The relationship of peroxidases to an indudble disease-resistance mechanism involving lignification of leaf epidermal cell walls was studied. Reed canargrass (Phalaris arundinacea L.) leaf discs were inoculated with Helminthosporium avenae Eidam and floated on water. In inoculated discs, the activity of soluble, ionic wall-bound and covalent wal-bound peroxidases was about twice the level of activity in noninoculated discs. The increase was attributable to increases in activity of three cathodic isoperoxidases and to the appearance of a new cathodic isoperoxidase. Peroxidase activity in cryostat microtome sections of inoculated discs was histochemically localized in the wail near the site of attempted penetration. When inoculated discs were floated on solutions of cydoheximide (25 ,lg/ml), increases in peroxidase activity were inhibited, and the fungus penetrated the tissue. The inhibition of peroxidase activity was related to inhibition of cathodic isoperoxidase activity. Anodic isoperoxidase activity did not show changes in response to inoculation or cydoheximide treatment.It was suggested that the resistance mechanism in P. arundinacea involves an induction of cathodic isoperoxidases in challenged tissue. These peroxidshes may function in the biosynthesis of lignin at the site of attempted penetration.Plant peroxidases have been extensively investigated with regard to their role in resistance to fungal, viral, and bacterial diseases (14, 15). Peroxidases have also been strongly implicated in lignin biosynthesis (5, 13). Recent studies have implicated lignin formation in the cell walls of reed canarygrass and wheat as a resistance response (11,12,16,17). Vance and Sherwood (17) showed that when the resistance response in reed canarygrass was inhibited by cycloheximide, lignin biosynthesis and activity of soluble enzymes (including peroxidase) involved in lignin biosynthesis were also inhibited. Since the cell wall provides the initial interface between the pathogen and the host, cell wall components such as lignins and peroxidases may be closely involved in the plant's response to a pathogen.In this study, we have investigated soluble and cell wall peroxidases of reed canarygrass. We have also investigated the localization of peroxidases around the attempted penetration site. The results are discussed in relation to lignin formation and disease resistance in reed canarygrass. (9) was grown in a glasshouse bed of peatmoss-vermiculite. Leaf discs (8-mm diameter) were cut with a corkborer from unblemished, expanded leaves. The discs were floated on distilled H20 or aqueous solutions of cycloheximide (25 ,ug/ml) in Petri dishes. The upper surface of the disc was thoroughly sprayed with a suspension of Helminthosporium avenae Eidam spores or a 0.05-ml drop of spore suspension was pipetted directly onto the disc. The preparation of inoculum was described previously (16).Cell-free extracts were obtained from samples collected 18 hr after inoculation. The samples were freeze dried. Cell wall and protoplas...

The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A-or It has become apparent that the distribution of "high energy" metabolites within a cell is under tight intracellular regulation. In particular, experimental evidence accumulating on the existence of numerous and highly specific transport and shuttle systems (8,20) indicates that there may be regulatory systems involving chloroplastic, mitochondrial, and cytoplasmic components. A necessary prerequisite for studying such systems is the ability to separate the various subcellular components in order to assess their individual functions and capabilities.Cyanide-insensitive respiration has often been measured by researchers primarily interested in mitochondrial electron transport and the energetics of ATP production (2, 19). It now seems clear (5, 13) that lipoxygenase activity may account for a substantial portion of total 02 consumption at certain times during development in soybean and wheat. Here, we report a series of respiratory studies carried out on conventionally isolated mitochondria and highly purified mitochondria from germinating wheat seedlings 12 through 64 h after imbibition. Data are presented which demonstrate the possible confusion that can arise when attempting to assay mitochondrial cyanide-insensitive res-

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A mitochondrial fraction was isolated from durum wheat seedlings via differential centrifugation using three different tissue grinding buffers. One buffer contained bovine serum albumin (BSA), a protein proven effective in protecting mitochondria from damage during isolation. The other two buffers contained either polyethyleneglycol of M.W. 20,000D (PEG-20), a phenolic binding compound or PEG-20 plus XAD-2, a non-ionic adsorbent material. Organelles isolated with the BSA exhibited an average ADP/O ratio of 2.3 and an average respiratory control ratio of 5.1, substantially higher than those obtained with the other media. Addition of the XAD-2 to a buffer containing PEG-20 resulted in an increase in the respiratory control ratio to an average value of 3.7 vs 2.7 for organelles isolated with PEG-20 alone without causing a concomitant rise in the ADP/O ratio which remained an average of 1.9. Protein values for the mitochondrial fraction isolated with BSA ranged from 2.5 to 3 times greater than those obtained from the PEG-20 preparations. This difference in organelle protein is discussed in relation to the accurate measurement of mitochondrial respiration.

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