Supplemental Figure 2. Foliar content of antinutritive proteins inMedicago truncatula ecotypes in response to caterpillar herbivory. Foliar levels of A) trypsin inhibitor (TI) and B) polyphenol oxidase (PPO) were compared in in two M. truncatula ecotypes, TN1.11 (least deterrent, LD) and F83005.5 (most deterrent, MD), after two weeks of continuous Spodoptera exigua caterpillar herbivory. Ecotype- or herbivory-associated changes were not observed (Supplemental Table IIC).

Antinutritive proteins: Trypsin inhibitor and polyphenol oxidase activity

Plants were treated as described in the “Constitutive and induced plant defense response” section. Samples were taken at the same time as for metabolite analysis (at caterpillar pupation, approx. 2 weeks after caterpillars were placed on the plant). Two plants were for each ecotype and treatment (control and insect) were taken per replication and the experiment was repeated three times (total n = 6).

Trypsin inhibitor (TI) assay. M. truncatula leaves (100 mg) that were finely ground in liquid nitrogen were extracted in 300 μL of ice-cold extracting buffer (0.1 M sodium carbonate, sodium bicarbonate buffer, pH 9.8 containing 0.1% Triton X-100 and 7% polyvinylpyrrolidone). An alkaline pH was used was to mimic the caterpillar gut (pH 9-10) (Dow, 1992). After vigorous vortexing and centrifugation at 13,000 rpm for 10 min. to remove cellular debris, the supernatant was transferred to a new tube on ice.

TI activity was measured as described by Lara (2000) and Weech et al. (2008). Bovine trypsin (0.5 μg, ~10,000 U/mg) was added to triplicate samples and incubated at 25°C with gentle shaking for 30 min. After addition of the trypsin substrate, N-benzoyl-DL-arginyl-β-naphthylamine (0.613 mM), the reaction was incubated for one hour when the reaction was then stopped with 2% hydrochloric acid. p-Dimethyl-amino cinnamaldehyde (24% in ethanol) was added and the resultant productmonitored at 540 nm using a microplate spectrophotometer (BioTek Synergy HT). Controls were: buffer without leaf extract to determine background absorbance and a negative control without bovine trypsin. A soybean TIstandard curve (0 -1000 ng/μL) was used to calculateTI levels in leaf extracts. TI levels were normalized by total soluble protein levels. Constitutive and induced TI levels were compared between F83005.5 and TN1.11 ecotypes by 2-factor ANOVA (Supplemental Table IIC).

Polyphenol oxidase (PPO) assay. Finely ground M. truncatula leaves (100 mg) were extracted in the same buffer as the TI extraction, except the buffer also contained proteinase inhibitor cocktail (Sigma). After vortexing and centrifugation at 13,000 rpm for 10 min, the supernatant was transferred to a new tube on ice.

PPO activity was measure according to Espín (1997) and Weech et al. (2008), with minor modifications. o-Diphenol dopamine dihydrochloride (35 mM), the substrate for catecholases or laccases (Anisezewski et al., 2008), was added to triplicate samples. N,N-dimethylformamide (40 mM) and 4 mM 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (0.2 mM, prepared in methanol) were added to prevent the reactive quinones produced by this reaction from reacting with the enzymes. Enzyme activity was monitored every 30 s for 5 min at 476 nm. A positive control using commercial tyrosinase and a blank control containing leaf extract where the enzymes had been inactivated by adding 20% sodium dodecyl sulfate followed by boiling for 5 minutes were included on every plate. PPO levels were normalized by total soluble protein levels (see below). Constitutive and induced PPO levels were compared between F83005.5 and TN1.11 ecotypes by 2-factor ANOVA (Supplemental Table IIC).

Soluble protein concentration. Soluble protein levels were measured by a modified Bradford assay (Bradford, 1976; Zor and Selinger, 1996). Coomassie blue Bradford reagent (Thermoscientific) was added to triplicate protein samples. After an incubation period of 10 minutes, readings were taken at 595 nm and 450 nm and the ratio was used to calculate protein concentration compared to a bovine serum albumin standard curve (5 to 100 µg/ml).

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