The synthesis andaccumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by fungal endo-a-1,4-polygalacturonase digestion of a pectic polysaccharide (Mr 5000-10,000) isolated from tomato leaves. Active oligosaccharides were-also released from isolated tomato leaf cell walls by endopolygalacturonases partially purified from tomato plants. It.is suggested that oligosaccharides, released from plant cell wall pectic polysaccharides by either endogenous or exogenous endopolygalacturonases at a wound or infection site, may have hormone-like roles in regulating plant defense responses in unwounded tissues many centimeters away from the site of release.Severe wounding of tomato or potato leaves by attacking pests or other mechanical damage releases a putative wound hormone that is transported throughout the plants where it induces leaves to initiate synthesis and accumulation of two proteinaceous inhibitors of serine endopeptidases (1). This proteinase inhibitorinducing factor (PIIF) has been consistently associated with polysaccharides ofwidely varying sizes (2), suggesting that PIIF activity may be intrinsic to a specific saccharide sequence or structure.Recently, a highly active tomato PIIF fraction of Mr 5000-10,000 was shown to be pectic polysaccharide. It has a composition similar to that of an enzymically produced fragment of sycamore cell walls, of Mr 200,000, that was shown to have activity similar to that oftomato PIIF (3). This evidence suggested that PIIF activity may be associated with a structural component of plant cell walls. However, in view.of the large sizes of.both the tomato pectic polysaccharide and the sycamore cell wall fragment, it is questionable whether they would be rapidly transported through the plant vascular system after wounding in vivo.-In this communication we report that an endopolygalacturonase purified from.the fungus Rhizopus stolonifer (4) degrades tomato PIIF into oligosaccharides that are active inducers of proteinase inhibitor I when supplied to excised tomato leaves. We also show that a mixture of two endopolygalacturonases, partially purified from. tomato fruit, degrades both tomato PIIF and purified tomato cell walls into PIIF-active oligosaccharides. These results suggest that PIIF activity produced in vivo by cell damage resides in small hydrolytic fragment(s) of plant cell walls.
MATERIALS AND METHODSTomato PIIF is a partially purified, highly methylated soluble pectic polysaccharide of Mr 5000-10,000 on the basis of its elution profile from Sephadex G-50 when compared to protein standards (unpublished data). Acid-demethylated tomato PIIF polysaccharide was prepared by dissolving 50 mg oftomato PIIF in 5 ml of 2 M trifluoroacetic acid and heating under reduced pressure at 859C for 1 hr. The reaction mixture was chilled in an ice bath and then centrifuged at 25,000 X g for 10 min. The supernatant was lyophilized to remove the trifluoroacetic acid. The residue was dissolved in water, adsorbed on DE-52 (Whatman),...