Biochemical Tests

Activities of specific enzymes can provide rapid and sensitive indicators of nutrient deficiencies in plants (54). Deficiencies of micronutrients can lead to inhibited activities of enzymes for which the nutrient is part of the specific enzyme molecule. Assays of enzymatic activity can help identify deficiencies when visual diagnosis does not distinguish between deficiencies that produce similar symptoms (55), when soil analysis does not determine if nutrients enter plants, or when plant analysis does not reflect the concentration of a nutrient needed for physiological functions (56). The enzymatic assays do not give concentrations of nutrients in plants, but the enzyme activity gives an indication of sufficiency or deficiency of a nutrient. The assay can be run on deficient tissue or on tissue into which the suspected element has been infiltrated to reactivate the enzymatic system. The assays are run on crude extracts or leaf disks to provide quick tests (57).

Peroxidase assays have been used to distinguish iron deficiency from manganese deficiency in citrus (Citrus spp. L.) (55,58). Peroxidases are heme-containing enzymes that use hydrogen peroxide as the electron acceptor to catalyze a number of oxidative reactions. In this application, during iron deficiency, peroxidase activity is inhibited, whereas during manganese deficiency peroxidase activity may be increased. Iron is a constituent of peroxidase, but manganese is not. Kaur et al. (59) reported associations of limited catalase and peroxidase activities with iron deficiency in chickpeas (Cicer arietinum L.). Leidi et al. (60) evaluated catalase and peroxidase activities as indicators of iron and manganese nutrition for soybeans (Glycine max Merr.). Nenova and Stoyanov (61) reported that intense iron deficiency resulted in low activities of peroxidase, catalase, and nitrate reductase in corn (Zea mays L.). Ranieri et al. (62) observed a suppression of peroxidase activity in iron-deficient sunflower (Helianthus annuus L.). On the other hand, carbonic anhydrase has been employed to identify zinc deficiency in citrus (63), sugarcane (Saccharum officinarum L.) (64), black gram (Vigna mungo L.) (65), and pecan (Carya illinoinensis Koch) (66). Zinc deficiency was associated with a decrease in messenger RNA for carbonic anhydrase along with a decrease in carbonic anhydrase activity in rice (Oryza sativa L.) (67). In another assay, alcohol dehydrogenase was twice as high in roots of zinc-sufficient rice as in zinc-deficient rice, and activity of alcohol dehydrogenase in roots was correlated with zinc concentration in leaves (68). Ascorbic acid oxidase assays have been used in the identification of copper deficiency in citrus (69). Molybdenum deficiency has been associated with low levels of nitrate reductase activity in citrus (70). Polle et al. (71) reported that the activities of superoxide dismutase and some other protective enzymes increased in manganese-deficient leaves of Norway spruce (Picea abies L.).

Applications of enzymatic assays for the micronutrient status of plants have not been adopted widely in agronomic or horticultural practice, although interest in usage may be increasing as is shown by the number of investigations associating enzymatic activity with plant nutrients. The peroxidase test in the assessment of iron deficiency has perhaps been employed more than other assays (57,72). Macronutrients have numerous functions in plants, and association of specific enzymatic activity with deficiencies of macronutrients is difficult. However, some assays have been developed, such as nitrate reductase activity for assessment of nitrogen deficiency, glutamate-oxaloacetate aminotransferase for phosphorus deficiency, and pyruvic kinase for potassium deficiency (54). Measurement of pyruvic kinase activity may also be useful for establishing the optimum balance between potassium, calcium, and magnesium concentrations in tissues (73).