The problem
Plants growing under natural conditions unavoidably face episodic situations of environmental stress in the course of their life times. They have developed numerous strategies to survive in such adverse conditions. Crops, in contrast, are selected by humans for their high productivity in agriculture, but this is usually not accompanied by increasing resistance to hostile environments. Diseases, unfavorable climates, or inappropriate soils are responsible for most agricultural losses. Analysis of major crops with economically valuable reproductive or vegetative structures (corn, soybeans, barley, potatoes, among others) shows potential record yields 3- to 7-fold greater than average yields¹.

One approach to obtain plants adapted to unfavorable environments may be to improve essential nutrient (nitrogen, phosphorus, or iron) acquisition. Although iron is abundant, plants need to solubilize it from insoluble oxides in alkaline, calcareous soils, which cover approximately one-third of the earth’s surface and represent a major deterrent for agriculture². Plants deprived of iron develop interveinal chlorotic symptoms in young leaves and a general decrease in photosynthetic activity that can lead to death. Chlorosis has been attributed to inhibition of chlorophyll synthesis, which requires the function of Fe-containing enzymes, but chlorophyll-binding proteins and other photosynthetic components are down-regulated with relative independence of pigment levels. Chloroplasts are therefore primary targets of iron deficiency.

On the other hand, plants sustain different types of environmental hardships, such as drought, flood, chilling, salinity, and radiation. Being motionless organisms, a plant’s defense to adverse conditions is limited to physiological and biochemical responses, including stomatal closure, osmotic adjustment, ion pumping, etc. In response to regulated changes in gene expression, central and secondary metabolisms are redirected to cope with the undesired effects of the hostile situation. This is achieved by up-regulating the synthesis of proteins and metabolites involved in protection (i.e., compatible solutes, antioxidant enzymes and compounds, heat-shock proteins, etc.). Whenever these defensive mechanisms are overcome by the intensity of the adverse condition, the plant is under environmental stress.

Although different types of nutritional and environmental adversities have their own features and display idiosyncratic responses, they all have in common a significant perturbation of the electron transfer network of the stressed cells, leading to electron derivation to non-productive routes, breakage of redox homeostasis, and eventually to the generation of reactive oxygen species (ROS) such as singlet oxygen, the superoxide and hydroxyl radicals, and hydrogen peroxide.

The cyanobacterial/algal solution
Photosynthetic microorganisms respond to adverse environments by eliciting a replacement strategy, which involves substitution of sensitive enzymes and proteins by resistant isofunctional versions. Most conspicuous among them is the induction of flavodoxin expression to take over the functions of the ubiquitous iron-sulfur protein ferredoxin, whose levels are down-regulated under conditions of environmental stress or iron starvation^3,4. Flavodoxins are small soluble electron carrier proteins containing flavin mononucleotide that shuttle electrons among a plethora of donors and acceptors. Their properties as redox carriers largely match those of ferredoxin. In heterotrophic bacteria, flavodoxins (and ferredoxins) are reduced enzymatically (largely but not exclusively by ferredoxin-NADP⁺ reductases) to provide low potential reducing equivalents to many different metabolic pathways such as dinitrogen fixation, sulfate and nitrate assimilation, nucleotide synthesis, and C2 carbon metabolism⁵. In cyanobacteria, the prokaryotic ancestors of modern-day chloroplasts, flavodoxin and ferredoxin are reduced photosynthetically at the level of photosystem I in vegetative cells. Ferredoxin is normally the preferred electron carrier, whereas flavodoxin acts as a backup whose expression is induced under adverse conditions.

While ferredoxins are present in virtually all types of organisms, the flavodoxin gene displays a rather limited distribution. Within photosynthetic species, flavodoxin is common, but not universal, among cyanobacteria. The gene entered the algal world during the endosymbiotic event that gave origin to photosynthetic eukaryotes and spread into all major algal lineages, with the corresponding product being targeted to chloroplasts. However, flavodoxin presence is less frequent in algae than in cyanobacteria and generally confined to species living in the open oceans, where nutritional and environmental conditions are usually extreme.

The plant solution
Substitutive strategies as those displayed by photosynthetic microorganisms are absent in higher plants. Indeed, the flavodoxin gene is not found in the plant genome and the considerable adaptive advantages derived from its expression were irreversibly lost somewhere during the evolution from green algae to vascular plants. Instead, plants have developed complex and multi-step responses to both adverse environments and iron deficit. In the first case, the response operates at various levels, including avoidance of the hostile conditions, scavenging of toxic compounds, and damage repair. In the second case, the strategy focuses on optimization of iron intake from scarcely available sources.

Noteworthy, cyanobacterial flavodoxin is still able to productively interact in vitro with plant enzymes whose prokaryotic ancestors used this flavoprotein as usual or occasional substrate. These observations prompted us to evaluate if reintroduction of this flavoprotein in plants could improve tolerance to abiotic stress and iron deficit as it occurs in microorganisms^3,4.

Design and characterization of transgenic tobacco plants expressing flavodoxin in chloroplasts
Preparation of the transformants The flavodoxin gene from the cyanobacterium Anabaena PCC7119 was cloned between the promoter and terminator regions of the cauliflower mosaic virus 35S, which directs constitutive expression in tobacco. A presequence, encoding a chloroplast transit peptide, was fused in-frame to the N-terminal end of the gene to direct the product to plastids. Several independent lines were obtained via Agrobacterium transformation and made homozygous by self-pollination followed by selection of the segregants³. Lines expressing ≥ 60 µM flavodoxin in chloroplasts were selected because levels of the foreign protein were similar to that of endogenous ferredoxin (~80 µM). When grown under normal conditions with water and iron ad libitum, transgenic plants displayed the same phenotypic features as wild-type (WT) non-transformed siblings with respect to growth rate, flowering time, and seed production, indicating that integration and expression of the foreign gene was inconsequential in phenotypic terms whenever plants were not stressed.

Flavodoxin-expressing plants could grow in iron-limited soils

Flavodoxin expression in chloroplasts confers tolerance to abiotic stress

Mechanisms underlying flavodoxin protection
As anticipated, both iron deficit and abiotic stress caused an imbalance in the distribution of electrons originated in the photosynthetic electron transport chain, due to ferredoxin decrease and augmented delivery of reducing equivalents to adventitious acceptors such as oxygen. Flavodoxin restores the redox homeostasis of the plastid by productively interacting with endogenous routes and partners, including ferredoxin-NADP⁺ reductase and thioredoxin reductase, that are crucial for optimal rates of CO₂ assimilation, and ferredoxin-dependent reactions of amino acid synthesis. Flavodoxin also contributes significantly to antioxidant protection by providing the reducing power needed to regenerate the active form of peroxiredoxin, the most abundant peroxidase of chloroplasts. The collected results indicate that flavodoxin maintains the welfare of plants under stress by avoiding electron misrouting and ROS formation, rather than by combating the deleterious consequences of these processes.

Conclusions and Perspectives
Manipulation of tolerance to iron deficit and hostile environments in crop plants represents a particularly demanding challenge for breeders and geneticists, due to its agronomical importance and the complexities of the plant responses. So far, strategies pursued to accomplish this task have been based on boosting endogenous protective systems such as those involved in ROS scavenging and iron uptake and storage. The success of this type of approach has been variable, as could be expected from the complexities and multigenic nature of the responses involved. The strategy described here is based on a different conceptual background. It relies on the assumption that most of the damage suffered by stressed or iron-starved plants is caused by faulty electron distribution within plastids and cells, resulting in impairment of key metabolic, regulatory, and dissipative pathways and/or ROS accumulation. Introduction of an alien protein that restores electron transfer to productive routes was expected to significantly ameliorate the undesired consequences of growth under sub-optimal conditions. The results obtained^3,4 largely confirmed the working hypothesis, leading to transgenic plants that displayed a remarkable tolerance to iron starvation and to various sources of abiotic stress of agronomical importance. Since flavodoxin-expressing plants took up the same amounts of iron as their WT siblings⁴, the incorporated metal could be reallocated to other demanding sources in these lines.

Besides increased tolerance, the transformants displayed other features of potential agronomical relevance: i) tolerant lines could be obtained by transformation with a single gene; ii) the bacterial origin of the transgene rules out unwanted complications such as silencing, co-suppression, and/or endogenous regulatory loops; and iii) flavodoxin expression remains unnoticed, in phenotypic terms, when plants are grown under normal conditions. Then, (re)introduction of flavodoxin into chloroplasts restored some of the selective advantages that allowed photosynthetic microorganisms to thrive in hostile media. Engineering of this ancient trait, either alone or in combination with other protective devices, opens entirely novel possibilities for the design of valuable crops with greater plasticity to face the challenges of environmental hardships.

Transformation of related Solanaceae (tomato, potato) yielded essentially the same tolerant phenotypes, and protocols to incorporate flavodoxin into several other crops, including cereals, are currently underway. The use of available experimental resources such as custom-ready promoters, tissue-specific expression, transplantomics, etc., could make the flavodoxin gene-based strategy a most useful biotechnological tool to improve crop yields and to gain at least some wastelands for agriculture.

References

1. Boyer JS (1982) Science 218, 443-448

2. Mori S (1999) Curr Opin Plant Biol 2, 250-253

3. Tognetti VB, Palatnik JF, Fillat MF, Melzer M, Hajirezaei MR, Valle EM, Carrillo N (2006) Plant Cell 18, 2035-2050

4. Tognetti VB, Zurbriggen MD, Morandi EN, Fillat MF, Valle EM, Hajirezaei MR, Carrillo N (2007) Proc Natl Acad Sci USA 104,11495-11500

5. Zurbriggen MD, Tognetti VB, Carrillo N (2007) IUBMB Life 59, 355-360

CLEANING-UP CROP GENOMES THROUGH INTRAGENIC MODIFICATION
Caius Rommens

Awareness that fruits and vegetables are excellent sources of nutrients gradually evolved during the last three centuries and is currently reinforced through USDA-backed promotion programs such as "5 A Day." However, there are still many issues associated with today’s food crops. Two recent reviews discuss the importance of removing lingering toxins and allergens while enhancing the levels of health-promoting antioxidants^1,2. Such improvements may be accomplished efficiently through intragenic modification, a new approach to genetic engineering that transforms plants with native genetic elements only.

Today’s crops as a work-in-progress
Crop improvement programs are primarily directed toward traits that have always been at the forefront of the breeder’s mind: ever more yield with, in some cases, a little added taste. In an often desperate effort to accomplish these goals effectively, breeders create as much DNA diversity as possible. They recombine entire genomes of cultivated crops with those of wild relatives to capture at least some of the diversity that evolved within species boundaries. Additionally, genes are modified or inactivated through chemical mutagenesis programs that induce extensive substitutions, deletions, or rearrangements. New varieties derived from introgression or mutation breeding may, indeed, display powerful new trait combinations. However, they also express many less-desirable characteristics that were not considered during the selection process (Fig. 1).

Genomes of crops such as peanut, wheat, soybean, rice, and apple are peppered with allergen-encoding genes. Transfer of these genes from existing to new varieties is often considered inevitable. Consequently, consumption of a single peanut can be life-threatening to people predisposed to developing allergic reactions, and bread intake continues to damage the intestinal lining of about 0.8% of Americans who suffer from Celiac disease. Food crops also contain numerous genes involved in the biosynthesis of natural toxins and antinutritional compounds, including glycoalkaloids, cyanogenic glycosides, glucosinolates, coumarins, and gossypol. Indeed, the majority of pesticides in the human diet are naturally produced in the edible parts of crops, often at levels at or near those known to cause health issues³. Additional toxins may be produced upon heat processing. High levels of asparagine and reducing sugars in potato tubers and wheat flour trigger the heat-induced formation of the toxic compound acrylamide⁴. Furthermore, the polyunsaturated fatty acids in frying oils are rapidly oxidized upon heating to produce carcinogens.

The functional activity of toxin- and allergen-associated genes is contrasted by the frequent inactivity of potentially beneficial genes. For instance, crops such as tomato and potato express the key gene in flavonol biosynthesis in anthers but not in fruits and tubers, respectively. This tissue specificity limits the dietary availability of some of the most powerful antioxidants. Similarly, levels of carotenoids, anthocyanins, and phenolic compounds are often much lower than could potentially be attained.

Thirteen years after the European Patent Office (EPO) granted Monsanto’s patent EP301749B1, the agency revoked it. The EPO’s decision—released on 07/07/07—hinged upon a definition.

Agracetus had filed the original patent application in 1988 with claims for genetically altered soybean plants. Two years after the patent’s grant in 1994, Monsanto acquired Agracetus and rights to the patent. The Agracetus patent came with baggage: organizations have opposed the patent on the grounds that it gives the patent owner de facto control over all genetically engineered soybeans. The latest, and last, fight focused on a claim for a "soybean seed which will yield upon cultivation a soybean plant comprising in its genome a foreign gene effective to cause the expression of a foreign gene product in the cells of the soybean plant."

Opponents argued that the claim embraced genetically modified soybean seeds regardless of the method used to produce them. The Bedford cultivar, derived by traditional cross-breeding from the soybean line Forrest, carries DNA from the soybean line PI 88788. The patent’s opponents urged that Bedford cultivar seeds, which pre-dated the patent application, destroyed the novelty of the patent claim.

Monsanto argued that their patent claim focused on seeds that contained a "foreign gene"—that is, a gene from a source other than the plant species transformed with that gene. The claimed soybean seeds, therefore, could not include seeds of the Bedford cultivar.

The EPO’s Technical Board of Appeal disagreed with Monsanto. Unable to find an explicit definition of "foreign gene" in the patent, the Board decided that the term could refer to DNA not previously present in a particular soybean plant’s genome.

The Board highlighted the following explanation from the patent: "The DNA sequence can be chimeric, in the sense of being constructed from DNA sequences from different organisms but full intact non-chimeric genes from other plant species or lines of the same species may also be used." The passage makes it clear, the Board maintained, that the claims encompass soybean seeds with DNA from a line of soybean species other than the line used to generate the seeds. Accordingly, Bedford seeds fall within the scope of the claim and destroy novelty.

Back in the Day, Biotech Inventors Won a Deuel
The novelty requirement reflects an important patent law policy: a patent should not eliminate rights to use something in the public domain. US patent law also prohibits the granting of patent claims to a novel invention that could have been readily deduced from publicly available information—prior art—at the time the invention was made, by a person knowledgeable about the relevant technological field. In patent jargon, an invention must have been nonobvious. Often, the nonobviousness requirement has proved difficult to apply and has incited much litigation, especially for patent claims to nucleic acid molecules and proteins.

During the early 1990s, US Patent and Trademark Office (PTO) examiners framed an obviousness determination of claimed biomolecules as a question of whether one skilled in the art could have isolated the claimed biomolecule using standard techniques. For example, an examiner would reject as obvious a claim to an isolated gene by combining two references: a document that described a related gene (such as a homologous gene), and a textbook of molecular biology protocols.

The Court of Appeals for the Federal Circuit condemned this approach in the 1995 decision, In re Deuel. "Even if," the court decided, "the existence of general cloning techniques, coupled with knowledge of a protein’s structure, might have provided motivation to prepare a cDNA or made it obvious to prepare a cDNA, that does not necessarily make obvious a particular claimed cDNA. ‘Obvious to try’ has long been held not to constitute obviousness."

For years, examiners had to bolster an allegation of obviousness for a claimed gene or protein by citing prior art that disclosed a related gene or protein, and a suggestion to modify the known molecule to obtain the claimed molecule. A recent Supreme Court case might have changed this.

Supremes Find Driving Force for Invention in Common Sense
KSR International Company developed an adjustable pedal system for cars equipped with cable-actuated throttles. General Motors Corporation contracted with KSR to supply adjustable pedal systems for trucks that had computer-controlled throttles. This presented a problem: computer-controlled throttles do not operate through force transferred from the pedal by a mechanical link, but rather open and close valves in response to electronic signals. To make their pedals compatible with the trucks, KSR added a sensor.

Teleflex Incorporated holds an exclusive license to a patent of Steven J. Engelgau, which covers a pedal assembly with an electronic pedal position sensor. The company sued KSR for patent infringement.

In district court, KSR depicted the Engelgau patent claims as invalid for obviousness. The court agreed, finding one prior art document that taught everything described in the Engelgau patent claim except a sensor that detected the pedal’s position and transmitted data to a computer controlling the throttle. Other prior art documents described uses of sensors. After reviewing the state of the industry, the judge concluded that the combination of electronic sensors and adjustable pedals had been inevitable. Teleflex appealed.

The Federal Circuit reversed the district court, because the court had failed to apply the "teaching, suggestion, or motivation" test. According to the TSM test, a patent claim is only proved obvious if the prior art, the nature of the problem solved by the invention, or the knowledge of a person having ordinary skill in the art reveals some motivation or suggestion to combine prior art teachings.

Federal courts have relied on the TSM test as a check against hindsight-based obviousness analysis. That is, a judge who fails to seek evidence of a suggestion, teaching, or motivation to combine references may unintentionally use a patent application as a blueprint for piecing together the prior art to defeat patentability.

The Federal Circuit decided that the cited references focused on problems sufficiently different from the problem that KSR solved by attaching a sensor, such that a person skilled in the art would not have been motivated to look at those references. The court did not find any evidence that the references taught, suggested, or motivated the combination of a pedal and a sensor. That it might have been obvious to try the combination, the court said, was irrelevant.

KSR appealed the decision to the US Supreme Court. On April 30, the Court reversed the Federal Circuit, scolding that court for analyzing obviousness in such a narrow, rigid manner. Prior art need not contain an explicit motivation or suggestion to combine elements of an invention.

Demands in the relevant field or present in the marketplace can illuminate the obviousness of an invention. "When there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill in the art has good reason to pursue the known options within his or her technical grasp," the Court said. "If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense." Mounting the sensor on the pedal, the Court concluded, had been within the grasp of a person of ordinary skill in the relevant art, and the benefit of doing so would have been obvious.

Reactions to the KSR decision varied. Some biotech patent experts predicted trouble for patents on drugs, nucleic acid molecules, and proteins. Others shrugged off such worries. Biotech inventions tend to be much more complex than a gas pedal. This complexity would shelter the inventions from KSR. Wouldn’t it?

PTO Uses KSR to Hammer NAIL Patent Claims
On May 31, the PTO’s Board of Patent Appeals and Interferences showed that biotech inventions have no immunity against the KSR decision. In Ex parte Kubin, the Board reviewed a patent examiner’s rejection of claims to nucleic acid molecules that encode natural killer cell activation inducing ligand (NAIL) polypeptides, which bind with membrane glycoprotein CD48. The NAIL receptor proteins modulate the activity of natural killer cells, which in turn, stimulates or inhibits the immune response.

The examiner rejected claims as obvious by combining the following references: (1) a patent that disclosed the NAIL protein and suggested a way to isolate NAIL cDNA, (2) a textbook of standard molecular biology protocols, and (3) a scientific report that discloses the nucleotide sequence of the mouse version of the NAIL protein.

Patent applicants argued that the examiner presented the discredited Deuel-type rejection. The argument failed to impress the Board. "The state of the art had unquestionably advanced significantly," the Board declared, "during the ten year period between the time the Deuel application was filed in 1990 and Appellants’ application was filed in 2000." The Board decided that one of ordinary skill in the art would have recognized the value of isolating NAIL cDNA, and would have been motivated to apply conventional techniques to do so.

What about Deuel? The Board took the position that KSR cast doubt on the viability of Deuel. "Under KSR," the Board ominously wrote, "it’s now apparent ‘obvious to try’ may be an appropriate test in more situations than we previously contemplated." The isolation of NAIL cDNA, the Board concluded, was not the product of innovation, but of ordinary skill and common sense.

Sources

Dates: December 12 & 13, 2007

Location: The Hyatt Regency, St. Louis, Missouri.

Purpose: The purpose of the meeting is to bring together academic, industry, government, and other interested scientists to discuss recent and ongoing research on topics related to gene flow from transgenic plants.

The meeting will focus on: 1) within-species gene flow; 2) hybridization and gene introgression between transgenic plants and their sexually compatible relatives; 3) consequences of gene flow from transgenic and non-transgenic plants; 4) approaches to managing gene flow; and 5) modeling gene flow.

Research papers and posters on transgenic plant species of agronomic, horticulture, forestry, and bio-fuels are being solicited.