Inhibitors of Digestion

The insect gut is the obvious target for orally acting insecticidal agents to be expressed in plants (e.g., Ref. 50). The membranes lining the gut appear to be the target for the Cry toxins and probably the Vips, through their pore-forming or lytic activities. The insect's digestive biochemistry is another target. Disruption should reduce nutrient intake, resulting in mortality or at least in slowing the growth of larvae to such an extent that they are subject to a greater degree of parasitism and predation in the field. Different components of the digestive system can be targeted depending on whether the food source is rich in protein or carbohydrate. Plants already appear to have adopted this as a defense strategy, as many accumulate high levels of pro-teinaceous inhibitors of digestive enzymes in their storage organs, such as seeds, fruits, tubers, and corms. A number of plants contain wound-inducible inhibitors of insect digestive enzymes that are switched on during herbivory.

1. Protease Inhibitors

To utilize the proteins present in their diets, insects rely predominantly on the serine proteases trypsin, chymotrypsin, and elastase. Trypsin is the dominant protease type in lepidopteran larvae. Plants accumulate high levels of trypsin inhibitors in their seed. These are thought to act as insect antifeedants as well as nitrogenous seed reserves. A number of genes for trypsin-chy-motrypsin inhibitors have been cloned and expressed in plants (reviewed in Refs. 51 and 52). These transgenic plants demonstrated antifeedant activity against a number of lepidopteran larvae. Larval growth rates were reduced, and in some cases mortality was markedly increased. Levels of the inhibitors had to be over 1% of leaf soluble protein in order to afford any protection. Similar results have been reported in transgenic rice, wheat, tobacco, lucerne, and poplar plants with a variety of serine and cysteine protease inhibitors from both plants and insects (reviewed in Ref. 7).

For example, a gene for a trypsin-chymotrypsin inhibitor from the giant taro plant, Alocasia mycrorrhiza (53), was expressed in transgenic tobacco. This inhibitor retarded the growth of Helicoverpa armigera, but the larvae rapidly altered their digestive physiology to compensate for the presence of the inhibitor in their diet. Although very active against mammalian chymotrypsin, the giant taro inhibitor turned out to be active only against H. armigera trypsin; its chymotrypsin inhibitor domain proved to be inactive in the insect. Insects fed on transgenic plants containing the giant taro inhibitor (or artificial diets containing purified inhibitor) adapted by elevating the levels of chymotrypsin and elastase in the midgut to counter the reduction in trypsin activity (54). Similar responses have been noted for other protease inhibitors, although the mechanisms of adaptation can differ (55-57). This process of adaptation may be responsible for the failure of any transgenic plants containing protease inhibitors to reach the marketplace, although there are a couple of reports of field tolerance, e.g., to stem borers in rice expressing the cowpea trypsin inhibitor genes (58). It will be important to include multiple protease inhibitors with differing specificities in order to develop robust insect tolerance in plants. For example, the stigma-specific protease inhibitor from Nicotiana alata (ornamental tobacco) is produced as a single polyprotein that is subsequently processed into six different trypsin and chymotrypsin inhibitors (59,60) and the gene confers tolerance to H. punctigera and H. armigera when expressed in transgenic plants (61,62).

2. Alpha Amylase Inhibitors

Some insects have diets rich in starch and utilize the enzyme «-amylase to digest the starch to simple sugars, a-Amylase inhibitors (aAIs) are produced in the seeds of many plants and may confer some insect tolerance (63). Seeds of many domesticated crops have been selected for reduced levels of their inhibitors of digestion, as they are antinutritional components for human or animal consumption. This has made some grain crops more vulnerable to attack, particularly in storage. Field peas are widely grown across southern Australia for both human and animal feed, but the green pods are prone to attack by the bruchid beetle, pea weevil (Bruchus pisorum). Some legumes are highly tolerant to attack by beetles and produce high levels of aAI, among other antinutritional compounds in their seeds. A gene for aAI from the common bean Phaseolus vulgaris was linked to a strong seed-specific promoter and used to generate transgenic field peas expressing levels of aAI as high as those in bean seeds (64). The inhibitor was stably expressed through to at least the T5 generation and the pea seeds were resistant to damage by pea weevil (Fig. 3), both in the glasshouse and in the field (65,66), as well as to a number of pests of stored grain (cowpea weevil, Callosobruchus maculatus and azuki bean weevil, C. chinensis) (64). The seeds were shown to have no detectable antinutritional effects in animal feeding trials (67). The same construct in transgenic azuki beans confers resistance to three stored grain pests, C. chinensis, C. maculatus, and C. analis, but not to the South American bruchid Zabrotes subfasciatus (68). This technology therefore holds promise for protecting grain legumes against certain coleopteran pests.

Figure 3 Field peas in Australia are attacked by a number of pests including the pea weevil, Bruchus pisorum (A), which also affects the stored grain. Larvae burrow into the seed and consume much of the internal tissues. They complete their development in the seed, emerging as adults and leaving a large exit hole. This reduces the nutritional value of the seed for animals and the economic value of stored grain. (B) Transgenic peas (cultivar "Laura") expressing the bean a-amylase gene (right) prevent larval development in the seed and very few adults emerge from seed grown in an infested field and stored for a few months compared with the control nontransformed variety (left) that is almost completely infested with larvae and emerged adults. (C) Closeup of the stored grain showing the extensive damage to the conventional variety and limited damage to the transformed "Laura" (left).

Figure 3 Field peas in Australia are attacked by a number of pests including the pea weevil, Bruchus pisorum (A), which also affects the stored grain. Larvae burrow into the seed and consume much of the internal tissues. They complete their development in the seed, emerging as adults and leaving a large exit hole. This reduces the nutritional value of the seed for animals and the economic value of stored grain. (B) Transgenic peas (cultivar "Laura") expressing the bean a-amylase gene (right) prevent larval development in the seed and very few adults emerge from seed grown in an infested field and stored for a few months compared with the control nontransformed variety (left) that is almost completely infested with larvae and emerged adults. (C) Closeup of the stored grain showing the extensive damage to the conventional variety and limited damage to the transformed "Laura" (left).

3. Lectins and Assorted Insecticidal Proteins

Few insecticidal proteins have been found that are active against sap-sucking insects such as plant hoppers, leafhoppers, and aphids, yet these insects represent a significant component of the pest problem in many crops. Fre quently, they are vectors for serious viral diseases devastating world agriculture. Sugar binding proteins, or lectins, have been purified from many plants, and a number of lectins have been reported (69) to have toxic effects on sap-sucking insects that derive most of their energy reserves from the sugars being transported in the phloem. The mannose binding lectin from the bulb of snowdrop (Galanthus nivalis) is toxic to the peach-potato aphid (Myzus persicae), the glasshouse potato aphid (Aulacorthum solani), and the rice brown planthopper (Nilaparvata lugens). When the snowdrop gene is expressed in transgenic tobacco from either a constitutive or a phloem-specific promoter, the lectin has been shown to be ingested by the aphids, as it can be detected in their honeydew. The gene confers some tolerance to aphids in whole plant bioassays (70). Wang and Guo (71) have expressed both the gene for the CrylAb Bt toxin and the gene for snowdrop lectin in transgenic tobacco and have generated plants showing high insecticidal activity to both II armigera and M. persicae, suggesting that it may be possible to stack genes active against both lepidopteran and sucking pests.

When three genes (CrylAc, Cry2A, and the gene for snowdrop lectin) were stacked in transgenic rice, protection against three important pests (a leaf folder, a stem borer, and a plant hopper) was achieved (72). Similarly, a pea lectin in transgenic tobacco was thought to have an additive effect with protease inhibitors in conferring tolerance to lepidopteran larvae, again highlighting the importance of multiple components of the defensive system (73). Snowdrop lectin in transgenic plants was also reported to have anti-nutritional effects on some lepidopterans (74), as was wheat germ agglutinin and jacalin (reviewed in Ref. 75). Many of these lectins are generally toxic, and experiments with transgenic potatoes containing the snowdrop lectin have been implicated in downstream effects on predators of the target sucking insect pests (76). Subsequent analyses, however, indicate that this is due not to the toxicity of the lectin to the insect predator but to the lowered nutritional value of the affected aphids (77). No effects were reported for the development of a wasp parasitoid fed on Lacanobia oleracea larvae raised on GNA-expressing potatoes, and there was a significant reduction in plant damage, at least in the glasshouse, using the combination of transgenic plant and parasitic wasp (78). However, as yet, no lectin-expressing transgenic plant has reached the marketplace. The biotin binding proteins avidin and streptavidin have also been shown to have strong insecticidal activity and have been expressed at high levels in the kernels of maize (79). Although originally produced for the commercial production of these proteins, the plants show good levels of resistance to a number of stored grain pests of corn (80). A thorough analysis of the human food safety issues associated with avidin and streptavidin corn will be necessary before the grain can enter the food chain.

Cholesterol oxidase (EC 1.1.3.6) from a Streptomyces species is a potent oral inhibitor of the cotton boll weevil (Anthonomonas grandis grandis Boheman) and has reduced activity against a number of lepidopteran species. It is thought to act through the oxidation of cholesterol in the membranes of the midgut brush border, and low concentrations cause mortality in larvae and reduced fertility in adults. The reason for the specificity of the protein is unclear as both susceptible and tolerant insect species have similar cholesterol levels. Gut pH may be involved, as the pH optimum of the enzyme does not favor its activity in the high pH found in the midguts of lepidopteran larvae (81). The gene for cholesterol oxidase has been cloned and expressed as enzymatically active protein in plant cells (82). The specificity of cholesterol oxidase makes it an attractive target for expression in transgenic cotton to enhance the current conventional approaches to boll weevil control. However, the success of the current boll weevil eradication program in the United States, which relies on conventional technology, may be a contributing factor in the lack of commercial incentive to progress the cholesterol oxidase gene as an insect control option in cotton. Other enzymes, chitinase and lipoxygenase, have also been reported to confer some insec-ticidal activity, but this has not been demonstrated in transgenic plants (referenced in Ref. 7).

C. Secondary Metabolites

Plants produce an abundance of secondary chemicals that serve as a defense against insects. In some cases, they act as olfactory or oral cues to both beneficial and pest species. Some of the defensive chemicals are being evaluated with the aim of manipulating the genes for their biosynthesis in transgenic plants to enhance the host plant resistance or for the biological production of commercial pesticides (83,84). Thomas et al. (85) created transgenic tobacco plants expressing the Catharanthus roseus gene for tryptophan decarboxylase (TDC), which converts tryptophan to the insecticidal indole alkaloid tryptamine. Sweet potato whiteflies (Bemisia tabaci) fed on these plants showed a dramatic decrease in fertility. However, others have reported undesirable plant phenotypes as well as elevated tryptamine levels (86).

Alterations in metabolite profiles brought about by overexpressing key branch point enzymes, such as TDC, can have both desirable and undesirable pleiotrophic effects, as has been reported in transgenic potatoes and canola (87,88). In potato, overexpression of TDC resulted in an altered balance of key substrates in the shikimate and phenypropanoid pathways, leading to reduced levels of phenolics and other defense compounds. These plants were more susceptible to fungal pathogens. In canola, reductions in the available tryptophan pool resulted in reductions in the levels of tryptophan-derived indole glucosinolates as well as increased tryptamine. Complex regulation of secondary metabolic pathways may also have allowed Smigocki et al. (89,90) to inadvertently elevate secondary metabolites in transgenic tobacco plants expressing the cytokinin-producing ipt gene from Agrobacterium tu-mefaciens driven by a wound-inducible promoter, either through the activation of cytokinin-regulated biosynthetic genes or through changes in pools of core metabolites shared between secondary metabolism and cytokinin biosynthesis. Their plants showed a considerably enhanced tolerance to tobacco hornworm (Manduca sexta) and the green peach aphid (Myzus persi-cae).

Transgenic plants with altered disease tolerance were generated by Hain et al. (91) when they introduced into tobacco two genes from grapevine encoding the enzyme stilbene synthase that converts 4-coumaroyl CoA and malonyl CoA into the toxic phytoalexin resveratrol. Plants that produced resveratrol constitutively showed enhanced tolerance to the fungal pathogen Botrytis cinerea, but this was associated with altered flower color and male sterility (92), highlighting the importance of regulating the production of toxic defense chemicals. Using the native grapevine genes, however, with their own highly regulated pathogen and wound-inducible promoters has allowed the production of transgenic tomatoes without the deleterious side effects (93). These plants were protected from infection by the fungus Phy-tophthora infestans, but not by B. cinerea and Alternaria solani, even though these pathogens induced the accumulation of resveratrol.

Before we can routinely engineer pest and pathogen tolerance by manipulating secondary metabolism in plants, we must develop a better understanding of the complex interactions between different metabolic pathways and their regulation.

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Detox Diet Basics

Detox Diet Basics

Our internal organs, the colon, liver and intestines, help our bodies eliminate toxic and harmful  matter from our bloodstreams and tissues. Often, our systems become overloaded with waste. The very air we breathe, and all of its pollutants, build up in our bodies. Today’s over processed foods and environmental pollutants can easily overwhelm our delicate systems and cause toxic matter to build up in our bodies.

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