The contribution of plant-derived proteinases to the breakdown of fresh pasture protein in the rumen.

Pastoral agriculture in New Zealand has a long history of improving the efficiency with which fresh pasture is converted into meat, wool and dairy products. New Zealanders live in a temperate climate where rainfall and pasture growth are the main driving forces in low-input pastoral systems. The process whereby protein in fresh pasture is broken down in the rumen and reassimilated into microbial protein for use by the animal is consequently of fundamental importance to ruminant nutrition in New Zealand, and indeed elsewhere. The article appearing in a previous issue of the British Journal of Nutrition by Kingston-Smith et al. (2005) provides further evidence for a role of plant proteinases in protein breakdown in the rumen and is therefore of interest. A scan of the literature in the area of ruminal protein degradation reveals numerous reports from the points of view of animal nutrition and rumen microbiology but very few on the contributions of plants to their own demise once ingested into the rumen. Up until 1996, the generally accepted version of events in the initial stages of degradation of soluble plant protein in the rumen involved the release of proteins from masticated plant tissue, the adsorption of protein to the surface of rumen bacteria and their subsequent hydrolysis by mainly cysteine proteinases (Wallace et al. 1997). Much of the information on this process was derived from the study of animals receiving dried or ensiled forage or concentrate diets (Brock et al. 1982; Kopecny & Wallace, 1982; Prins et al. 1983; Falconer & Wallace, 1998), and protein solubility, degree of folding and cross-linking were found to be important determinants of degradability. Under these circumstances, protein breakdown was mediated by rumen microbes, principally the bacteria, while protozoa were also important in degrading insoluble protein and recycling N within the rumen by predation of bacteria (Nugent & Mangan, 1981; Brock et al. 1982). Detailed studies on rumen proteinases followed and defined the main enzymatic types found in rumen contents and correlated these with their occurrence in rumen microbial species (summarised in Wallace et al. 1997). Since 1996, a series of publications from the Institute of Grassland and Environmental Research in Aberystwyth, Wales, have promoted the concept that plant proteinases may play an important role in the breakdown of protein in fresh forage diets. Theodorou et al. (1996) first raised this possibility, arguing that pasture-grazed ruminants ingest plant material that is largely composed of intact cells that are still metabolically active. Plant cell vacuoles contain proteinases that are known to be involved in proteolysis during ensilage and it was proposed that conditions found in the rumen (elevated temperature, pH and lack of O2) favoured plant cell death and increased plant proteinase activity. They also pointed out that those rumen microbes most closely associated with plant material in the rumen (cellulolytic bacteria and fungi) were either not proteolytic or had very low proteolytic activity. Subsequently, Zhu et al. (1999) examined ryegrass, red clover, white clover and bird’s-foot trefoil in vitro in the presence and absence of rumen micro-organisms and measured volatile fatty acid (VFA) production and the disappearance of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). They found similar patterns of protein degradation both in the presence and absence of rumen micro-organisms and concluded that plant proteinases contributed significantly to plant protein breakdown in the rumen. However, significant amounts of VFA (mainly acetate) were produced during these incubations, suggesting at least some growth of microbes associated with the plant material had occurred, thus raising the possibility of protein degradation by the epiphytic microbial population. In response to these observations, Wallace et al. (2001) tested the plant proteinase hypothesis by autoclaving fresh ryegrass to rule out the action of plant-associated microbes and to inactivate plant proteinases. They found that autoclaving markedly decreased the rate of NH3 release in vitro from both chopped and blended grass compared with non-autoclaved material. However, the rate of protein breakdown in blended grass as measured by C-labelled casein hydrolysis was approximately 10 % of that usually observed in rumen fluid. They concluded that although plant proteinases had low activity, their co-location with their protein substrate within plant cells gave them a disproportional contribution to the initial stages of protein breakdown in the rumen. Further in vitro experiments by Beha et al. (2002) examined a number of plant protein and senescence parameters in ryegrass under temperature and anoxic stresses. Leaf blades incubated under rumen-like conditions in the absence of microbes released protein and amino acids, which subsequently appeared in the incubation buffer. The amount of amino acids appearing in the buffer exceeded that originally observed in the plant material, suggesting breakdown of protein in the plant tissue and release of amino acids. Proteinase zymograms indicated that the main activity in mature leaf samples was a non-cysteine proteinase. Measurement of in situ protein degradation using Rubisco antisera indicated that some of the large and small subunit proteins were lost from the chloroplast after 6 h incubation. The authors concluded that ingested plant cells in the rumen entered a metabolic state similar to, but not exactly the same as, senescence, where protein was lost at a greater rate than either cell nuclei or chlorophyll. More recently, Kingston-Smith et al. (2003a) made similar observations in white and red clover leaves. The protein content of leaves decreased rapidly over the first 6 h of incubation and was accompanied by increased proteinase activity and more proteinase isoforms. These clovers also British Journal of Nutrition (2005), 93, 421–423 DOI: 10.1079/BJN20041360 q The Authors 2005