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Polyphenol Oxidase and o-Diphenols Inhibit Postharvest Proteolysis in Red Clover and Alfalfa

U.S. Dairy Forage Research Center, ARS-USDA, 1925 Linden Drive West, Madison, WI 53705

View larger version (25K): [in a new window]   Fig. 1. Proteolytic inhibition in red clover extracts requires o-diphenols. Proteolysis, as measured by amino acid release over time, was measured in red clover leaf extracts that were unfractionated (Crude, ), desalted by gel filtration to remove low molecular weight compounds (Desalted, ), or desalted and supplemented with the o-diphenol caffeic acid at a final concentration of 3 mM (Desalted+CA, •). Results are the average ± SEM of three experiments using independently prepared extracts.

View larger version (63K): [in a new window]   Fig. 2. RNAi reduces PPO expression in red clover leaves. Leaf extracts of nontransformed control (C) or independently derived silenced (numbered) plants from NEWRC27 and NEWRC30 red clover genotypes were analyzed for extract browning, PPO protein by immunoblotting (5 µg protein per lane) with a PPO antibody, or for PPO activity by a quantitative PPO activity assay. PPO activity is expressed as nkat mg–1. For NEWRC27–1 and NEWRC30–0 plants transformed with the silencing construct, PPO activity was not detectable (ND) and no browning was seen, even after 24 h.

View larger version (20K): [in a new window]   Fig. 3. Proteolytic inhibition in red clover extracts requires PPO. Proteolysis, as measured by amino acid release over time, was measured in leaf extracts from untransformed control (•) or PPO-Silenced () red clover. Results are the average ± SEM of three experiments using independently prepared extracts from NEWRC27–1 (2 replicates) and NEWRC30–0 and their corresponding controls.

View larger version (29K): [in a new window]   Fig. 4. PPO inhibits proteolysis in alfalfa extracts in an o-diphenol-dependent manner. Proteolysis, as amino acid release over time, was measured in leaf extracts from Control- (Control-Alf, , •) or PPO-Alfalfa (PPO-Alf, , ) in the absence (, ) or presence (•, ) of added caffeic acid (CA, 3 mM final concentration). Results are the average ± SEM of three experiments using independently prepared extracts.

View larger version (18K): [in a new window]   Fig. 5. Proteolytic inhibition in alfalfa extracts requires relatively little PPO or o-diphenol. (A) Alfalfa extracts (2 mg mL–1 protein) of varying PPO concentration were prepared by mixing PPO- and Control-Alfalfa extracts to give the desired level of PPO activity. The resulting extracts were incubated with (•) or without () 3 mM caffeic acid as indicated and proteolysis, as amino acid release after 4 h, was measured. PPO activity (caffeic acid substrate) is given as nkat mL–1. (B) Control- () or PPO- containing (•, 4.17 nkat mL–1) alfalfa extracts (2 mg mL–1 protein) were incubated with various levels of caffeic acid and proteolysis, as amino acid release after 4 h, was measured. Results for both experiments are the average 6 SEM of three experiments using independently prepared extracts.

View larger version (74K): [in a new window]   Fig. 6. A variety of o-diphenol compounds can function with PPO to inhibit proteolysis. Control- (gray bars) or PPO-containing (black bars, 4.17 nkat mL–1) alfalfa extracts (2 mg mL–1 protein) were incubated with various o-diphenols (3 mM final concentration) and proteolysis, as amino acid release after 4 h, was measured. The tested o-diphenols were caffeic acid (CA), hydrocaffeic acid (HCA), chlorogenic acid (CGA), catechol (CAT), and (–)-epicatechin (EPI). Addition of ethanol (the solvent used for the o-diphenol stock solutions) served as a negative control (NONE). Results are the average ± SEM of three experiments using independently prepared extracts.