Rapid generation of superoxide and accumulation of H2O2 is a characteristic early feature of the hypersensitive response following perception of pathogen avirulence signals. Emerging data indicate that the oxidative burst reflects activation of a membrane-bound NADPH oxidase closely resembling that operating in activated neutrophils. The oxidants are not only direct protective agents, but H2O2 also functions as a substrate for oxidative cross-linking in the cell wall, as a threshold trigger for hypersensitive cell death, and as a diffusible signal for induction of cellular protectant genes in surrounding cells. Activation of the oxidative burst is a central component of a highly amplified and integrated signal system, also involving salicylic acid and perturbations of cytosolic Ca2+, which underlies the expression of disease-resistance mechanisms.

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Recognition of an avirulent pathogen triggers the rapid production of the reactive oxygen intermediates superoxide (O2-) and hydrogen peroxide (H2O2). This oxidative burst drives crosslinking of the cell wall, induces several plant genes involved in cellular protection and defence, and is necessary for the initiation of host cell death in the hypersensitive disease-resistance response. However, this burst is not enough to support a strong disease-resistance response. Here we show that nitric oxide, which acts as a signal in the immune, nervous and vascular systems, potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen intermediates and functions independently of such intermediates to induce genes for the synthesis of protective natural products. Moreover, inhibitors of nitric oxide synthesis compromise the hypersensitive disease-resistance response of Arabidopsis leaves to Pseudomonas syringae, promoting disease and bacterial growth. We conclude that nitric oxide plays a key role in disease resistance in plants.

Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border. Among ‫ف‬ 5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development. Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes. INTRODUCTIONEthnobotany and limited screens of medicinal plants indicate that the huge repertoire of chemical diversity in plants contains many potentially useful bioactive structural principles for developing novel drugs, flavors, fragrances, and other specialty chemicals. Unfortunately, these complex natural products usually occur in very low abundance and with a restricted tissue distribution. Moreover, almost all of this phytochemical biodiversity resides in exotic, uncultivated species. Whereas drugs such as taxol, vinblastine, and vincristine illustrate the potential of plants as sources of new drugs, the development of rational approaches for the generation of useful amounts of complex natural products for industrial evaluation remains an unsolved problem. In particular, an intense 30-year effort using cell and tissue cultures from medicinal plants has failed to generate useful quantities of complex products for the commercial production of established drugs in vitro or for high-throughput, multiplex screening of phytochemicals (Facchini and Deluca, 1995;McCaskill and Croteau, 1998). This failure probably reflects the stringent spatial and temporal transcriptional controls governing the biosynthesis of specific natural products during plant development (Fowler, 1983;Robins, 1994;Facchini and Deluca, 1995). Transgenic manipulation to override these genetic controls thus may provide the key to enhancing natural product biosynthesis for industrial evaluation and exploitation.Activation tagging with the enhancer from the cauliflo...