developing world, as well as global health and environmental issues.

BIO 2006 was the 14th annual meeting of the Biotechnology Industry Organization and is the largest international biotechnology conference in the world. BIO represents more than 1,100 biotechnology companies, academic institutions, state biotechnology centers, and related organizations across the United States and 31 other nations. BIO 2006 attracted a record 19,479 attendees from 62 countries. Nearly one-third of the attendees were international participants. Twelve U.S. governors attended. Members of BIO are involved in the research and development of healthcare, agricultural, industrial, and environmental biotechnology products.

First introduced in 1996, biotech crops came of age during the Clinton Administration. The benchmark of one billion acres of commercial biotech crop production experience across the world was reached in 2005.

The 42^nd President of the United States said he supports the development of biotechnology applications in agriculture, with results evaluated under proper testing and continuous monitoring. "I think that we should be driven in America by science, evidence, and argument, not by assertion and fear," he said, drawing one of the stronger outbursts of applause during his speech.

"I did support the development of genetically engineered crops. If anybody could give me any evidence why I shouldn’t do it, I’d be happy to change my position. But failing evidence, I think the use of agricultural technology, which uses less fertilizer, takes better care of the soil, increases productivity, and could be transferred at low cost through seeds to poor farmers in developing countries is a good thing," Clinton said. "We need more people to be able to grow their own food and feed themselves." Clinton noted that he did not believe there’s anything inconsistent with organic farming and "supporting genetic developments."

Clinton said that he sees an enormous role to play for biotechnology in health care. One key health concern, he said, is the rising rate of obesity in America, including children, which can result in myriad problems, including the onset of diabetes. "First, you’ve got to get people to eat more sensibly. Then you’ve got to get people to exercise more; but we’ve also got to make sure that we’re operating at maximum metabolic efficiency, and I think it’s unlikely that this problem would be solved by anyone other than someone in your line of work (biotechnology)".

Biotechnology also promises to play a major role in building sustainable, clean energy. "We need to deal with the fact that we’re wasting tons of money every year on energy costs, much of it from imported energy, contributing 25% of the greenhouses gases in the world even though we’re (the U.S.) only 4% of the world’s population with 20% of its wealth," he said.

Climate change, and the potential applications of biotechnology in managing it, was clearly a theme Clinton stressed in his speech. "What you do in agriculture is important because if the climate continues to change, we will see a continued erosion of the top soil, more dust storms; and in the Northern Hemisphere, agricultural production being pushed north and the Southern Hemisphere being pushed south. And since the first obligation all societies have is to be able to feed themselves, we have to explore every conceivable alternative not only to reduce the rate of the climate’s warming and to reduce the human contribution to it by restricting greenhouse gas emissions, but also to make the best of what will inevitably be a more difficult situation," he said.

Clinton said he favors the development of biofuels, but favors a biofuel future based more on cellulistic fuels than corn, which is a principal contributor to ethanol now. "Why? Because the conversion ratio is better. If the goal is not only to have clean fuels but to reduce the use of greenhouse gases, then you want stuff that’s lying around anyway, that you didn’t have to burn oil to produce in the first place. And there’s all kinds of agricultural waste that can be used."

He pointed to the aftermath of last year’s Hurricane Katrina as an example. "I could have fueled every shrimp boat in the Gulf with the fallen trees and shrubs and other greenery that was all over the ground from Alabama to New Orleans if we’d had the plants available and the distribution mechanisms available. And at $65 [per barrel] oil, diesel for those shrimpers is $3.00 a gallon. With the tax subsidy available in the law now, we could sell biofuels for about $1.80. And if you’re in the shrimp boat business, the difference between spending 18% of your revenues on fuel and 33% is the difference between making a living and going broke."

In the growing global economy, where more and more jobs are in competition with others, the U.S. will need to keep finding a source of new jobs that pay well and create strong consumers. "The new source is right there before us. It’s converting to a clean energy, an alternative energy future." In fact, in the next decade, Clinton said he believes biotechnology can replace energy as the main source of new jobs. "It’ll take us about a decade to reach the full implications of the sequencing of the human genome, so that we will be able to apply it to all kinds of diseases and conditions, develop vaccines, develop preventive strategies, be producing all kinds of products and services that we never even dreamed of before."

Two of the more well-known names to speak at BIO 2006 were actor Bernie Mac and NBA legend Earvin "Magic" Johnson, both of whom related how biotech-driven medical and health care advancements have made a difference in their lives. Mac suffers from sarcoidosis, a rare autoimmune disease, and Johnson stunned the sports world in 1991 when he announced his retirement because of the AIDS virus.

Jim Mullen, chairman of the BIO organization and CEO of Biogen Idec, gave an overview of how the biotechnology industry has advanced over the decades, beginning in the 1970’s as a "technology with many dreams of practical applications in a wide range of areas."

Early commercialized products included human insulin, recombinant vaccine for hepatitis B, and interferon alpha for cancer, as well as advances in agriculture and new fuels. To date, Mullen says the global biotech industry has grown to approximately 4,500 companies that employ more than 200,000 people, spend more than $18 billion on R&D, and generate more than $60 billion in revenue.

Former CIA director James R. Woolsey discussed the role of biofuels to U.S. energy security, and Mike Leavitt, U.S. Secretary of Health and Human Services, discussed how health care will evolve with biotechnology. Genetic information will improve as more becomes known about the human genome. Paired with family history data, "the next decade will be viewed by future generations as the time when treatments became preventive, predictive, and personalized," he said.

The BIO program featured breakout sessions under two dozen topic areas, from bioethics and nanotechnology to biodefense and regenerative medicine. The Food and Agriculture program at BIO 2006 was the largest and most comprehensive agbiotech venue in BIO’s history. For the first time, the conference included an animal biotechnology subtrack, with six sessions that specifically addressed animal biotechnology issues.

Program topics under the food and agriculture track included:

Our model on risk assessment for pest resistance transgenes in natural communities was recently featured in the ISB News Report.^1,2 Since then, we have published a modification of our original model to allow estimation of the impact of multiple seasons of transgene input into a non-crop population; input seasons may be at either continuous or discontinuous intervals.³ As in the original model, the modification is illustrated using oil seed rape (OSR) and its common predator, diamondback moth (Plutella macropennis). In the context of ecological interaction in a naturally fluctuating environment, we come to much the same conclusion with repeated as with single-season transgene input: the transgene need not have a large impact on the natural community, and our suggestions for assessing and mitigating any threat still stand.

We are in the process of setting-up a website so that programs for implementing our model may be downloaded.

References

1. Kelly CK et al. (2005) An analytical model assessing the potential threat to natural habitats from insect resistance transgenes. Proceedings of the Royal Society of London B 272(1574), 1759-1767

2. Kelly CK. (2006) Risk assessment for insect resistance transgenes. In ISB News Report, January 2006. p. 5-7

3. Kelly CK, Bowler MG, & Breden FM. (2006) An analytical model assessing the potential threat to natural habitats from insect resistance transgenes: continuous transgene input. Biology Letters 2(2). DOI:10.1098/rsbl.2006.0444

Spiders and insects have been able to spin silk for at least 380 million years. It has been estimated that the strength of spider silk is at least five times as strong as steel, twice as elastic as nylon, waterproof, and stretchable. Scientists have long envied the strength and elasticity of spider’s silk but have been unable to synthesize it. The dragline silk of the Golden Orb-weaving Spider is the most studied in scientific research. Dragline silk is the fiber from which spiders make the scaffolding of their webs. Of late, demonstrations of cloning and expression of silk fibroin protein, including spider dragline silk, represent an exciting scientific opportunity and technological challenge.

Spider silk is a natural polypeptide, polymeric protein and is in the scleroprotein group, which also includes collagen (in ligaments) and keratin (in nails and hair). Spider silk genes encode proteins composed of iterated peptide motifs, and the consensus sequences are multiply repeated throughout the silk proteins. Patterns of ‘Ala-rich’ blocks and ‘Gly-rich’ amorphous blocks provide both strength and elasticity, and such biomaterials might therefore be valuable for industrial and medical purposes.

Although spider silk was initially produced in animal cells, mass production from animal cells is expensive and the quantity produced is limited, mainly due to the requirement for large fermenters, sterile conditions, and expensive media. In order to overcome these factors, plants (tobacco, potato, Arabidopsis, and soy somatic cells) have been evaluated for the production of spider silk proteins. These studies demonstrated the feasibility of plant based silk-like protein (SLP) production. However, high yield and low production costs remain a challenge.

Jianjun Yang and co-authors, from Central Research Development and Crop Genetics Research, Du Pont de Nemours & Co, USA, report high yield recombinant SLP production through protein targeting in transgenic Arabidopsis. The researchers used a synthetic DP1B gene for producing SLP, achieving variable accumulation levels through protein targeting into a special cellular components.¹ DP1B is a synthetic analogue of spider dragline silk protein, which can be spun to form silk fiber. An average DP1B accumulation level in plant cells below 1.5% of total soluble protein has been previously reported. However, Yang et al. showed that ER and vacuole targeting in seeds and apoplast, and ER targeting in leaves, can dramatically increase SLP accumulation. Many transgenic plants engineered with the ER targeting mechanism were able to accumulate DP1B SLP in their seeds to a level greater than 15% of total soluble protein.

A high yield of SLP was achieved by utilizing DP1B-8P, which is a plant-optimized, synthetic 8-mer DP1B gene that encodes a 64-kD DP1B SLP. Targeting of SLP to the designated cellular components was enabled by adding a 21-amino acid sporamin signal peptide, 16-amino acid sporamin propeptide, and 4-amino acid ER retention peptide to appropriate positions on the DP1B molecule. To express the resultant DP1Ba, DP1Be, and DP1Bv fusion proteins in Arabidopsis, expression plasmids pGYV501, pGYV502, and pGYV503 were introduced into Arabidopsis thaliana by an Agrobacterium-mediated floral transformation method. Healthy T1 transgenic plants were grown in soil and T2 seeds were harvested from individual T1 plants to represent independent transformation events. Later, T2 seeds were germinated on selective plates to obtain the next generation. All T2 transgenic seedlings originating from the same parents were grown together in soil. T3 seeds were collected from these plants and pooled together to represent independent transformation events. Standard molecular assays were performed to confirm transgene expression in the independent transformation events. DP1B production yield was calculated based on DP1B content and relative to total soluble protein content in the extract.

The transformation was confirmed by PCR amplification of sporamin signal peptide coding sequences from the genomic DNA. Leaf protein extract made from a pGY401 Arabidopsis plant was used to show control levels of unmodified, cytosol-accumulated 64-kD DP1B. The results of immunoblot assay showed that in leaf tissues of Arabidopsis, apoplast and ER targeting are suitable approaches for DP1B production, but the vacuole targeting is not. Results also showed that accumulation of the unmodified DP1B in leaf tissues of pGY401 plants ranged from 0.01% to 1.7% of total soluble protein (TSP), with an average yield of 0.3%, while DP1Ba accumulation in leaf tissue of pGYV501 plants was from 2.1% to 8.5% TSP, with an average yield of 4%. DP1Be accumulation in leaf tissues of pGYV502 plants was from 0.3% to 6.7% TSP with an average yield of 1.5%. Vacuole targeting did not result in accumulation of correctly-sized DP1Bv molecules. This comparison of accumulation levels between the non targeted and targeted DP1Bs revealed that DP1B productivity in leaf tissues was increased 13-fold by targeting to apoplast and 5-fold by targeting to ER lumen. Apoplast targeting is the best approach.

Characterization of seed-specific DP1B fusion proteins showed accumulation of unmodified DP1B in pGY411 seeds ranging from 1% to 1.4% TSP with an average yield of 1.2%. The accumulation of the ER-targeting DP1Be in pGYV512 seeds ranged from 1.8% to 18% TSP (avg. 9.4%), and the vacuole-targeting DP1Bv in pGYV513 seeds ranged from 4.8% to 8.2% TSP (avg. 6.5%). Thus a comparison between the targeted and non-targeted DP1Bs suggested that the ER and vacuole targeting significantly enhanced DP1B productivity in seeds by 7.8- and 5.4-fold, and at the highest levels, up to 15-fold increase was observed in ER targeting.

Importantly, DP1B transgenic plants were heritable after one and two cycles of sexual reproduction, as confirmed by transgene insertion and expression studies in T2 leaves or T3 seeds, and the accumulation level of DP1B fusion protein was stable. Thus this research demonstrates the highest level of DP1B accumulation among all protein targeting strategies. It was concluded that combining seed-specific synthesis and ER targeting is the best method of production. Though Arabidopsis is not a good candidate for commercial production, the approach may be tried in an industrially important crop that supports the large-scale plant based production of DP1B SLP.

Sources:

Yang J, Barr LA, Fahnestock SR & Liu Z-B (2005) High yield recombinant silk-like protein production in transgenic plants through protein targeting. Transgenic Research, 14: 313-324

Huemmerich D et al. (2004) Novel assembly properties of recombinant spider dragline silk proteins. Current Biology 14, 2070–2074

Brandle J & Menassa R. (2004) Silk purse from a sow’s ear? Spider silk production in tobacco. ISB News Report, December

Scheller J, Guhrs K-H, Grosse F, & Conrad U (2001) Production of spider silk proteins in tobacco and potato. Nature Biotech 19: 573-577

ROOT ENGINEERING: A STRATEGY FOR AGRICULTURE IN MARGINAL AREAS OF CULTIVATION
Roberto A. Gaxiola

Water availability is a major concern for agriculture in both developed and developing countries. Global water strategies focus on reducing overall agricultural use and increasing availability for human consumption. However, population growth and global warming are driving regional shifts in production and increased demand for irrigation. Improvement of plant water utilization is therefore critical. Some drought resistant plants develop deep and dense root systems. These natural adaptations suggest that manipulation of developmental mechanisms to enhance lateral root proliferation can be an effective strategy to engineer drought resistant plants. Despite their obvious role in water uptake, roots have not been targeted in genetic engineering strategies to improve crop performance under drought conditions.

We have shown that overexpression of the H⁺-pyrophosphatase (H⁺-PPase) AVP1 results in salt and water stress tolerant Arabidopsis plants.¹ Both phenotypes were initially explained by an enhanced uptake of ions into their vacuoles. Significantly, further characterization of these AVP1 overexpressing plants revealed a dramatic enhancement of their root development with obvious implications for their ability to withstand drought. Moreover, root and shoot development in avp1-1 loss-of-function mutants was impaired.²

Our data indicated that although classically thought of as a tonoplast resident proton pump responsible for acidifying the vacuole, AVP1 also contributes to the regulation of apoplastic pH and to auxin transport, likely by mediating the trafficking of the PM P-ATPase and associated proteins, including PIN1.² Changes in intracellular auxin levels are also known to alter the expression of P-ATPase genes, establishing a feedback loop where AVP1 activity can regulate both targeting and level of the PM proton pump. Thus, in addition to its established role in the maintenance of vacuolar pH, our new data reveal a novel role for AVP1 in facilitating auxin transport and the regulation of auxin-related processes, such as root development.² Of note, the high degree of identity at the amino acid level among the type I H⁺-PPases in the plant kingdom³, together with the striking phenotypes that its overexpression produced in Arabidopsis^1,2, suggested that the H⁺-PPase AVP1 could be a potential target for genetic engineering of root systems in agriculturally important crop plants.

To test if the water stress resistance phenotype triggered by the overexpression of AVP1 in Arabidopsis could provide a more universal strategy to improve the performance of crops under water deficit conditions, we engineered plants of a commercial cultivar of tomato (Lycopersicon esculentum) to express the Arabidopsis AVP1 H⁺-PPase. This approach resulted in a) greater pyrophosphate-driven cation transport into root vacuolar fractions, b) increased root biomass, and c) enhanced recovery of plants from an episode of soil water deficit stress.⁴ We concluded that more robust root systems allowed transgenic tomato plants to take up greater amounts of water during the imposed water deficit stress, resulting in a more favorable plant water status and less injury.

This study documents a novel general strategy for improving drought resistance of crops. Furthermore, it is tempting to speculate that enhanced development of root systems will also have a positive impact in plant’s mineral nutrition. We are currently testing the latter hypothesis.

1. Gaxiola RA et al. (2001) PNAS 98, 11444

2. Li J et al. (2005) Science 310, 121

3. Drozdowicz YM, Rea PA (2001) Trends in Plant Science 6, 206

4. Park S et al. (2005) PNAS 102, 18830

CropLife International is making publicly available a database of published papers and reviews demonstrating the benefits and safety implications associated with the use of agricultural biotechnology products.

Agricultural biotechnology today is realizing economic, environmental, health, and social benefits for farmers and society in both industrial and developing countries. In 2004, 8.25 million farmers in 17 countries planted biotech crops – 90 per cent in developing countries. While studies recording, demonstrating, and quantifying these benefits exist, they can be difficult to locate and access.

The purpose of this database is to enable you quickly and easily to locate and access credible scientific information about the demonstrated benefits associated with the use of agricultural biotechnology products, and about their safety.

VIRGINIA BIOINFORMATICS INSTITUTE TO DEVELOP TOMATO METABOLITE DATABASE

A researcher at the Virginia Bioinformatics Institute (VBI) at Virginia Tech, Blacksburg, Virginia, is developing a database and computational tools to help scientists learn more about how certain genes in tomatoes affect the crop’s flavor and nutritional value.

The Tomato Metabolite Database, which is being implemented by Zhangjun Fei, a senior bioinformatics scientist in VBI’s Cyberinfrastructure Group, will be used to store a wide range of information and data about tomato, including microarray and metabolite profiling data as well as information on metabolic pathways. This resource will be used to identify key genes involved in the synthesis of essential metabolites that impact tomato flavor and the quality of its nutrients.

Fei’s work is part of a collaboration with Harry Klee, professor of horticultural science at the University of Florida and principal investigator for the project, and Jim Giovannoni, adjunct professor of plant biology at the Boyce Thompson Institute at Cornell University and research molecular biologist at the United States Department of Agriculture (USDA) Agricultural Research Service’s Plant, Soil and Nutrition Laboratory. The work is funded by a $2 million grant from the National Science Foundation.

Research groups led by Klee and Giovannoni will use the information provided by the Tomato Metabolite Database to characterize the functions of those genes identified as being responsible for genetic variation in tomato using state-of-the-art RNA interference (RNAi) technology. RNAi technology allows scientists to silence or turn down the specific function of a gene within a cell and represents a powerful approach to accurately establish gene function on a large scale.

Professor Klee remarked: "The development of this database will allow researchers the world over to develop and test hypotheses regarding the regulation of flavor, nutrition and quality metabolites in edible crop tissues."

By identifying the highly complex traits that control the flavor and nutritional value of the tomato, this work will not only help to improve the way tomatoes are grown but will also contribute to how this crop is being used and developed in other biotechnology research programs. Researchers will also be able to directly apply the findings from this project to other important crops grown in the United States and around the world.