Acidolysis and hot water extraction provide new insights into the composition of the induced ‘‘lignin-like’’ material from squash fruit Richard R. Stange Jr. a, *, John Ralph b , Junpeng Peng b , James J. Sims c , Sharon L. Midland c , Roy E. McDonald a a USDA-ARS, Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945, USA b USDA-ARS, US Dairy Forage Research Center, 1925 Linden Drive West, Madison, WI 53706-1108, USA c Department of Plant Pathology, University of California, Riverside, CA 92521, USA Received 28 October 1999; received in revised form 6 June 2000 Abstract Accumulation of ‘‘lignin-like’’ material (L-LM) by plant tissues in response to injury or disease has been observed in a wide variety of plant taxa. The most intensively studied L-LM is that produced by members of the Cucurbitaceae; this material is thought to be an unusual lignin rich in p-coumaryl alcohol derived subunits. Employing acidolysis we found the primary degrada- tion product of L-LM from squash fruit was p-coumaryl aldehyde. These findings conflict with the current concept of L-LM, but would be consistent with L-LM being a polymer derived directly from p-coumaryl aldehyde or a gum containing this compound. Results of hot water extraction support the latter possibility. Further, we report on a simple TLC method useful for rapid quali- tative characterization of acidolysis degradation products. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cucurbita maxima; Cucurbitaceae; Squash; Composition of induced lignin; p-Coumaryl alcohol; p-Coumaryl aldehyde 1. Introduction The lignin-like material (L-LM) produced by many plants in response to injury or disease has been studied by numerous investigators and is frequently associated with resistance to fungal or bacterial pathogens (Ride, 1975; Vance et al., 1980; Nicholson and Hammersch- midt, 1992). Lignins are a complex class of compounds derived via the free radical polymerization of the three primary lignols (Fig. 1), p-coumaryl 1b, coniferyl 2b and sinapyl 3b alcohols (Harkin, 1967; Freudenberg and Neish, 1968; Whetten et al., 1998). Lignins are classified based on the ratio of the different lignols present. Because of their high molecular weight and insolubility, precise knowledge of the composition of lignins cannot be obtained directly, but must be gained indirectly by NMR of soluble lignin fractions or by identification of lignin degradation products (Lewis and Yamamoto, 1990). Degradation methods used include pyrolysis-GC– MS, hydrolysis, and oxidation (Tanahashi and Higuchi, 1988; Ralph and Hatfield, 1991). In addition to provid- ing subunit composition, a portion of the products from the first two methods retain their C-3 side chains, pro- viding information about the interunit linkages in the parent lignin (Lundquist, 1992; Ralph and Hatfield, 1991). However, the sheer number of possible products complicates routine analysis of lignins by these meth- ods. In contrast, oxidative methods degrade lignins to the benzaldehyde or benzoic acid analogues of their parent lignols. Because there are only three primary products, analyses of oxidative degradation products are relatively simple, (Tanahashi and Higuchi, 1988), but information on the nature of the sidechains is lost. Deposition of L-LM has been reported in a wide variety of plant taxa (Vance et al., 1980; Rittinger et al., 1987). However, only in a relatively small number of plants has the composition of the L-LM been examined. The L-LM from members of the Cucurbitaceae is per- haps the best characterized, having been studied by four independent research groups using alkaline cupric oxide or nitrobenzene oxidization methods. L-LM induced by fungal infection in five species of cucurbits was 0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00096-6 Phytochemistry 57 (2001) 1005–1011 www.elsevier.com/locate/phytochem * Corresponding author. Tel.: +1-561-462-5838; fax: +1-561-462- 5986. E-mail address: rstange@ushrl.ars.usda.gov (R.R. Stange Jr.).