Acidolysis and hot water extraction provide new insights into the composition of the induced "lignin-like" material from squash fruit.

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 degradation 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 qualitative characterization of acidolysis degradation products.

Characterization of a 34-kDa soybean binding protein for the syringolide elicitors.

Syringolides are water-soluble, low-molecular-weight elicitors that trigger defense responses in soybean cultivars carrying the Rpg4 disease-resistance gene but not in rpg4 cultivars. 125I-syringolide 1 previously was shown to bind to a soluble protein(s) in extracts from soybean leaves. A 34-kDa protein that accounted for 125I-syringolide 1 binding activity was isolated with a syringolide affinity-gel column. Partial sequences of internal peptides of the 34-kDa protein were identical to P34, a previously described soybean seed allergen. In soybean seeds, P34 is processed from a 46-kDa precursor protein and was shown to have homology with thiol proteases. P34 is a moderately abundant protein in soybean seeds and cotyledons but its level in leaves is low. cDNAs encoding 46-, 34-, and 32-kDa forms of the soybean protein were cloned into the baculovirus vector, pVL1392, and expressed in insect cells. The resulting 32- and 34-kDa proteins, but not the 46-kDa protein, exhibited ligand-specific 125I-syringolide binding activity. These results suggest that P34 may be the receptor that mediates syringolide signaling.

Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems.

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.

Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves.

Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D. The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene. A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained. The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating. A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s). Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity. However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves. Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene. Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.

Syringolide 1 Triggers Ca2+ Influx, K+ Efflux, and Extracellular Alkalization in Soybean Cells Carrying the Disease-Resistance Gene Rpg4.

Alleles of avirulence gene D (avrD) specify the production by bacteria of syringolides that elicit the hypersensitive response in soybean (Glycine max) plants carrying the disease-resistance gene Rpg4, but not rpg4 plants. Syringolide 1 caused extracellular alkalization, K+ efflux, and Ca2+ influx about 30 min after addition to suspension-cultured cells of two Rpg4 cultivars, Harosoy and Flambeau, but not in two rpg4 cultivars, Acme and Merit. All responses were sustained for at least 1.5 h and were inhibited by La3+, which blocks certain Ca2+ channels. These results suggest that syringolide 1 activates a Ca2+ influx-dependent signaling pathway only in Rpg4 soybean cells.

Phthalide-based host-plant resistance toSpodoptera exigua andTrichoplusia ni inApium graveolens.

A chemical basis for the difference in suitability between two celeriac (Apium graveolens var.rapaceum) cultivars for the survival and growth ofSpodoptera exigua (Hübner) andTrichoplusia ni (Hübner) was identified as sedanenolide (3-n-butyl-4,5-dihydro-isobenzofuranone). Sedanenolide was isolated using a bioassay-driven extraction and purification procedure and was identified using several spectrometric methods. Foliar concentrations of sedanenolide were negatively correlated with larval performance and were significantly higher in the cultivar less suitable for larval survival and growth. Sedanenolide andBacillus thuringiensis Berliner acted additively in reducing larval growth when combined in artificial diets, a result that is consistent with previous studies in which the combined effect of host plant cultivar andB. thuringiensis on larval survival and growth were additive.

An antifungal compound produced by grapefruit and Valencia orange after wounding of the peel.

Wounding citrus induces the production of the antifungal compound 1, isolated from peel of injured grapefruit, Citrus paradisa, and injured orange, Citrus sinensis. Interpretation of ms, uv, and 1D and 2D nmr spectroscopic data led to the identification of the new compound as 3-[4-hydroxy,3-(3-methyl-2-butenyl)-phenyl]-2-(E)-propenal.

Allelochemical resistance of bald cypress,Taxodium distichum, heartwood to the subterranean termite,Coptotermes formosanus.

The heartwood of bald cypress,Taxodium distichum (L.) Rich., resisted feeding attack by the Formosan subterranean termite,Coptotermes formosanus Shiraki. Hexane-extracted heartwood, however, was consumed at > 12 times the amount of sound heartwood eaten. A bioassay usingT. distichum sapwood as a feeding substrate was employed to assess the antitermitic activity of successive hexane, acetone, and methanol extracts of heartwood shavings and isolates derived from the active hexane extract. Two fractions, eluted from the crude hexane extract by liquid chromatography, significantly reduced termite feeding compared to the parent extract, while a third fraction was less active than the original hexane extract. Each fraction contained one major component. All three components were structurally related diterpenes. The two most active heartwood constituents were identified by GC-MS and NMR as ferruginol and manool, while the third and least active, but most prevalent, compound in heartwood was identified as nezukol. Results of bioassays suggest that these allelochemicals act principally as feeding deterrents with accompanying termite mortality due to starvation.

Degradation of crystalline ergocalciferol [vitamin D2, (3 beta,5Z,22E)-9,10-secoergosta-5,7,10(19),22-tetra en-3-ol].

The products of the degradation of crystalline ergocalciferol were investigated. These studies showed that numerous acidic and neutral oxidation products were formed resulting in the complete destruction of the triene functionality. Separation of the neutral products by preparative TLC led to material identified as the Windaus ketone IIa, 2,3,3a,4,5,6,7,7a beta-octahydro-7a alpha-methyl-1R-(1 alpha,1R,4R,5-trimethyl-2E-hexenyl)-4H-inden-4-one.