Proteinase inhibitor-inducing factor activity in tomato leaves resides in oligosaccharides enzymically released from cell walls

Abstract

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),...

Positional Specificity of a Phospholipase A Activity Induced by Wounding, Systemin, and Oligosaccharide Elicitors in Tomato Leaves

“…OGAs derived from plant cell walls (Bishop et al, 1981) and chitosan, which is a mixture of glucosamine oligomers derived from fungal cell walls , are elicitors of defensive genes in a variety of plant systems (Ryan and Farmer, 1991;John et al, 1997). The inducing activities of both OGA and chitosan are considered to be receptor mediated (Felix et al, 1993;Ebel, 1998) and, in tomato leaves, like systemin, they activate genes through the octadecanoid signaling pathway (Doares et al, 1995).…”

Analysis of Transport Activity of Arabidopsis Sugar Alcohol Permease Homolog AtPLT5

“…During wounding, oligosaccharides are released from the wounding site in two ways: 1) by mechanical destruction of the cell, followed by the discharge of the contents of the cell, and 2) by induction of endogenous polygalacturonases. Oligosaccharides released from wounding sites have been suggested to have a role in signaling, but aside from that they are also an important resource for the plant that needs to be recovered (35,36). In addition, invertases are also induced in wounded tissues, for instance, in wounded taproots of carrots (37).…”

Variation potential in higher plants: Mechanisms of generation and propagation

“…This substance must: (i) be synthesized quickly in response to damage, (ii) induce electrical reactions, and (iii) propagate easily in plants. Oligosaccharides of broken cell walls, 66 systemin, 67 jasmonate, 18,68 salycilic acid, 69 ethylene 19,70 and abscisic acid 1,2 are considered to be potential wound substances. In recent studies, 71 hydrogen peroxide (H 2 O 2 ) has been shown to propagate from the damage zone and induce an electrical reaction.…”