The effects of PPO activity on the proteome of ingested red clover and implications for improving the nutrition of grazing cattle

Abstract

Increasing the rumen-stable protein content of feed would lead to improved nitrogen utilisation in cattle, and less nitrogenous waste. Red clover (Trifolium pratense L.) is a high protein ruminant feed containing high polyphenol oxidase (PPO) activity. PPO mediated protein-quinone binding has been linked to protecting plant proteins from proteolysis. To explore the mechanism underlying the effect of PPO on protein protection in fresh forage feeds, proteomic components of feed down-boli produced from wild-type red clover and a low PPO mutant, at point of ingestion and after 4 h in vitro incubation with rumen inoculum were analysed. Significant differences in proteomic profiles between wild-type and mutant red clover were determined after 4 h incubation, with over 50% less spots in mutant than wild-type proteomes, indicating decreased proteolysis in the latter. Protein identifications revealed preferentially retained proteins localised within the chloroplast, suggesting that PPO mediated protection in the wild-type operates due to the proximity of target proteins to the enzyme and substrates, either diffusing into this compartment from the vacuole or are present in the chloroplast. This increased understanding of protein targets of PPO indicates that wider exploitation of the trait could contribute to increased protein use efficiency in grazing cattle.Biological significanceOne of the main challenges for sustainable livestock farming is improving capture of dietary nitrogen by ruminants. Typically up to 70% of ingested protein-N is excreted representing a loss of productivity potential and a serious environmental problem in terms of nitrogenous pollution of lands and water. Identification of key characteristics of rumen-protected protein will deliver target traits for selection in forage breeding programmes. The chloroplastic enzyme PPO catalyzes the oxidation of phenols to quinones, which react with protein. Little is currently known about the intracellular protein targets of the products of PPO activity or the mechanism underlying protein complexing, including whether there is any specificity to the reaction. Here we have determined significant differences in the proteomes of freshly ingested down boli corresponding to the presence or absence of active PPO. These results show that in the presence of PPO the forage protein is less amenable to proteolysis and provide the novel information that the protected proteins are putatively chloroplastically located. These data also contribute to a growing evidence base that a chloroplastic PPO substrate exists in red clover in addition to the currently known vacuolar substrates.

Characterization of nitrogen transformation dynamics in alfalfa and red clover and their mixture silages

“…It was more likely to be influenced by the PPO‐mediated proteolytic inhibition in red clover. The role of PPO in reducing proteolysis has been well demonstrated by numerous studies (Hart, Onime, Davies, Morphew, & Kingston‐Smith, ; Sullivan & Hatfield, ). The proposed mechanism of PPO‐reduced proteolysis is through the generation of o ‐quinones that react with nucleophilic sites on cellular proteins, reforming a phenol covalently bound to protein.…”

“…The proposed mechanism of PPO‐reduced proteolysis is through the generation of o ‐quinones that react with nucleophilic sites on cellular proteins, reforming a phenol covalently bound to protein. Proteolysis thus is reduced either directly, by effective inhibition of proteases, or a reduction in general protein solubility through protein–phenol binding (Hart et al., ). This PPO‐mediated protein protection is only effective for a limited time post‐sealing because of its oxygen dependence (Lee, Scott, Tweed, Minchin, & Davies, ).…”

The use of red clover (Trifolium pratense) in soil fertility-building: A Review

“…Breeding in North America has also reduced the drying time of RC forage by removing its stem hairs (Taylor, 2008b), and incorporation of this into other breeding programs, particularly in areas in which drying times are problematic, may alleviate this concern. Increased expression of polyphenol oxidase (PPO) should also be a key breeding target, as PPO activity in ensiling inhibits proteolysis, enhancing N-use efficiency in ruminants and reducing losses in excreta (Hart et al, 2016). The forage value of new varieties is further increased by im-provements in persistence, as these can produce high protein yields for over three years (Marshall et al, 2017), which requires consistent amounts of N from BNF, ultimately contributing to soil fertility.…”

A route to decreasing N pollution from livestock: Use of Festulolium hybrids improves efficiency of N flows in rumen simulation fermenters

“…The rumen can be considered to be a semi-continuous flow fermenter in which the microbial consortium degrades the ingested feed, the products of which such as volatile fatty acids (VFA) and protein breakdown products are used to drive microbial growth (Huws et al, 2018). Flow-through of rumen liquor containing a proportion of the microbiota and nondegraded plant protein (Hart, Onime, Davies, Morphew, & Kingston-Smith, 2016) from the rumen to the small intestine allows absorption of nutrients into the animal's bloodstream driving animal productivity (Firkins, 1996). It has previously been demonstrated that endogenous plant enzymes responses to rumen conditions could contribute to inefficient protein use during fermentation by the rumen microbiota (Beha, Theodorou, Thomas, & Kingston-Smith, 2002;Kingston-Smith, Davies, Edwards, Gay, & Mur, 2012;Kingston-Smith, Merry, Leemans, Thomas, & Thoeodorou, 2005).…”