Genes containing the cauliflower mosaic virus 35S promoter fused to open reading frames coding for tomato proteinase inhibitor I, tomato inhibitor II, and potato inhibitor II were expressed in transgenic tobacco plants. Inhibitor I and II proteins were identified by immunoblotting and quantified by immunoradial diffusion. Both inhibitors exhibited the molecular weights found for the native proteins in their natural environments. Extracts of leaves from transformed plants contained inhibitory activities against trypsin and chymotrypsin that reflected the levels of inhibitor I or H protein present. The results demonstrate that in tobacco leaves the introns of both inhibitor I and inhibitor II genes were excised correctly and that pre and prepro inhibitor I and II proteins were correctly processed. Growth of Manduca sexta larvae (tobacco hornworms) feeding on leaves of transgenic plants containing inhibitor II, a powerful inhibitor of both trypsin and chymotrypsin, was significantly retarded, compared to growth of larvae fed untransformed leaves. Levels of inhibitor H protein as low as 50 jg/g of tissue moderately affected larval growth, whereas levels above 100 ,ug/g severely reduced growth. The presence of tomato inhibitor I protein, a potent inhibitor of chymotrypsin but a weak inhibitor of trypsin, in transgenic tobacco leaves had little effect on the growth of the larvae. These experiments indicated that trypsin inhibitory activity, but not chymotrypsin inhibitory activity, was mainly responsible for the inhibition of larval growth.Potato and tomato plants contain two small multigene families that code for two powerful inhibitors of serine proteinases, called inhibitor I (monomer Mr 8100) and inhibitor II (monomer Mr 12,300) (1). Inhibitor I is an inhibitor of chymotrypsin that only weakly inhibits trypsin at its single reactive site (1), whereas inhibitor II contains two reactive sites, one of which inhibits trypsin and the other of which inhibits chymotrypsin (1). Members of both gene families are expressed in leaves in response to chewing insects or other severe mechanical damage (2). Both inhibitors are synthesized as precursors and undergo posttranslational modification (3)(4)(5) to form the mature proteins, which are sequestered in the vacuole (6). These inhibitors are thought to help defend the plant, by reducing the digestibility and nutritional quality of the leaves, against insect predators (7). Both cDNAs (4, 5) and genes (8, 9) that encode inhibitors I and II have been isolated and characterized. These are now being employed to further investigate the role of proteinase inhibitors in plant defense.It was previously shown (10) that transformation of tobacco plants with a gene encoding a cowpea trypsin inhibitor was able to confer increased resistance against predation by Heliothis virescens larvae. In order to assess the potential of inhibitor I and inhibitor II for increasing the natural defenses of crop plants through transformation, genes encoding these inhibitors were stably introduced ...

A full understanding of an elementary reaction must include the functional dependence of the reaction rate (kinetics) on the driving force (thermodynamics), which should include all pertinent variables such as stereoelectronic factors and medium effects. Unimolecular dissociations of radical ions into radicals and ions (mesolytic scissions) ['] provide an opportunity to develop such a quantitative theory for a bond-breaking process. We present here the experimentally determined free-energy relationship for mesolytic cleavages of C-C bonds in 7c radical ions spanning a range of over 40 kcalmol-' for the driving force and over 17 powers of ten for the rate constants. We show that these fragmentations have low intrinsic barriers if the stereoelectronic factors are optimal, and that a parabolic, Marcus-type free-energy relation may be fitted to the data.The unpaired electron in the x radical ion resides in a x-type orbital localized on one bonding partner of the scissile bond. The fragmentation reaction is accompanied by the redistribution of electron density to the forming fragments according to one of the modes of electron apportionment"] [Equations (a)-(d)]. As illustrated by the curved arrows, the processes described by Equations (a) and (c) formally correspond to homolysis; the alternatives (b) and (d) are equivalent to heterolysis.We have prepared['. 3 * 4 1 a series of directly observable radical ions of substrates containing two benzylic units (1-5) that undergo unimolecular C-C bond scissions according to Equations (a)-(d). The radical ions were designed to provide optimal overlap of the x system bearing the unpaired electron with the scissile C-CIn all cases p r~b e d ,~~.~] the C-C bond cleavage reactions were irreversible under the reaction conditions, as was found in experiments with stereochemically pure evythrojthreo or mesoldl radical ion precursors. The kinetic data (Table 1) photolysis, pulse radiolysis, time-resolved fluorescence spectroscopy, and cyclic voltammetry (CV). The primary fragments observed corresponded to the thermodynamically predicted apportionment of electrons.[39 41 In all cases, the isolated products were consistent with fragmentation of the central C-C bond. In our systems AGh has been approximated by experimentally accessible AGZ. The approximation is valid (k 3 kcalmol-') if the radical coupling has only a small activation barrier (that is, the reaction is diffusion-limited) and the thermolysis is carried out under conditions selected to minimize recombination within the solvent cage. The kinetic and thermodynamic data at 300 K (Table 1) were used to construct the free-energy relationships of Figure 1. The slope of the solid line corresponds to unity; that is, it depicts endergonic reactions with no kinetic "overhead". The overhead is defined here as the difference between AG: and AG,,, and represents the barrier to the reverse (exergonic) reaction (that is,

In order to test the generality of the method for the synthesis of substituted 3-tert-butyldioxindoles 6, a range of ringsubstituted N-pivaloylanilines 5 was subjected to identical reaction conditions without optimization of individual cases. The yields of isolated products were good (Table 1). It is known that dioxindoles such as 2 and 6 can be reduced to the corresponding indoles.['.

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