Insect entrapment

We have cloned the 59 kD trichome PPO from a cDNA library constructed from epidermal mRNA (H. Yu and J. Steffens, unpublished data) and are currently using this clone as a probe to isolate the genomic DNA encoding the S. berthaultii trichome PPO (S. Newman and J. Steffens, unpublished data). These studies may allow us to increase the insect entrapping abilities of cultivated potato, which has retained low Type A trichome densities and no longer possesses the biochemical machinery... 

Cytochrome P-450 enzymes have been isolated from a variety of mammalian tissues, insects, plants, yeasts and bacteria. The P-450 cytochromes (Gunter and Turner, 1991) are membrane bound mono-oxygenase enzymes which catalyse oxygen atom transfer to entrapped non-polar substrates. The binding of carbon monoxide to the enzyme produces a split in the 420 nm Soret band to give bands at 364 and 450 nm. The absorption at 450 nm distinguishes the hemoprotein from all others and hence provides... 

Polyphenol Oxidases. Plant trichomes and their exudates confer resistance to a variety of insects (54-56). In solanaceous plants, such as the tomato and potato, trichomes contain polyphenol oxidases and catecholic phenolics (e.g., caffeic and chlorogenic acids), which contribute to resistance to a variety of insect pests. In the potato plant, the polyphenol oxidases and phenolics are separated in different trichomes. When insects, such as aphids or leaf hoppers, walk across the surface of the plant they break the two types of trichomes. Trichomal fluids are liberated and, upon mixing, polymerize as a result of polyphenol oxidase activity on catechols, forming an often lethal adhesive trap for the insects (52,58) In tomato plants, the polyphenol oxidase and chlorogenic acid are separated by intracellular compartments, but upon breakage of trichomes by insects, polymerization and physical entrapment occurs (54). 

Adheslve entrapment. Mortality and immobilization of aphids increases with a rise in density of Type A and Type B trichomes and with increased volume of Type A trichome glands. Insects landing on the foliage first encounter Type B trichome exudate which forms an adhesive coating on the tarsi. This accelerates... 

Trichome PPO appears to be an example of an evolutionary modification of an existing plant enzyme for use as an insect defense mechanism. PPO enzymes, of molecular weight 45,000, are localized in the thylakoid and are nearly ubiquitous in tissues of plants (12,18.). Unlike other known nuclear encoded chloroplast proteins, the 45 kD thylakoid PPO, whose function is unknown, is translated at its mature size of 45 kD, and does not possess a transit peptide sequence (19). In contrast, the 5 9 kD trichome PPO is translated as a 67 kD precursor and localized in the leucoplasts of trichome and outer epidermal cells. Immunological and primary sequence similarities between the 59 kD trichome PPO and the 45 kD thylakoid PPO underscore the close evolutionary relationship between these two proteins. The apparent advantage of the very high concentration of PPO in the trichome is the high initial rate of catalysis which results upon trichome rupture, facilitating the entrapment of mobile insects. 

The second important event in this discovery is an observation that 4 and certain of its close analogs, notably RH-5849, had a profound effect on insect molting. Examination of intoxicated caterpillars revealed that these insects were somehow totally imable to shed their old cuticles at the conclusion of the molt. Instead, they remained entrapped within their old unshed cuticle, ultimately dying of starvation and/or blood loss, the so-called "double headcapsule" state. 

Techniques of attachment, entrapment, and encapsulation are most widely used for cell immobilization with support materials, which are illustrated in Figure 7.1. These techniques can be applied to essentially all the viable or nonviable wholecell systems of potential interest microorganisms, plant cells, and mammalian and insect cells [2]. Although most of the principles associated with enzyme immobilization are directly applicable to cell immobilization, due to the complete difference in size and biochemical properties between enzymes the cells, the relative importance of these methods is considerably different [10]. 

Immobilized insect cells -Galactosidase Spodopterafrugiperda (Sf-9) Entrapped within reticulated [114,115]... 

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