Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40-to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae , the FaNES1 -expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants.

The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as a-pinene and b-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of a-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of a-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.

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Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of P12-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by P12, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by P12. P12-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.The involvement of endogenous proteinaceous proteinase inhibitors (PIs) in plant defense against leaf-feeding insects has been well recognized (1, 2). Experiments with artificial diets and a wide range of insects confirmed the antinutritional effects of proteinaceous PIs (3-7), although some negative results were also reported (8). The expression of heterologous PIs in transgenic tobacco plants provided final confirmation in planta for their roles as resistance factors, although protection was only partial (9, 10).Recently, the effectiveness of proteinase inhibitors was suggested to depend on the affinity or specificity of an inhibitor for the main gut proteinases of an insect (4,11,12), but the mechanism of action and effect of proteinase inhibitors are only partially understood. Broadway and Duffey (3) showed that gut proteinase activities of Spodoptera exigua and Heliothis zea were similar or increased when larvae were chronically exposed to high levels of potato proteinase inhibitor II (PI2) or soybean trypsin inhibitor in artificial diets. The simple scenario that growth rates were reduced due to reduced rates of proteolysis (13) was, therefore, dismissed. Instead, these results were interpreted by Broadway and Duffey (3) to suggest that a feedback mechanism was leading to the hyperproduction of proteinases to compensate for the loss of activity, which in turn led to the depletion of essential amino acids and finally resulted in retarded growth rates.Our aim was to establish to what extent and how PI2 expressed in tobacco leaves would affect larval growth and digestive physiology of S. exigua. Our data show that S. exigua larvae adapt to PIs by induction of gut proteinase activity that is insensitive to inhibition.t MATERIALS AND METHODSPlant Transformation. A full-length PI2 cDNA clone, p303.5 1, was selected from a tuber-specific Solanum tuberosum cv. Bintje cDNA library by using p303 as a probe (14, 15). The region encoding the N-terminal part was amplified by PCR using primers 152FO (5'-GCGGGATCCACCATGGC...

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