Planta (1992)187:295-300 P l a n t a 9 Springer-Verlag 1992 Chitinase and peroxidase in effective (fix +) and ineffective (fix-) soybean nodules Christian Staehelin, Joachim Miiller, Robert B. Mellor, Andres Wiemken, and Thomas Boiler* Botanisches Institut der Universiffit Basel, Hebelstrasse 1, CH-4056 Basel, Switzerland Received 19 July; accepted 28 December 1991 Abstract. Chitinase and peroxidase, two enzymes thought to be involved in the defense of plants against pathogens, were measured in soybean (Glycine max L. Merr.) roots and in nodules colonized by Bradyrhizobium japonicum strains differing in their symbiotic potential. Activities of both enzymes were higher in nodules than in roots. In "effective", nitrogen-fixing nodules, col- onized by wild-type bacteria, chitinase and peroxidase activities had low levels in the central infected zone and were enhanced primarily in the nodule cortex. An ascor- bate-specific peroxidase, possibly involved in radical scavenging, had similarly high activities in the infected zone and in the cortex. "Ineffective" nodules colonized by bacteria unable to fix nitrogen symbiotically showed a similar distribution of chitinase and peroxidase. In another type of "ineffective" nodule, colonized by a B. japonicum strain eliciting a hypersensitive response, activities of both enzymes were enhanced to a similar degree in the infected zone as well as in the cortex. Tissue prints using a direct assay for peroxidase and an anti- serum against bean chitinase corroborated these results. The antiserum against bean chitinase cross-reacted with a nodule protein of Mr 32 000; it inhibited most of the chitinase activity in the nodules but barely affected the chitinase in uninfected roots. It is concluded that proteins characteristic of the defense reaction accumulate in the cortex of nodules independently of their ability to fix nitrogen, and in the entire body of hypersensitively react- ing nodules. Key words: Bradyrhizobium - Chitinase - Glycine (nod- ules) - Hypersensitive reaction- Peroxidase (isoenzymes) Introduction The interaction of legumes with rhizobia normally cul- minates in the formation of a symbiotic organ, the nod- * To whom correspondence should be addressed Abbreviations: PAGE = polyacrylamide gel electrophoresis; SDS = sodium dodecyl sulphate ule, which functions in nitrogen fixation (Rolfe and Gresshoff 1988; Sprent 1989). The early stages of infec- tion by the rhizobia resemble a pathogen attack; the process of nodulation has therefore been viewed as a "beneficial disease", (Vance 1983) caused by a "refined pathogen" (Djordjevic et al. 1987). It is interesting to study the extent to which inducible defenses against pathogens come into play during nodule formation. A classical defense reaction, the accumulation of phytoalexins, has been examined in soybean roots infected by Bradyrhizobium japonicum (Werner et al. 1985). Wild-type bacteria, which caused the formation of effective, nitrogen-fixing nodules, induced the formation of only small amounts of the soybean phytoalexin glyce- ollin at the earliest stages of interaction. However, cer- tain strains which lead to the formation of ineffective nodules not fixing nitrogen, induced a sort of hypersen- sitive reaction and caused accumulation of large amounts of glyceollin in a way similar to roots infected by patho- gens (Werner et al. 1985; Parniske et al. 1990). We have thus studied further defense reactions in effective and ineffective nodules, focussing our attention on the activities of two enzymes often associated with plant defense reactions, namely chitinase (Boiler 1988) and peroxidase (Hammerschmidt et al. 1982; Svalheim and Robertsen 1990). Plant chitinases inhibit the growth of certain fungi (Schlumbaum et al. 1986) and often have lysozyme activity as well, indicating that they may func- tion in defense against bacteria (see Boiler 1988). Peroxidases have been suggested to be involved in the activation of preformed toxins and in lignification, two typical elements of the plant defense reaction (Lewis and Yamamoto 1990). We report that chitinase and peroxidase are present at elevated levels in the cortex but not in the central region of effective root nodules and in the whole body of certain ineffective nodules. These find- ings indicate that the constitutive expression of defense- related genes in cortical ceils of effective root nodules may function in protection of the symbiotic organ from pathogen attack.