Publisher Summary This chapter describes the preparation and extraction of nitrate reductase from higher plants. For preparation of nitrate reductase, chlorophyllous lamina tissue from illuminated plants well supplied with nitrate is normally used as a source material because of its high activity. However, monchlorophyllous organs, such as corn scutella or roots, have been used. Soybean leaves, corn scutella, and cultured rice seedlings are the best known sources of NAD(P)H nitrate reductase. For extraction of the enzyme, the material is homogenized for 30 to 90 seconds in a medium of 1 mM EDTA, 1 to 25 mM cysteine, and 25 mM potassium phosphate, adjusted to a final pH of 8.8 with KOH. Six milliliters of grinding medium are added for each gram of fresh weight of tissue. The homogenate is pressed through four layers of cheesecloth or a single layer of Miracloth and the filtrate centrifuged for 15 minutes at 30,000 g. The supernatant fluid is then decanted through glass wool and used for assays. The homogenates and extracts are kept cold (2° to 3°) throughout. The optimum concentration of cysteine must be verified for each type of tissue and homogenization technique. Glutathione and dithiothreitol are usually slightly more effective (10%) than cysteine.

[24] Nitrate reductase from higher plants

Increasing interest in the stress physiology of higher plants has, in the last few years, greatly expanded our knowledge concerning the effects of stresses such as drought, freezing, nutrient deficiencies, disease and insect attack on plant metabolism. This work, however, has concentrated on the influences of stress on the basic processes of growth and development (primary metabolism). Very little attention has been paid to the changes in secondary metabolism that may be occurring. Information on the effects of stress conditions on secondary metabolites has come mainly from research efforts to maximize the yield of active constituents from herb, spice and drug plants and from attempts to reduce the levels of toxins in certain food and forage crops. Advances in our understanding of the functional roles of secondary metabolites in plants, however, and new information about how pest resistance is altered by stress should stimulate additional interest in the responses of secondary metabolites to stress.

Changes in the Levels of Plant Secondary Metabolites Under Water and Nutrient Stress

Atmospheric ozone: Formation and effects on vegetation

Weeds are known to cause enormous losses due to their interference in agroecosystems. Because of environmental and human health concerns, worldwide efforts are being made to reduce the heavy reliance on synthetic herbicides that are used to control weeds. In this regard the phenomenon of allelopathy, which is expressed through the release of chemicals by a plant, has been suggested to be one of the possible alternatives for achieving sustainable weed management. The use of allelopathy for controlling weeds could be either through directly utilizing natural allelopathic interactions, particularly of crop plants, or by using allelochemicals as natural herbicides. In the former case, a number of crop plants with allelopathic potential can be used as cover, smother, and green manure crops for managing weeds by making desired manipulations in the cultural practices and cropping patterns. These can be suitably rotated or intercropped with main crops to manage the target weeds (including parasitic ones) selectively. Even the crop mulch/residues can also give desirable benefits. Not only the terrestrial weeds, even allelopathy can be suitably manipulated for the management of aquatic weeds. The allelochemicals present in the higher plants as well as in the microbes can be directly used for weed management on the pattern of herbicides. Their bioefficacy can be enhanced by structural changes or the synthesis of chemical analogues based on them. Further, in order to enhance the potential of allelopathic crops, several improvements can be made with the use of biotechnology or genomics and proteomics. In this context either the production of allelochemicals can be enhanced or the transgenics with foreign genes encoding for a particular weed-suppressing allelochemical could be produced. In the former, both conventional breeding and molecular genetical techniques are useful. However, with conventional breeding being slow and difficult, more emphasis is laid on the use of modern techniques such as molecular markers and the selection aided by them. Although the progress in this regard is slow, nevertheless some promising results are coming and more are expected in future. This review attempts to discuss all these aspects of allelopathy for the sustainable management of weeds.

Allelopathic Interactions and Allelochemicals: New Possibilities for Sustainable Weed Management

Phenolics are one of the many secondary metabolites implicated in allelopathy To establish that allelopathy functions in a natural ecosystem, the allelopathic bioassay must be ecologically realistic so that responses of appropriate bioassay species are determined at relevant concentrations It is important to isolate, identify, and characterize phenolic compounds from the soil However, since it is essentially impossible to simulate exact field conditions, experiments must be designed with conditions resembling those found in natural systems It is argued that allelopathic potential of phenolics can be appreciated only when we have a good understanding of 1) species responses to phenolic allelochemicals, 2) methods for extraction and isolation of phenolic allelochemicals, and 3) how abiotic and biotic factors affect phenolic toxicity

Plant phenolics in allelopathy

Stress modification of allelopathy of helianthus annuus l. debris on seed germination

Foliar sensitivity of soybeans to ozone as related to several leaf parameters

When added to soil,Helianthus annuus debris from plants grown under various nutrient stresses in the greenhouse and field had a significant negative effect onAmaranthus retroflexus seedling dry weight production. The regression models for dry weight versus total phenolic compounds and the variation in N, P, and K contents of theHelianthus debris were significant. It was possible to partially simulate debris inhibition ofA. retroflexus seedling dry weight production when chlorogenic acid alone was added to the soil instead ofHelianthus debris. The inhibition ofAmaranthus growth byHelianthus debris and chlorogenic acid was not evident when nutrient solution was applied to the soil.

Stress modification of allelopathy ofHelianthus annuus L. debris on seedling biomass production ofAmaranthus retroflexus L.

Soybeans [Glycine max (L.) Merr.] were harvested at various periods after a 2 h exposure to determine the effects of ozone on nitrate reductase and selected metabolites related to nitrate reduction. Ozone initially depressed the concentrations of reducing sugars, amino acids, and nitrate reductase activity. On subsequent days following exposure, amino acid and protein levels were higher than the respective controls while the levels of reducing sugars and nitrate reductase activity returned to control levels. The ozone depression of nitrate reduction was not the result of a direct ozone reaction with the nitrate reductase protein but rather an interference with reactions that supplied the NAD(P)H needed for nitrate reduction.

Ozone Alteration of Nitrate Reduction in Soybean

Soybean [Glycine max (L.) Merr.] cultivars Dare and Hood were exposed to ozone (980 μg/m3) for 2 h to determine if differences in cultivar sensitivity were associated with differential activation of selected enzymes. The first trifoliate leaves of the cultivars were in similar stages of development (14 days from sowing) when exposed. The ozone treatment increased the activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, polyphenol oxidase and peroxidase above the control in both cultivars within 30 h after exposure. However, the activity of all these enzymes increased several hours sooner in Dare (sensitive) than in Hood (tolerant). Our data suggest that the differential sensitivity of these two cultivars to ozone could be based on the differential ozone induced activation of the enzymes involved in the lesion formation process.

Roger C. Fites

Papers

Stress modification of allelopathy of helianthus annuus l. debris on seed germination

Foliar sensitivity of soybeans to ozone as related to several leaf parameters

Stress modification of allelopathy ofHelianthus annuus L. debris on seedling biomass production ofAmaranthus retroflexus L.

Ozone Alteration of Nitrate Reduction in Soybean

Differential Foliar Sensitivity of Soybean Cultivars to Ozone Associated with Differential Enzyme Activities