J. PlantPhysiol. Vol. 133. pp. 336-339(1988) Invertases from Rust-infected Wheat Leaves HARI B. KRISHNAN and STEVEN G. PUEPPKE Department of Plant Pathology, 108 Waters Hall, University of Missouri, Columbia, MO 65211, USA Received March 3,1988 . Accepted May 24,1988 Summary Total invertase activity increases three-fold in leaves of Triticum aestivum L. infected with Puccinia strii- formis. Levels of invertase activity are elevated about 17 days after infection, when fungal spores can be detected on the leaves. DEAE-cellulose chromatography resolves the activity into two different peaks, termed Peak I and Peak II. The two forms differ in their pH optima, KM values for sucrose, and their re- sponse to inhibition by citrate. The KM and pH optima of Peak I invertase from healthy leaves are differ- ent than those from infected leaves. The relative amounts of the two forms of the enzyme also vary in healthy versus infected leaves. The stimulation of total invertase activity in rust-infected leaves is due to a net increase in Peak I activity. Key words: DEAE-cellulose chromatography, invertase, Puccinia striiformis, rust infection, Triticum aes- tivum. Abbreviations: DTT, DL-dithiothreitol; EDTA, ethylenediaminetetraacetic acid; DEAE-cellulose, diethylaminoethyl cellulose. Introduction Infection of higher plants by fungal pathogens often results in drastic changes in plant metabolism (Misaghi, 1982). One of the striking changes associated with fungal infection is the accumulation of host photosynthate, e.g., starch and hexose sugars. Starch generally appears as granules in chloroplasts of photosynthetic tissues (Akai et aI., 1967). Starch accumula- tion in infected plants has been attributed to an increase in ADP-glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis (MacDonald and Strobel, 1970). The increase in the enzyme activity is probably a response to fungal-induced changes in effector molecules, e.g_, inorganic phosphate or 3-phosphoglyceraldehyde. Accumulation of re- ducing sugars in plants infected with certain biotrophic fungi probably reflects an increase in acid invertase, an enzyme which cleaves sucrose into glucose and fructose (Long et aI., 1975; Billett et aI., 1977; Callow et aI., 1980). Detailed in- formation, however, is not available on the physical and kinetic properties and the possible regulation of this enzyme in diseased tissue_ We consequently undertook this study to investigate the kinetic properties of invertases and explore © 1988 by Gustav Fischer Verlag, Stuttgart their role in the accumulation of reducing sugars in diseased tissue. Materials and Methods Triticum aestivum L. cv. Tyee (obtained from the Department of Agronomy, Washington State University, USA) was used in our studies. Plants were grown in a greenhouse under controlled tem- perature, relative humidity and light, essentially as described by MacDonald and Strobel (1970). Primary leaves of 10-day-old plants were inoculated with uredospores of Puccinia striiformis (wheat yel- low rust) as described by Sharp (1965). This isolate of Puccinia strii- formis was obtained from Dr. A. Qayoum, Department of Plant Pathology, Washington State University, USA. The host plants then were placed in a dark dew chamber for 48 h to facilitate spore germination, prior to return to the greenhouse. Control plants were similarly treated except that they were inoculated with water in- stead of uredospores. Extraction of Invertase Five g of leaf tissue was ground to a fine powder under liquid ni- trogen with a mortar and pestle. The powder was extracted with