By Martina Newell-McGloughlin

Agricultural innovation has always involved new, science-based products and
processes that have contributed reliable methods for increasing productivity
and sustainability.
Biotechnology has introduced a new dimension to such innovation, offering
efficient and cost-effective means to produce a diverse array of novel,
value-added products and tools.

The first generation of biotechnology products commercialized were crops
focusing largely on input agronomic traits whose value was largely opaque to
consumers. The coming generations of crop plants can be grouped into four
broad areas, each presenting what, on the surface, may appear as unique
challenges to regulatory oversight.

The present and future focus is on continuing improvement of agronomic
traits such as yield and abiotic stress resistance in addition to the biotic
stress tolerance of the present generation; crop plants as biomass
feedstocks for biofuels and ''biosynthetics''; value-added output traits
such as improved nutrition and food functionality; and plants as production
factories for therapeutics and industrial products. From a consumer
perspective, the focus on valueadded traits, especially improved nutrition,
is of greatest interest.

Developing plants with these improved traits involves overcoming a variety
of technical, regulatory, and indeed perception challenges inherent in the
perceived and real challenges of complex modifications. Both traditional
plant breeding and biotechnologybased techniques are needed to produce
plants with the desired quality traits. Continuing improvements in molecular
and genomic technologies are contributing to the acceleration of product
development. Table I presents examples of crops that have already been
genetically modified with macronutrient and micronutrient traits that may
provide benefits to consumers and domestic animals.

NUTRITION VERSUS FUNCTIONALITY

At a fundamental level, food is viewed as a source of nutrition to meet
daily requirements at a minimum in order to survive but with an ever greater
focus on the desire to thrive. In the latter instance, there is an
evergrowing interest in the functionality of food. Functional foods have
been defined as any modified food or food ingredient that may provide a
health benefit beyond the traditional nutrients it contains. The term
nutraceutical is defined as ''any substance that may be considered a food or
part of a food and provides health benefits, including the prevention and
treatment of disease'' (Goldberg, 1994).

From the basic nutrition perspective, there is a clear dichotomy in
demonstrated need between different regions and socioeconomic groups, the
starkest being overconsumption in the developed world and undernourishment
in less developed countries. Dramatic increases in the occurrence of obesity
and related ailments in developed countries are in sharp contrast to the
chronic malnutrition in many less developed countries.

Both problems require a modified food supply, and the tools of biotechnology
have a part to play. Worldwide, plant-based products comprise the vast
majority of human food intake, irrespective of location or financial status
(Mathers, 2006). In some cultures, either by design or default, plant-based
nutrition actually comprises 100% of the diet. Therefore, it is to be
expected that nutritional improvement can be achieved via modifications of
staple crops.

While the correlative link between food and health is still open to debate,
a growing body of evidence indicates that food components can influence
physiological processes at all stages of life. Functional food components
are of increasing interest in the prevention and/or treatment of at least
four of the leading causes of death in the United States: cancer, diabetes,
cardiovascular disease, and hypertension. The U.S. National Cancer Institute
estimates that one in three cancer deaths are diet related and that eight of
10 cancers have a nutrition/diet component (Block et al., 1992; Potter and
Steinmetz, 1996). Inverse relationships have been observed between
carotenoid-rich foods and certain cancers (Botella- Pavia and Rodriguez-
Conception, 2006).

Other nutrient-related correlations link dietary fat and fiber to the
prevention of colon cancer, folate to the prevention of neural tube defects,
calcium to the prevention of osteoporosis, psyllium to the lowering of blood
lipid levels, and antioxidant nutrients to the scavenging of reactive
oxidant species and protection against oxidative damage of cells that may
lead to chronic disease, to list just a few (Mutch et al., 2005; Mathers,
2006).

Many food components are known to influence the expression of both
structural genes and transcription factors (Tfs) in humans (Go et al., 2005;
Mazzatti et al., 2007). Examples of these phytochemicals are listed in Table
II. The large diversity of phytochemicals suggests that the potential impact
of phytochemicals and functional foods on human and animal health is worth
examining as targets of biotechnology efforts.

On the functionality side, there is a mirror component from the perspective
of the genetic makeup of the individual doing the consuming. This field of
personal response to nutrients is further divided into two thematic subsets
with subtle differences. Nutrigenomics is the prospective analysis of
differences among nutrients in the regulation of gene expression, while
nutrigenetics is the analysis of genetic variations among individuals with
respect to the interaction between diet and disease. These spheres of
enquiry are designed to provide nutritional recommendations for personalized
or individualized nutrition (Brigelius- Flohe and Joost, 2006).

Haplotyping studies are beginning to indicate gender- and ethnicity-specific
polymorphisms that are implicated in susceptibilities to polygenic disorders
such as diabetes, cardiovascular disease, and some cancers (Corthesy-Theulaz
et al., 2005; Mutch et al., 2005; Brigelius-Flohe and Joost, 2006). For
example, several studies have reported some evidence to suggest that the
risks from high intake of well-done meat are higher in fast or presumed fast
acetylator haplotypes (NAT1 and/or NAT2) or in rapid NAT2 (haplotypes) and
CYP1A2 phenotypes. During cooking of muscle meat at high temperature, some
amino acids may react with creatine to give heterocyclic aromatic amines.

Heterocyclic aromatic amines can be activated through acetylation to
reactive metabolites, which bind DNA and cause cancers. Only NAT2 fast
acetylators can perform this acetylation. Studies have shown that the NAT2
fast acetylator genotype had a higher risk of developing colon cancer in
people who consumed relatively large quantities of red meat. Understanding
individual response is at least as complex a challenge as the task of
increasing or decreasing the amount of a specific protein, fatty acid, or
other component of the plant itself (Brigelius-Flohe and Joost, 2006). It is
of little use producing a plant with a supposed nutritional benefit unless
that benefit actually improves the health of humans or animals.

From a health perspective, plant components of dietary interest can be
broadly divided into four main categories, the first two to be enhanced and
the latter two to be limited or removed: macronutrients (proteins,
carbohydrates, lipids [oils], fiber); micronutrients (vitamins, minerals,
functional metabolites); antinutrients (substances such as phytate that
limit the bioavailability of nutrients); and allergens (intolerances and
toxins).

THE TECHNOLOGY

As noted, plants are a treasure trove of interesting and valuable compounds,
since they must glean everything from the spot on earth where they are
rooted and they cannot escape when threatened; therefore, they have evolved
a most impressive panoply of products to thrive in ever-changing
environments despite these limitations.

It is estimated that plants produce up to 200,000 phytochemicals across
their many and diverse members (Oksman-Caldenty and Inze, 2004); obviously,
a more truncated subset of this number is available on our food palate, with
approximately 25,000 different metabolites in general plant foods (Go et
al., 2005). The quality of crop plants, nutritionally or otherwise, is a
direct function of this metabolite content (Memelink, 2004). This brings
metabolomic approaches front and center both in better understanding what
has occurred during crop domestication (lost and silenced traits) and in
designing new paradigms for more targeted crop improvement that are better
tailored to current needs (Hall et al., 2008). In addition, of course, with
modern techniques we have the potential to trawl the rest of that
biochemical treasure trove to find and introgress traits of value that were
outside the scope of previous breeding strategies.

Research to improve the nutritional quality of plants has historically been
limited by a lack of basic knowledge of plant metabolism and the compounding
challenge of resolving the complex interactions of thousands of metabolic
pathways. Both traditional plant breeding and biotechnology techniques are
needed to metabolically engineer plants with desired quality traits.
Metabolic engineering is generally defined as the redirection of one or more
enzymatic reactions to improve the production of existing compounds, produce
new compounds, or mediate the degradation of undesirable compounds. It
involves the redirection of cellular activities by the modification of the
enzymatic, transport, and regulatory functions of the cell. Significant
progress has been made in recent years in the molecular dissection of many
plant pathways and in the use of cloned genes to engineer plant metabolism.

With the tools now being harnessed through the many ''omics'' and
''informatics'' fields, there is the potential to identify genes of value
across species, phyla, and kingdoms. Through advances in proteomics and
glycomics, we are beginning to quantify simultaneously the levels of many
individual proteins and to follow posttranslational alterations that occur
in pathways. Ever more sophisticated metabolomic tools and analysis systems
allow the study of both primary and secondary metabolic pathways in an
integrated fashion (Hall et al., 2008). However, the increasing
sophistication of this tool also demonstrates some anomalies in relying on
this approach.

For example, in potato (Solanum tuberosum), flow injection mass spectrometry
analysis of a range of genotypes revealed genotypic correlations with
quality traits such as free amino acid content (Beckman et al., 2007). Yet,
matrixassisted laser desorption/ ionization chemotyping and gas
chromatography-mass spectrometry profiling of tomato (Solanum lycopersicum)
cultivars have revealed extensive differences in metabolic composition
(sugars, amino acids, organic acids) despite close specific/ genotypic
similarities (Carrari et al., 2006; Fraser et al., 2007). Likewise, with
regard to metabolomic analysis on the consumer side, little is known of the
extent to which changes in the nutrient content of the human diet elicit
changes in metabolic profiles. Moreover, the metabolomic signal from
nutrients absorbed from the diet must compete with myriad nonnutrient
signals that are absorbed, metabolized, and secreted.

Although progress in dissecting metabolic pathways and our ability to
manipulate gene expression in genetically modified (GM) plants has
progressed apace, attempts to use these tools to engineer plant metabolism
have not quite kept pace. Since the success of this approach hinges on the
ability to change host metabolism, its continued development will depend
critically on a far more sophisticated knowledge of plant metabolism,
especially the nuances of interconnected cellular networks, than currently
exists.

This complex interconnectivity is regularly demonstrated. Relatively minor
genomic changes (point mutations, single gene insertions) are regularly
observed following metabolomic analysis, leading to significant changes in
biochemical composition (Bino et al., 2005; Long et al., 2006;
Davidovich-Rikanati et al., 2007). Giliberto et al. (2005) used a genetic
modification approach to study the mechanism of light influence on
antioxidant content (anthocyanin, lycopene) in the tomato cv Moneymaker.
However, other, what on the surface would appear to be more significant,
genetic changes unexpectedly yield little phenotypic effect (Schauer and
Fernie, 2006).

Likewise, there are unexpected outcomes, such as the fact that significant
modifications made to primary Calvin cycle enzymes (Fru- 1,6-bisphosphatase
and phosphoribulokinase) have little effect while modifi- cations to minor
enzymes (e.g. aldase, which catalyzes a reversible reaction) seemingly
irrelevant to pathway flux have major effects (Hajirezaei et al., 1994; Paul
et al., 1995).

These observations drive home the point that a thorough understanding of the
individual kinetic properties of enzymes may not be informative as to their
role (Haake et al., 1998). They also make clear that caution must be
exercised when extrapolating individual enzyme kinetics to the control of
flux in complex metabolic pathways. With these evolving tools, a better
understanding of the global effects of metabolic engineering on metabolites,
enzyme activities, and fluxes is beginning to be developed.

Attempts to modify storage proteins or secondary metabolic pathways have
also been more successful than have alterations of primary and intermediary
metabolism (DellaPenna and Pogson, 2006). While offering great
opportunities, this plasticity in metabolism complicates potential routes to
the design of new, improved crop varieties. Regulatory oversight of
engineered products has been designed to detect such unexpected outcomes in
biotech crops, and as demonstrated by Chassy et al. (International Life
Sciences Institute, 2004a, 2004b, 2008), existing analytical and regulatory
systems are adequate to address novel metabolic modifications in
nutritionally improved crops.

One potential approach to counter some of the complex problems in the
metabolic engineering of pathways involves the manipulation of Tfs that
control networks of metabolism (Kinney and Knowlton, 1998; Bruce et al.,
2000). For example, expression of the maize (Zea mays) Tfs C1 and R, which
regulate the production of flavonoids in maize aleurone layers under the
control of a strong promoter, resulted in a high accumulation rate of
anthocyanins in Arabidopsis (Arabidopsis thaliana), presumably by activating
the entire pathway (Bruce et al., 2000). DellaPenna (Welsch et al., 2007)
found that Tf RAP2.2 and its interacting partner SINAT2 increased
carotenogenesis in Arabidopsis leaves.

Expressing the Tf Dof1 induced the up- regulation of genes encoding enzymes
for carbon skeleton production, a marked increase of amino acid content, and
a reduction of the Glc level in transgenic Arabidopsis (Yanagisawa, 2004),
and the DOF Tf AtDof1.1 (OBP2) up-regulated all steps in the glucosinolate
biosynthetic pathway in Arabidopsis (Skirycz et al., 2006). Such expression
experiments hold promise as an effective tool for the determination of
transcriptional regulatory networks for important biochemical pathways. In
summary, metabolic engineers must not only understand the fundamental
physiology of the process to be affected but also the level, timing,
subcellular location, and tissue or organ specificity that will be required
to ensure successful trait modification. Gene expression can be modulated by
numerous transcriptional and posttranscriptional processes. Correctly
choreographing these many variables is the factor that makes metabolic
engineering in plants so challenging.

As a corollary to these techniques, there are several new technologies that
can overcome the limitation of single gene transfers and facilitate the
concomitant transfer of multiple components of metabolic pathways. One
example is multiple transgene direct DNA transfer, which simultaneously
introduces all of the components required for the expression of complex
recombinant macromolecules into the plant genome, as demonstrated by
Nicholson et al. (2005), who successfully delivered into rice (Oryza sativa)
plants four transgenes that represent the components of a secretory
antibody.

More recently, Carlson et al. (2007) constructed a minichromosome vector
that remains autonomous from the plant's chromosomes and stably replicates
when introduced into maize cells. This work makes it possible to design
minichromosomes that carry cassettes of genes, enhancing the ability to
engineer plant processes such as the production of complex biochemicals.
Christou and Kohli (2005) demonstrated that gene transfer using minimal
cassettes is an efficient and rapid method for the production of transgenic
plants containing and stably expressing several different transgenes. Since
no vector backbones are required, thus preventing the integration of
potentially recombinogenic sequences, they remain stable across generations.
These groups' constructions facilitate the effective manipulation of
multigene pathways in plants in a single transformation step, effectively
recapitulating the bacterial operon model in plants.

More recently, Christou and colleagues (Agrawal et al., 2005; Christou and
Kohli, 2005) demonstrated this principle by engineering the entire
carotenoid pathway in white maize, visually creating a latter day rainbow
equivalent of Indian maize depending on the integrated transgene complement.
This system has an added advantage from a commercial perspective in that
these methods circumvent problems with traditional approaches that not only
limit the amount of sequences transferred but may disrupt native genes or
lead to poor expression of the transgene, thus reducing both the numbers of
transgenic plants that must be screened and the subsequent breeding and
introgression steps required to select a suitable commercial candidate.

The agronomically improved GM crops now being grown on more than 114 million
ha around the world are products of the application of these technologies to
crop plants (James, 2008). They generally involve the relatively simple task
of adding a single gene or a small number of genes to plants. These genes in
the main function outside of the plant's primary metabolic processes and
thus have little or no effect on the composition of the plants. In addition
to numerous success stories, some studies, as noted, even with these simpler
modifications, have yielded unanticipated results.

For example, the concept of gene silencing emerged from the unexpected
observation that adding a chalcone synthase gene to increase color in
Petunia spp. resulted instead in the switching off of color, producing white
and variegated flowers (Napoli et al., 1990). This initially unexpected
observation, now termed RNA interference, is one of the principal tools
applied in everything from the analysis of molecular evolution to designing
targeted therapeutics. In plants, it has now been turned to advantage in the
first generation, developing robust virus resistance through coat protein
posttranscriptional gene silencing, and in nutritional improvement, such as
switching off of expression of an allergen in soybean (Glycine max).

To summarize, omics-based strategies for gene and metabolite discovery,
coupled with high-throughput transformation processes and automated
analytical and functionality assays, have accelerated the identi- fication
of product candidates. Identifying rate-limiting steps in synthesis could
provide targets for genetically engineering biochemical pathways to produce
augmented amounts of compounds and new compounds. Targeted expression will
be used to channel metabolic flow into new pathways, while gene-silencing
tools can reduce or eliminate undesirable compounds or traits or switch off
genes to increase desirable products (Liu et al., 2002; Herman et al., 2003;
Davies, 2007). In addition, molecular marker-based breeding strategies have
already been used to accelerate the process of introgressing trait genes
into high-yielding germplasm for commercialization. In the interest of
space, Table I summarizes the work done to date on specific applications in
the categories listed above. The following sections provide brief examples
of some specific applications under those categories. MACRONUTRIENTS

Protein

Protein energy malnutrition is the most lethal form (Food and Agriculture
Organization, 2006) of malnutrition and affects every fourth child
worldwide, according to the World Health Organization (2006). The Food and
Agriculture Organization estimates that 850 million people worldwide suffer
from undernutrition, to which insufficient protein in the diet is a
significant contributing factor. Most plants have a poor balance of
essential amino acids relative to the needs of animals and humans.

The cereals (maize, wheat [Triticum aestivum], rice, etc.) tend to be low in
Lys, whereas legumes (soybean, pea [Pisum sativum]) are often low in the
sulfur-rich amino acids Met and Cys. Poultry, swine, and other nonruminant
animals have specific requirements for each of the essential amino acids.
The primary requirements for maize and soybean meal-based diets are Lys in
mammals and Met in avian species. High-Lys and high-Met maize and soybeans
could allow diet formulations that reduce animal nitrogen excretion by
providing an improved balance of essential amino acids. When they are out of
balance, the amino acid in excess results in increased nitrogen excretion.
That balance can be accomplished now, but only by adding costly synthetic
Lys and Met to the diet. Successful examples of improving amino acid balance
to date include high-Lys maize (Eggeling et al., 1998; O'Quinn et al., 2000)
canola (Brassica napus), and soybean (Falco et al., 1995). Free Lys is
significantly increased in high-Lys maize by the introduction of the dapA
gene from Corynebacterium glutamicum, which encodes a form of
dihydrodipicolinate synthase that is insensitive to Lys feedback inhibition.
As a cautionary tale in this successful system, substantial increases in Lys
only occurred in plants in which flux increased to such a level that the
first enzyme of the catabolic pathway became saturated (Brinch-Pedersen et
al., 2000), again illustrating the potential complexities of metabolic
regulation. Consumption of foods made from these crops potentially can help
to prevent malnutrition in developing countries, especially among children.

Another method of modifying storage protein composition is to introduce
heterologous or homologous genes that code for proteins containing elevated
levels of the desired amino acid, such as sulfur- containing Met and Cys or
Lys. An interesting solution to this is to create a completely artificial
protein containing the maximum number of the essential amino acids Met, Thr,
Lys, and Leu in a stable, helical conformation designed to resist proteases
to prevent degradation. This was done by Beauregard et al. (1995), who
created an 11-kD synthetic protein, MBI, with 16% Met and 12% Lys, which
they introduced into soybean using vectors targeted to seed protein storage
bodies using appropriate leader sequences and seed-specific promoters
(Simmonds and Donaldson, 2000). This was also achieved in a nonseed food
crop, sweet potato (Ipomoea batatas), modified with an artificial storage
protein gene (Egnin and Prakash, 1997). These transgenic plants exhibited 2-
and 5-fold increases in the total protein content in leaves and roots,
respectively, over that of control plants. A significant increase in the
level of essential amino acids, such as Met, Thr, Trp, Ile, and Lys, was
also observed (Egnin and Prakash, 1997; International Life Sciences
Institute, 2008). A key issue is to ensure that the total amount and
composition of storage proteins is not altered to the detriment of the
development of the crop plant when attempting to improve amino acid ratios
(Rapp, 2002). Since this is a completely novel protein to the human diet, it
will be subjected to extensive review; yet, as demonstrated by the
International Life Sciences Institute (2008), the existing regulatory and
analytic methods are appropriate and sufficient to achieve this aim.

Young et al. (2004) used a novel approach to indirectly increase protein and
oil content. They used a bacterial cytokinin- synthesizing isopentenyl
transferase enzyme, under the control of a self-limiting
senescence-inducible promoter, to block the loss of the lower floret,
resulting in the production of just one kernel composed of a fused endosperm
with two viable embryos. The presence of two embryos in a normal-sized
kernel leads to the displacement of endosperm growth, resulting in kernels
with an increased ratio of embryo to endosperm content. The end result is
maize with more protein and oil and less carbohydrate (Young et al., 2004;
International Life Sciences Institute, 2008).

Carbohydrates

As the somewhat disputed notion of a glycemic index has supplanted Atkins as
the indicator of choice when addressing carbohydrates in the diet, it has
become clear to the public that not all carbohydrates are created equal.
While it is still something of a value judgment to describe ''good'' versus
''bad'' carbohydrates, there are clear clinical indications of the value of
polymeric versus simple sugars. Plants are effective at making both
polymeric carbohydrates (e.g. starches and fructans) and individual sugars
(e.g. Suc and Fru). The biosynthesis of these compounds is sufficiently
understood to allow the bioengineering of their properties or to engineer
crops to produce polysaccharides not normally present.

Fructans are an important ingredient in functional foods because evidence
suggests that they promote a healthy colon (as a prebiotic agent) and help
reduce the incidence of colon cancer. Sevenier et al. (1998) reported
high-level fructan accumulation in a transgenic sugar beet (Beta vulgaris)
without adverse effects on growth or phenotype. This work has implications
both for the commercial manufacture of fructans and also for the use of
genetic engineering to obtain new products from existing crops. Fructans
consisting of linear b-(1/2)-linked Fru polymers are called inulins.
Hellwege et al. (2000) produced a transgenic potato synthesizing the full
spectrum of inulin molecules naturally occurring in globe artichoke (Cynara
scolymus) roots. A similar approach is being used to derive soybean
varieties that contain some oligofructan components that selectively
increase the population of beneficial species of bacteria (e.g.
bifidobacteria) in the intestines of humans and certain animals and inhibit
the growth of harmful species of bacteria (e.g. Escherichia coli 0157:H7,
Salmonella spp., etc.; Bouhnik et al., 1999) When colonic bacteria ferment
dietary fiber or other unabsorbed carbohydrates, the products are
short-chain saturated fatty acids. These short-chain fatty acids may enhance
the absorption of minerals such as iron, calcium, and zinc; induce
apoptosis, preventing colon cancer; and inhibit 3-hydroxy-3-
methylglutaryl-CoA reductase, thus lowering lowdensity lipoprotein (LDL)
production (Watkins et al., 1999; German et al., 2005).

The amylose-amylopectin ratio has the greatest in- fluence on the
physicochemical properties of the starch, and for many applications it is
desirable to have a pure or enriched fraction of either amylopectin or
amylose. Schwall et al. (2000) created a potato producing very high-amylose
(slowly digested) starch by inhibiting two enzymes that would normally make
the amylopectin type of starch that is rapidly digested. This ''resistant
starch'' is not digested in the small intestine but is fermented in the
large intestine by microflora. Clinical studies have demonstrated that
resistant starch has similar properties to fiber and has potential
physiological benefits in humans (Yue and Waring, 1998; Richardson et al.,
2000).

Fiber

Fiber is a group of substances chemically similar to carbohydrates, but
nonruminant animals including humans poorly metabolize fiber for energy or
other nutritional uses. Fiber is only found in foods derived from plants and
never occurs in animal products. Fiber provides bulk in the diet, such that
foods rich in fiber offer satiety without contributing significant calories.
Current controversies aside, there is ample scientific evidence to show that
prolonged intake of dietary fiber has various positive health benefits,
especially the potential for reduced risk of colon and other types of
cancer. Fiber type and quantity are undoubtedly under genetic control,
although this topic has been little studied. The technology to manipulate
fiber content and type by genetic engineering would be a great benefit to
the health status of many individuals who refuse, for taste or other
reasons, to include adequate amounts of fiber in their daily diet. For
example, fiber content could be added to more preferred foods or the more
common sources of dietary fiber could be altered for greater health
benefits.

Nonruminant animals do not produce enzymes necessary to digest
cellulose-based plant fiber. Plants low in fiber should yield more
digestible and metabolizable energy and protein and less manure and methane
when fed to monogastric species (North Carolina Cooperative Extension
Service, 2000). Vermerris and Bout (2003) cloned a Brown midrib gene that
encodes caffeic acid O-methyltransferase, a lignin- producing enzyme. They
generated mutants that give rise to plants that contain significantly lower
lignin in their leaves and stems, leading to softer cell walls compared with
the wild type. The plant- softening mutations improve the digestibility of
the food, and livestock also seem to prefer the taste. Such improved fiber
digestibility in ruminants should have significant beneficial effects,
because the efficiency of digestion of most high-fiber diets for ruminants
is far from optimized. Novel Lipids

Gene technology and plant breeding are combining to provide powerful means
for modifying the composition of oilseeds to improve their nutritional value
and provide the functional properties required for various food oil
applications. Genetic modification of oilseed crops can provide an abundant,
relatively inexpensive source of dietary fatty acids with wide-ranging
health benefits. Production of such lipids in vegetable oil provides a
convenient mechanism to deliver healthier products to consumers without the
requirement for significant dietary changes. Major alterations in the
proportions of individual fatty acids have been achieved in a range of
oilseeds using conventional selection, induced mutation, and, more recently,
posttranscriptional gene silencing. Examples of such modified oils include
low- and zero-saturated fat soybean and canola oils, canola oil containing
medium-chain fatty acids, high-stearic acid canola oil (for trans-fatty
acidfree products), high-oleic acid (monounsaturated) soybean oil, and
canola oil containing the polyunsaturated fatty acids gamma-linolenic acid
(GLA; 18:3 n-6) and stearidonic acid (SDA; C18:4 n-3), very-long-chain fatty
acids (Zou et al., 1997), and omega-3 fatty acids (Yuan and Knauf, 1997).
Many of these modified oils are being marketed, and a number of countries
have a regulatory systemin place for the premarket safety reviewof novel
foods produced through conventional technology.

Medium chain fatty acids range from 6 to 10 carbons long and are only minor
components of natural foods, with the exception of coconut and palm kernel
oils. When medium-chain triglycerides (MCTs) are substituted for long-chain
triglycerides (LCTs) in the diet, animals gain less weight, store less
adipose tissue, and experience an increase in metabolic rate (Baba et al.,
1982; Geliebter et al., 1983). Mice fed diets with MCTs have also been shown
to possess increased endurance in swimming tests over that of mice fed diets
with LCTs (Fushiki et al., 1995). Expression of an acyl-ACP thioesterase
cDNA from Cuphea hookeriana in seeds of transgenic canola, an oilseed crop
that normally does not accumulate any capric and caprylic acids, resulted in
a dramatic increase in the levels of these two MCTs (Dehesh et al., 1996).

Edible oils rich in monounsaturated fatty acids provide improved oil
stability, flavor, and nutrition for human and animal consumption. Oleic
acid (18:1), a monounsaturate, can provide more stability than the
polyunsaturates linoleic acid (18:2) and linolenic acid (18:3). From a
health aspect, the monounsaturates are also preferred. Antisense inhibition
of oleate desaturase expression in soybean resulted in oil that contained
more than 80% oleic acid (23% is normal) and had a significant decrease in
polyunsaturated fatty acids (Kinney and Knowlton, 1998). High-oleic-acid
soybean oil is naturally more resistant to degradation by heat and oxidation
and so requires little or no postrefining processing (hydrogenation),
depending on the intended vegetable oil application. In 2009, DuPont hopes
to introduce soybean oil composed of at least 80% oleic acid, linolenic acid
of about 3%, and over 20% less saturated fatty acids than commodity soybean
oil. Monsanto's Vistive contains less than 3% linolenic acid, compared with
8% for traditional soybeans. These result in more stable soybean oil and
less need for hydrogenation and concomitant reduction in trans-fatty acids.
For lower trans- fats in livestock products, Nicholas Roberts and Richard
Scott at AgResearch (New Zealand) are researching an ingenious method to
prevent plantderived cis-polyunsaturated fatty acids from being transformed
into saturated trans-fats in the rumen by borrowing an adaptation from
plants themselves. They are engineering forage crops, such as grasses and
legumes, with polyoleosin genes from sesame (Sesamum indicum), which should
result in triglycerides being encapsulated within self-assembling
polyoleosin micelles, thus sealing them off from bacterial activity during
transit though the rumen (O'Neill, 2007).

A key function of alpha-linolenic acid is as a substrate for the synthesis
of longer chain omega-3 fatty acids found in fish, eicosapentaenoic acid
(EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3), which play an
important role in the regulation of inflammatory immune reactions and blood
pressure, brain development in utero, and, in early postnatal life, the
development of cognitive function. SDA (C18:4n-3), EPA, and DHA also possess
anticancer properties (Christensen et al., 1999; Smuts et al., 2003; Reiffel
and McDonald, 2006). Research indicates that the ratio of n-3 to n- 6 fatty
acidsmay be as important to health and nutrition as the absolute amounts
present in the diet or in body tissues. Current western diets tend to be
relatively high in n-6 fatty acids and relatively low in n-3 fatty acids.
Production of a readily available source of long-chain polyunsaturated fatty
acids, specifically omega- 3 fatty acids, delivered in widely consumed
prepared foods could deliver much needed omega-3 fatty acids to large
sectors of the populationwith skewed n-6:n-3 ratios. In plants, the
microsomal omega-6 desaturase-catalyzed pathway is the primary route of
production of polyunsaturated lipids. Ursin (2003) has introduced the
Delta-6 desaturase gene from a fungus (Mortierella), succeeding in producing
&ohgr-3 in canola. In a clinical study, James et al. (2003) observed that
SDA was superior to alpha-linolenic acid as a precursor by a factor of 3.6
in producing EPA, DHA, and docosapentaenoic acid (C22:5n-3). Transgenic
canola oilwas obtained that contains more than 23%SDA, with an overall
n-6:n-3 ratio of 0.5.

However, not all &ohgr-6 fatty acids are created equal. GLA (C18:3n-6) is an
&ohgr-6 fatty acid with health benefits that are similar and complementary
to the benefits of &ohgr-3 fatty acids, including anti-inflammatory effects,
improved skin health, and weight loss maintenance (Schirmer and Phinney,
2007). Arcadia Biosciences (2008) has engineered GLA safflower oil, with up
to 40% GLA, essentially quadrupling the levels obtained in source plants
such as evening primrose (Oenothera biennis) and borage (Borago
officinalis).

Structural lipids also have positive health benefits; for example, in
addition to their effect in lowering cholesterol, membrane lipid
phytosterols have been found to inhibit the proliferation of cancer cells by
inducing apoptosis and G1/S cell cycle arrest through the
3-hydroxy-3-methylglutaryl-CoA reductase target mentioned previously (Awad
and Fink, 2000). In addition to this and the above, specialty oils may also
be developed with further pharmaceutical and chemical feedstock applications
in mind.

Vitamins and Minerals

Micronutrient malnutrition, the so-called hidden hunger, affects more than
half of the world's population, especially women and preschool children in
developing countries (United Nations System Standing Committee on Nutrition,
2004). Even mild levels of micronutrient malnutrition may damage cognitive
development, lower disease resistance in children, and increase the
incidence of childbirth mortality. The costs of these deficiencies, in terms
of diminished quality of life and lives lost, are enormous (Pfeiffer and
McClafferty, 2007). The clinical and epidemiological evidence is clear that
select minerals (iron, calcium, selenium, and iodine) and a limited number
of vitamins (folate, vitamins E, B6, and A) play a significant role in the
maintenance of optimal health and are limiting in diets.

Using various approaches, vitamin E levels are being increased in several
crops, including soybean, maize, and canola, while rice varieties are being
developed with the enhanced vitamin A precursor, beta-carotene, to address
vitamin A deficiency that leads to macular degeneration and affects
development. Ameliorating another major deficiency in less developed
countries, minerals such as iron and zinc have also been addressed. Other
targets include improved iron content, ferritin-rich lettuce (Lactuca
sativa), bioavailable phosphorus, and divalent ions released from phytate,
folate- enriched tomatoes, and isoflavonoids (DellaPenna, 2007; Yonekura-
Sakakibara et al., 2007).

As with macronutrients, one way to ensure an adequate dietary intake of
nutritionally beneficial phytochemicals is to adjust their levels in plant
foods. Until recently, such work had been hindered by the diffi- culty of
isolating the relevant genes (e.g. for vitamin biosynthesis). However, the
advent of genomics during the past few years has provided new routes for
such work. Using nutritional genomics, DellaPenna (Shintani and DellaPenna,
1998; DellaPenna, 2007) isolated a gene, gamma-tocopherol methyltransferase,
that converts the lower activity precursors to the highest activity vitamin
E compound, alpha-tocopherol. With this technology, the vitamin E content of
Arabidopsis seed oil has been increased nearly 10-fold and progress has been
made to move the technology to agricultural crops such as soybean, maize,
and canola (DellaPenna, 2007).

Rice is a staple that feeds nearly half the world's population, but milled
rice does not contain any beta-carotene or its carotenoid precursors.
Integrating observations from prokaryotic systems into their work enabled
researchers to clone the majority of carotenoid biosynthetic enzymes from
plants during the 1990s. Taking advantage of this, Golden rice, with
beta-carotene expression in the endosperm, was created (Ye et al., 2000).
The health benefits of the original Golden rice and, especially, Golden rice
II are well reviewed (International Life Sciences Institute, 2008). A
similar method was used by Monsanto to produce beta-carotene in canola. Iron
is the most commonly deficient micronutrient in the human diet, and iron
deficiency affects an estimated 1 to 2 billion people. Anemia, characterized
by low hemoglobin, is the most widely recognized symptom of iron deficiency,
but there are other serious problems, such as impaired learning ability in
children, increased susceptibility to infection, and reduced work capacity.
A research group led by Goto et al. (2000) and another led by Lucca et al.
(2002) employed the gene for ferritin, an iron-rich soybean storage protein,
under the control of an endosperm-specific promoter. Grain from transgenic
rice plants contained three times more iron than normal rice. To increase
the iron content in the grain further, the researchers focused on iron
transport within the plant (Lucca et al., 2002). Drakakaki et al. (2005)
demonstrated endosperm- specific coexpression of recombinant soybean
ferritin and Aspergillus phytase inmaize,which resulted in significant
increases in the levels of bioavailable iron. A similar result was achieved
with lettuce (Goto et al., 2000).

Functional Metabolites

Unlike for vitamins and minerals, the primary evidence for the
health-promoting roles of phytochemicals comes from epidemiological studies,
and the exact chemical identities of many active compounds have yet to be
determined. However, for select groups of phytochemicals, such as
nonprovitamin A, carotenoids, glucosinolates, and phytoestrogens, the active
compound or compounds have been identified and rigorously studied. A great
irony of nature is that the body's natural metabolism involving oxygen also
produces a host of toxic compounds called ''free radicals.'' These compounds
can harm body cells by altering molecules of protein and fat and by damaging
DNA. Antioxidants counteract, or neutralize, the harmful effects of free
radicals.

Epidemiologic studies have suggested a potential benefit of the carotenoid
lycopene in reducing the risk of prostate cancer, particularly the more
lethal forms of this cancer. Five studies support a 30% to 40% reduction in
risk associated with high tomato or lycopene consumption in the processed
form in conjunction with lipid consumption, although other studies with raw
tomatoes were not conclusive (Giovinazzo et al., 2005). As a nonpolar
carotenoid, lycopene is more soluble in a lipid base; in addition,
carotenoid- binding proteins are broken down during processing, leading to
greater bioavailability. While modifying polyamines to retard tomato
ripening, Mehta et al. (2002) discovered an unanticipated enrichment in
lycopene, with levels up by 2- to 3.5-fold compared with the conventional
tomatoes. This is a substantial enrichment, exceeding that so far achieved
by conventional means. This novel approach may work in other fruits and
vegetables.

Stilbenes, including resveratrol (3,5,4'-trihydroxystilbene), are phenolic
natural products that accumulate in a wide range of plant species, including
pine (Pinus spp.), peanut (Arachis hypogaea), rhubarb (Rheum spp.), and
grape (Vitis vinifera; Tropf et al., 1994). Resveratrol inhibits platelet
aggregation and eicosanoid synthesis and is thought to contribute to
improved heart function and lower blood cholesterol, based on
epidemiological studies (Frankel et al., 1993; Pace-Asciak et al., 1995;
Wieder et al., 2001). It was shown to have ''chemopreventive'' activity,
preventing the formation of tumors in mouse skin bioassays, and, therefore,
may help reduce cancer rates in humans (Jang et al., 1997). More recent
studies appear to demonstrate that it mimics the life-extending effect on
rodents of severe caloric restriction. This diet extends the life span of
rodents by 30% to 50%, and even if it is started later it has a benefit
proportionate to the remaining life span. The method of action is believed
to be in protecting the sirtuins, genes implicated in DNA modification and
life extension (Baur, 2006). The April 2008 purchase of Sirtris by Glaxo
Smith Kline (Pollack, 2008) demonstrates that big pharma is now showing an
interest in the arena of food functionality. Resveratrol glucoside
production has been achieved in alfalfa (Medicago sativa), wheat, kiwi
(Actinidia deliciosa), and tomato (Stark-Lorenzen et al., 1997; Hipskind and
Paiva, 2000; Kobayashi et al., 2000; Szankowski et al., 2003; Niggeweg et
al., 2004; Giovinazzo et al., 2005; Shin et al., 2006).

Other phytochemicals of interest include the flavonoids, such as tomatoes
expressing chalcone isomerase, which show increased contents of the
flavanols rutin and kaempferol glycoside; glucosinolates and their related
products, such as indole-3 carbinol; catechin and catechol; isoflavones,
such as genistein and daidzein; anthocyanins; and some phytoalexins (Table
I). A comprehensive list of phytochemicals is outlined in Table II. Although
there is a growing knowledge base indicating that elevated intakes of
specific phytochemicals may reduce the risk of disease, such as certain
cancers, cardiovascular diseases, and chronic degenerative diseases
associated with aging, further research and epidemiological studies are
still required to prove definitive relationships.

ANTINUTRIENTS, ALLERGENS, AND TOXINS

Plants produce many defense strategies to protect themselves from predators,
and many of these, such as resveratrol and glucosinate, which are primarily
pathogen-protective chemicals, also have demonstrated beneficial effects for
human and animal health. Many, however, have the opposite effect. For
example, phytate, a plant phosphate storage compound, is an antinutrient, as
it strongly chelates iron, calcium, zinc, and other divalent mineral ions,
making them unavailable for uptake. Nonruminant animals generally lack the
phytase enzyme needed for digestion of phytate. Poultry and swine producers
add processed phosphate to their feed rations to counter this. Excess
phosphate is excreted into the environment, resulting in water pollution.
When low-phytate soybean meal is utilized along with low-phytate maize for
animal feeds, the phosphate excretion in swine and poultry manure is halved.
A number of groups have added heat- and acid-stable phytase from Aspergillus
fumigatus to make the phosphate and liberated ions bioavailable in several
crops (Lucca et al., 2002). To promote the reabsorption of iron, a gene for
a metallothioneinlike protein has also been engineered. Low-phytate maize
was commercialized in the United States in 1999 (Wehrspann, 1998). Research
indicates that the protein in low-phytate soybeans is also slightly more
digestible than the protein in traditional soybeans. In a poultry feeding
trial, better results were obtained using transgenic plant material than
with the commercially produced phytase supplement (Keshavarz, 2003). Poultry
grew well on the engineered alfalfa diet without any inorganic phosphorus
supplement, which shows that plants can be tailored to increase the
bioavailability of this essential mineral.

Other antinutrients that are being examined as possible targets for
reduction are trypsin inhibitors, lectins, and several other heat-stable
components found in soybeans and other crops. Likewise, strategies are being
applied to reduce or limit food allergens (albumins, globulins, etc.),
malabsorption and food intolerances (gluten), and toxins (glycoalkaloids,
cyanogenic glucosides, phytohemagglutinins) in crop plants and undesirable
aesthetics such as caffeine (Ogita et al., 2003). Examples include changing
the levels of expression of the thioredoxin gene to reduce the intolerance
effects of wheat and other cereals (Buchanan et al., 1997). Using RNA
interference to silence the major allergen in soybean (p34, a member of the
papain superfamily of Cys proteases) and rice (14- to 16-kD allergenic
proteins by antisense; Tada et al., 1996), blood serum tests indicate that
p34- specific IgE antibodies could not be detected after consumption of
gene-silenced beans (Herman et al., 2003).

Biotechnology approaches can be employed to down-regulate or even eliminate
the genes involved in the metabolic pathways for the production,
accumulation, and/or activation of these toxins in plants. For example, the
solanine content of potato has already been reduced substantially using an
antisense approach, and efforts are under way to reduce the level of the
other major potato glycoalkaloid, chaconine (McCue et al., 2003). Work has
also been done to reduce cyanogenic glycosides in cassava through expression
of the cassava enzyme hydroxynitrile lyase in roots (Siritunga and Sayre,
2003). When ''disarming'' plant natural defenses in this way, we need to be
cognizant of potentially increased susceptibility to pests and diseases, so
the base germplasm should have input traits to counter this.

IMPLICATIONS FOR SAFETY ASSESSMENT

On the surface, it may appear that the greater complexity involved in
modifying the nutritional content of crop plants would necessitate more
rigorous oversight than the simpler modifications. However, extensive
research reported previously (International Life Sciences Institute, 2004a,
2004b) and updated in a more recent case study analysis (International Life
Sciences Institute, 2008) indicates that existing oversight systems are more
than adequate. Trait modifications with the addition of one or two genes
that do not act on central or intermediary metabolism produce targeted,
predictable outcomes, whereas major modi- fications of metabolic pathways
can produce unanticipated effects. Therefore, it is very encouraging that
the ever-evolving and increasingly sensitive and discriminating analytical
technologies have been able to detect and assess the safety of these
unanticipated effects. In addition, regulatory oversight of GM products has
been designed to detect such unexpected outcomes. At a very fundamental
level, a recent report (Baack and Rieseberg, 2007) on genome-wide analyses
of introgression from oak (Quercus spp.) to fruit flies indicates that a
substantial fraction of genomes are malleable. Hybridization gives rapid
genomic changes, chromosomal rearrangements, genome expansion, differential
expression, and gene silencing (transposable elements). In the context of
this sea of malleability, reports have demonstrated that GM crops have a
composition more similar to the isogenic parental strain used in their
development than to other breeding cultivars of the same genus and species
and in some instances even the location in which they are grown, and on
occasion the latter ''terroir'' effect demonstrated greater variation than
breeding strategy. This effect has been observed at the proteome level for
potato (Lehesranta et al., 2005), tomato (Corpillo et al., 2004), and wheat
(Shewry, 2003). Parallel results have been observed at the metabolomic level
for wheat (Baker et al., 2006) and potato (Catchpole et al., 2005). As more
metabolic modifications are introduced, we must continue to study plant
metabolism and the interconnected cellular networks of plant metabolic
pathways to increase the likelihood of predicting pleiotropic effects that
may occur as a result of the introduced genetic modification.

THE FUTURE OF CROP BIOTECHNOLOGY

Research to improve the nutritional quality of plants has historically been
limited by a lack of basic knowledge of plant metabolism and the almost
insurmountable challenge of resolving complex branches of thousands of
metabolic pathways. With the tools now available to us through the fields of
genomics and bioinformatics, we have the potential to fish in silico for
genes of value across species, phyla, and kingdoms and subsequently to study
the expression and interaction of transgenes on tens of thousands of
endogenous genes simultaneously. With advances in proteomics, we should also
be able to simultaneously quantify the levels and interactions of many
proteins or follow posttranslational alterations that occur. With these
newly evolving tools, we are beginning to get a handle on the global effects
of metabolic engineering on metabolites, enzyme activities, and fluxes.
Right now, for essential macronutrients and micronutrients that are limiting
in various regional diets, the strategies for improvement are clear and the
concerns, such as pleiotropic effects and safe upper limits, are easily
addressed. However, for many other health- promoting phytochemicals, clear
links with health benefits remain to be demonstrated. Such links, if
established, will make it possible to identify the precise compound or
compounds to target and which crops to modify to achieve the greatest
nutritional impact and health benefits. The achievement of this aim will be
a truly interdisciplinary effort, requiring expertise and input from many
disparate fields, ranging from the obvious human physiology and plant
research to the less obvious ''omics'' and analytic fields.

With these emerging capabilities, the increase in our basic understanding of
plant secondary metabolism during the coming decades will be unparalleled
and will place plant researchers in the position of being able to modify the
nutritional content of major and minor crops to improve many aspects of
human and animal health and well-being.

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