Published online 3 October 2006
Published in Agron J 98:1392-1399 (2006)
DOI: 10.2134/agronj2005.0265
© 2006 American Society of Agronomy
677 S. Segoe Rd., Madison, WI 53711 USA
Forages
Proteolysis and Characterization of Peptidases in Forage Plants
Gaston R. Pichard*,
Bruno R. Tesser,
Catalina Vives,
Catterina Solari,
Adolfo Hott and
Rafael E. Larraín
Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Vicuña Mackenna 4860, Macul, Santiago, Chile
* Corresponding author (gpichard{at}uc.cl)
Received for publication September 8, 2005.
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ABSTRACT
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Plant peptidases are involved in protein degradation in the rumen of grazing animals, but little is known about the variation in their activity under anaerobic conditions at 39°C. We investigated the activity of endogenous peptidases in 342 accessions of grass and legume forages and the molecular weight and class of peptidases in a subsample of them. Proteolytic activity was measured by in vitro incubation of tissue over a gelatin substrate. Molecular weight and class of peptidases were assessed by zymograms. A wide range in proteolytic activity index (PAI) was detected (mean PAI = 0.35, ranged from 0.00 to 1.30). More than 88% of the observed variance was due to differences in genus, species, and cultivar. Most of the species had one main peptidase, and their molecular weights ranged from 54 to 130 kDa. In the six species tested, proteases present corresponded to the serine class. The wide variation in proteolysis observed and the predominant occurrence of one peptidase of the same enzyme class support the idea that a reduction of protease activity in forages can be achieved by genetic improvement.
Abbreviations: PAI, proteolytic activity index
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INTRODUCTION
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HAVING AN ADEQUATE SUPPLY of protein-rich food, particularly animal meat and milk products, has been a major concern of human populations since the beginning of humankind. Such concerns continue today, but new constraints have been added with the demand that these high quality proteins be produced with environmentally sound production practices (Pichard and Larraín, 2001). A concern thus arises with pasture-based production systems because grazing ruminants fed high quality pastures have an inherently low efficiency of protein retention, and a large proportion of the nitrogen consumed is released to the environment in feces and urine.
When ruminants eat diets with a high proportion of fresh herbage, the release of energy to microorganisms is slow relative to the release of low-molecular-weight nitrogenous compounds. This asynchrony causes the microbial population to use amino acids for energy and liberate ammonia through deamination (Kingston-Smith and Theodorou, 2000). This asynchronous pattern also occurs when ruminants are fed ensiled forages because silages have low levels of readily fermentable sugars, and much of the protein has already undergone extensive breakdown during ensiling (McDonald et al., 1991).
Reduced protein degradation in polyphenol-containing forages can provide up to 50% more forage protein for digestion and absorption by cattle, reducing nitrogen losses and increasing profitability in the farm (Grabber et al., 2004). In the same way, lowering protein degradation by reduction of plant peptidases has the potential for increasing protein utilization efficiency in forage–livestock systems.
Plant peptidases are responsible for the release of low-molecular-weight peptides and nonprotein N compounds during the first days of ensiling. In alfalfa (Medicago sativa L.) silages treated with UV radiation, there were no differences in proteolysis between irradiated (sterile) and control silages in the first 4 d postensiling (Charmley and Veira, 1991). Inhibition of peptidase activity helps to preserve protein integrity in silages, thus reducing the release of soluble nonprotein N and increasing the concentration of peptides in the N fraction (Nsereko et al., 1998; Nsereko and Rooke, 1999).
Although most plant peptidases are located inside vacuoles, they are actively involved in protein degradation in the rumen of grazing animals. When fresh forage was incubated at 39.5°C under anaerobic conditions but in the absence of rumen bacteria, the enzyme ribulose 1,5-carboxilase oxygenase was degraded to a similar extent as in samples inoculated with rumen microbes (Merry et al., 1995; Zhu et al., 1999). Because fresh forages are rolled into a bolus and swallowed after a brief chewing by grazing cattle and sheep, more than 50% of ingested plant cells may remain intact (Kingston-Smith and Theodorou, 2000) and able to continue with their metabolic functions inside the rumen. The living plant cells are thus immersed in a stressing environment of high temperature, flooding, anaerobiosis, and a diverse microbial population before being destroyed by rumination and the action of fibrolytic microflora. During this incubation period, defense mechanisms against these stressors should be triggered. A common consequence may be the onset of senescence processes (Feller and Fischer, 1994; Buchanan-Wollaston, 1997; Crafts-Brandner et al., 1998; Herrmann and Feller, 1998), which exacerbate proteolytic activity and lead to programmed cell death.
Variations in proteolytic activity exist among species and among cultivars of the same species. In a study comparing protein degradation during wilting and ensiling of alfalfa, red clover (Trifolium pratense L.), birdsfoot trefoil (Lotus corniculatus L.), orchardgrass (Dactylis glomerata L.), smooth brome (Bromus inermis Leyss.), and timothy (Phleum pratense L.), the species with the greatest and least protein losses were alfalfa and red clover, respectively, in both conservation systems (Papadopoulus and Mckersie, 1983). Similar research with alfalfa, crownvetch (Coronilla varia L.), orchardgrass, perennial ryegrass (Lolium perenne L.), and tall fescue [Lolium arundinaceum (Schreb.) S.J. Darbyshire], established that alfalfa underwent the greatest protein degradation, particularly with ensiling (Messman et al., 1994). Significant variations in protein degradation rates and rumen protein escape were also detected among cultivars of red clover when evaluated using the inhibited in vitro method (Broderick et al., 2004).
Variation in proteolysis may involve multiple plant factors, such as peptidase concentration, substrate susceptibility to degradation, presence of peptidase inhibitors, spatial localization within the cell organelles, and resistance of the cell and its membranes to stressors and mechanical injury. Potential exists to improve animal performance and to reduce nitrogen losses to the environment by exploiting these sources of variation through plant selection and modification. However, doing so will first require assessment of the variability in proteolysis among a wide range of species and cultivars and characterization of the main peptidases involved in the protein degradation process.
Up to now, comparisons of proteolysis among species and cultivars have been made with limited numbers of germplasms. Thus, the aim of this study was to investigate the activity of endogenous peptidases in a large number of accessions of forage grasses and legumes and to determine the molecular weight and peptidase class of the main peptidases in a subset of these forages.
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MATERIALS AND METHODS
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Materials
Seeds from 342 accessions, representing two families (Poaceae and Fabaceae), 12 genera, and 65 species (Table 1) of forage plants were collected from commercial companies, research institutes, and plant germplasm banks. Seed sources included the Institute of Grassland and Environmental Research (IGER, United Kingdom), the South Australian Research and Development Institute (SARDI, Australia), Anasac (Chile), Ecsa (Chile), the Instituto de Investigaciones Agropecuarias (INIA, Chile), and SG2000 (Chile). Accessions included cultivars, wild types, and research lines. The complete list of accessions used and their sources are available on request from the authors.
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Table 1. Protein degradation of forage species in agar-gelatin films, expressed as estimated proteolytic activity index (PAI).
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Three plants of each accession were produced by sowing four to five seeds in one randomly selected cell of three independent polystyrene tree-nursery trays. After germination, the stand was manually thinned to one plant in each cell. Plants were grown in a greenhouse under a 13-h photoperiod, with a low temperature limit of 5°C and daily irrigation. Every 15 d, plants were fertilized with a macro- and micromineral mix (Plant Food Improved Formula, Phostrogen, Cambridge, UK) containing N, P, and K at a ratio of 14:10:27. Fertilizer was applied at an approximate rate of 300 kg N ha–1 yr–1 as N deficiency has been linked with changes in transcription of peptidases in common wheat (Triticum aestivum L.) (Crafts-Brandner et al., 1998). Nitrogen fertilization was applied to Fabaceae plants because the amount of soil in each nursery-tray cell was not considered enough for developing a Rhizobium spp. population able to fully meet the demands of the plants during regrowth.
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise.
Peptidase Activity over Agar-Gelatin Film
The methodology developed for measuring proteolytic activity in plant tissues was based on in vitro incubations of fresh, mechanically damaged leaf tissue placed over a standard protein substrate. The enzymatic activity was assessed by dye retention in the substrate after the peptidase action and was expressed as a proportion of the color disappeared from a purified peptidase used as standard.
The protein substrate was a thin film of agar and gelatin made with 0.32 mmol L–1 phosphate buffer (pH 6.5), 5.6 mmol L–1 cysteine, 2.4 g L–1 gelatin, and 30 g L–1 agar. Upon heating to 96°C, the solution became completely clear and was then poured on a leveled, prewarmed, flat-bottom, 80-mm-diam. Petri dish until the dish surface was completely covered with a near 2-mm-thick layer. Irregular films were discarded, and the most regular ones were sealed, stored, and kept overnight at 4°C to be used next morning.
With a sharp metal punch, three 3-mm discs were cut from a single leaf and considered as triplicate samples of one plant. Because leaf age is a key factor in peptidase activity (Morris et al., 1996; Nieri et al., 1998), leaf discs were cut from the youngest fully expanded leaf. To simulate the mechanical injury that occurs during the first chewing by an animal, each disc was placed in a glass plate with the main vein along a 0.5-mm channel, and a sintered glass rod, 5-mm diam. with a 400-g weight on top, was rolled once over the leaf tissue (Fig. 1
). To facilitate the diffusion of leaf enzymes to the protein substrate, the leaf discs were punctured against a glass surface with a set of entomology needles packed with 540 needles cm–2 and then quickly placed with the bottom side over the protein substrate. Three plants of each accession were evaluated. Plants were discarded if they had any sign of insect attack or disease. Each plant was evaluated by placing one of the triplicate discs in a different Petri dish. A set of three Petri dishes was used to evaluate 25 plants.
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Fig. 1. Schematic representation of tools used for mechanical damage of the leaf discs: (a) glass plate; (b) channel, 0.5-mm width; (c) leaf disc, 3-mm diam.; (d) sintered glass rod, 5-mm diam.; and (e) weight, 400 g.
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A peptidase standard was prepared with 125 mg L–1 papain in a water solution with 10 mmol L–1 tritiplex and 50 mmol L–1 cysteine (Worthington Biochem. Corp., 1972). Immediately after mechanical damage, samples and three discs of filter paper (Advantec 5A, Advantec MFS, Pleasanton, CA), 3 mm in diameter, the later with 1.5 µL of standard peptidase solution, were gently placed over the protein substrate in each Petri dish (Fig. 2
). The dishes with the plant tissue samples and controls were gassed with CO2, sealed, and kept in an anaerobic chamber for 4 h at 39.5°C.
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Fig. 2. Petri dishes with agar-gelatin films: (a) Petri dish with samples and papain standards before incubation and (b) Petri dish after peptidase action showing spots with variable activities over a dark background. ( ) Papain standards.
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After incubation, leaf and filter paper discs were removed from the Petri dish with a mild cold water flux, and the substrate film was gently rinsed. The agar-gelatin films were then covered with an aqueous solution of Amido-black (2 g L–1) and gently agitated for 10 min with constant lateral and frontal movements. The films were subsequently washed in water for 10 min, drained, and kept overnight at 4°C. Peptidase activity was evident by clear, round circles surrounded by a dark blue background (Fig. 2). Digital images of the agar-gelatin films and zymograms were made with an Agfa Duoscan T1200 scanner using a scale of 256 gray tones and a resolution of 150 dots per inch (dpi). Activity spot area and color measurements were made with Scion Image software (Scion Corp., Frederick, MD). The area (A) and inverse color intensity (I) value corresponding to each sample (i) was expressed as a proportion of the arithmetic mean of the three papain standard discs in each Petri dish (Eq. [1] and [2]).
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The PAI of each sample was calculated by multiplying its area and inverse color intensity proportions (Eq. [3]).
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The PAI of each plant was calculated as the arithmetic mean of the three samples obtained from it.
Main Peptidases in Extracts
A small piece of healthy, recently expanded leaf lamina was macerated in 0.5-mL Triz buffer (500 mmol L–1) with sodium dodecyl sulfate (SDS) (5 g L–1) at pH 6.8. Macerates were clarified by centrifugation (10 min at 20 000 g and 4°C). Denaturating polyacrylamide minigels (10 by 7 cm) were made according to Laemmli (1970) with 6 mg mL–1 of gelatin as substrate for peptidase activity. After running the gels, peptidases were renaturalized with a 1-h gentle wash in Triton X-100 (25 mL L–1). Peptidases were allowed to act 15 h by incubating the gels at 39.5°C in buffer (pH, 7.0; Triz, 50 mmol L–1; NaCl, 200 mmol L–1; and CaCl2, 5 mmol L–1). Following incubation, gels were rinsed and stained with a solution of Coomasie Blue (1 g L–1), methanol (400 mL L–1), and acetic acid (100 mL L–1) and finally washed in 100 mL L–1 acetic acid. Clear bands over a blue background were observed in the sites where peptidases degraded the gelatin incorporated in the gels. Molecular weight of each peptidase was estimated at the middle line of each band using a wide-range standard of 13 proteins in the range of 2 to 212 kDa (Winkler, Santiago, Chile).
Peptidase Class Identification
Peptidase class was determined using zymograms as described above, with the addition of various specific peptidase inhibitors in the incubation buffer. Inhibitors and concentrations utilized are listed in Table 2. Serine- and aspartate-peptidase inhibitors were first dissolved in ethanol. The incubation buffer for those classes of inhibitors did not contain CaCl2, and the final concentration of ethanol in the buffer was 50 mL L–1. Controls for each treatment were incubated in the same buffer, but without the addition of the peptidase inhibitor. Following incubation, gels were rinsed, stained, and washed as described, and the color intensity of the bands was measured.
Statistical Analysis
Assays were performed on the three individual plants grown of each accession. Each plant was considered a replicate. Plants were discarded if they showed signs of insect attack or disease; thus, some accessions had only two replicates. All 342 accessions grown were evaluated for PAI. Type III ANOVA and estimation of variance components for the PAI was performed with the MIXED procedure of SAS (SAS Inst., 2001) using a randomized complete block design with a nested structure. Nursery trays were considered blocks. Components of the nested structure were family, genus within family, species within genus, and cultivars within species. Family was considered a fixed effect while genus, species, and cultivar were considered random effects. Denominator degrees of freedom for tests of fixed effects were estimated using a general Satterthwaite approximation. Mean PAI for each genus, species, and cultivar was estimated.
Molecular weight of the main peptidases in plant extracts were evaluated in a selection of 52 Poaceae and 34 Fabaceae germplasms. Selection was made based on PAI results and agronomic interest on the accession. The class of the main peptidases present in leaf extracts was identified in alfalfa (‘Maris Kabul’), red clover (‘Redqueli’), white clover (Trifolium repens cv. Pitau), tall fescue (cv. ‘Quantum’), perennial ryegrass (‘Nui’), and Italian ryegrass (Lolium multiflorum Lam. cv. Ajax). Differences among peptidase-inhibitor treatments were estimated using one-way analysis of variance and Tukey's Studentized Range Test. Differences were considered significant at the 0.05 probability level.
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RESULTS
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A wide range of proteolytic activity was detected among the 65 species evaluated (Table 1). Species in the high-proteolysis decile were lopsided oat (Avena strigosa Schreb.), water medic (Medicago littoralis Rohde ex Loisel.), alfalfa, sphere medic (Medicago sphaerocarpos Bertol.), disc medic [Medicago tornata (L.) Miller], and bulbous canarygrass (Phalaris aquatica L.). The presence of several Medicago species within the high activity group is remarkable. In addition, the species in the lower decile were: hybrid fescue [Festulolium loliaceum (Huds.) P. Fourn.], birdsfoot trefoil, common wheat, Egyptian clover (Trifolium alexandrinum L.), suckling clover (Trifolium dubium Sibth.), and subterranean clover (Trifolium subterraneum L.).
The PAI values for the 342 accessions ranged from no detectable activity to 1.30, with an overall mean of 0.35 (data not shown, available on request from the authors). Accessions with no detectable proteolysis over the agar-gelatin films in all three assessed plants were common oat (Avena sativa L. cv. Flaemings), common barley (Hordeum vulgare L. cv. Lina), hybrid ryegrass (Lolium x hybridum Hausskn. cv. Sabrina), birdsfoot trefoil (‘Leo’ and ‘San Gabriel’), big trefoil (Lotus pedunculatus Cav. cv. Maku), red clover (‘Redqueli’), white clover (‘Sustain’), subterranean clover (‘Tallarok’ and ‘Gosse’), and common wheat (‘Metrenco’).
ANOVA revealed that genus within family, species within genus, and cultivar within species were significant factors affecting PAI (Table 3). More than 88% of the observed variance was due to differences in genus, species, and cultivar (Table 4). Because genus was the factor with the highest variance component, the average PAI for each genus is presented in Fig. 3
. Caution is needed to interpret these results as the number of species and plants evaluated in each genus was different (Table 1).
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Fig. 3. Protein degradation of forage genera in agar-gelatin films, expressed as proteolytic activity index (PAI). Lines over the bars represent standard error of the estimated mean.
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Zymogram analysis revealed that most species had only one clearly detectable peptidase in the plant extracts that was capable of degrading protein in the polyacrylamide gels (Table 5). A second, clearly detectable peptidase was also observed in most common oat and in all triticale (x T. rimpaui) plants. The molecular weight of the peptidases detected was in the range from 54 to 130 kDa.
Treatments with serine-peptidases inhibitors significantly reduced the activity of the peptidases in all evaluated germplasm except TPCK in Italian ryegrass and perennial ryegrass (Table 6). The PMSF treatment completely inhibited peptidase activity in all six evaluated species, which failed to exhibit any clear band in the gels. The inhibitors for other peptidase classes did not reduce the activity of any of the enzymes. In perennial ryegrass, metallo-peptidase inhibitors significantly increased total peptidase activity.
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Table 6. Changes in proteolytic activity (PA) in zymograms of six forage species after peptidase inhibition treatment.
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DISCUSSION
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The methodology used to evaluate protein degradation was designed to partially conserve integrity of the plant tissue because many plant cells remain intact after being swallowed by grazing cattle (Kingston-Smith and Theodorou, 2000), and the release of cell contents is affected by the extent of mechanical damage (Boudon et al., 2002). However, the results obtained in this study should not be extrapolated directly to animals consuming fresh forages. Rather, they can be used as references of protein degradation by means of plant peptidases.
As gelatin is not of plant origin, specificity between enzyme and substrate might introduce a source of error in the PAI assay. However, our results are in good agreement with other studies in which endogenous protein degradation was evaluated (Papadopoulus and Mckersie, 1983; Messman et al., 1994). Gelatin was chosen as a protein substrate because of its open and heterogeneous structure in gels, which is easily accessible to proteases; also, it is widely available, very soluble, does not coagulate after heating, and is inexpensive. Previous evaluation of the assay showed that its coefficient of variation was 12% and that the PAI of the youngest fully expanded leave was the best estimate of the PAI for the whole plant (Larraín and Vives, 1998).
The absence of detectable activity in some accessions does not mean that these plants have no proteases or that their activity is completely inhibited. Activity may be detected in these plants if the incubation time is extended. Our assay was intended to focus on the early protein breakdown because of its significance in terms of proteolysis during rumen fermentation. For this reason, we selected shorter incubation times knowing that delayed measurements would yield a complete hydrolysis in many entries. Four hours of incubation time was used to estimate PAI as this time maximized the range in PAI among accessions of high and low proteolytic activity (data not shown).
Lower protein degradation was expected among some accessions of Lotus species and red clover because peptidases inhibitors have been found in them. In red clover, a polyphenol oxidase is responsible for browning the tissue and preventing proteolysis by polymerization of proteins and phenols (Jones et al., 1995). The low PAI observed in some Lotus species was probably due to the presence of condensed tannins (Zhu et al., 1999). Condensed tannins (proanthocyanidins) may bind to proteins, changing their conformation and inducing steric interference with peptidases (McMahon et al., 2000). Condensed tannins can also bind directly to hydrolytic enzymes reducing their activity (Makkar et al., 1988).
High proteolysis within alfalfa was expected because it has been the species with the greatest protein degradation under various conditions in several studies (Papadopoulus and Mckersie, 1983; Messman et al., 1994). In this study, average value for alfalfa was within the decile of highest PAI (mean PAI = 0.773). However, a wide range of variation was observed among cultivars (from 0.263 in ‘Sundor’ to 1.083 in ‘Daisy’). Similarly, common oat accessions had high PAI (mean PAI = 0.678) with the exception of the cultivar Flaemings, which had no detectable activity. These observations indicate that genotypes with low proteolytic activity are already available even within species with overall high PAI. Thus, germplasms with reduced proteolysis may be a valuable genetic resource in the improvement of forage cultivars of known high agricultural value or in the development of new lines selected for this attribute.
With most species, only one peptidase enzyme was detectable by the gelatin zymograms (Table 5). In contrast, two peptidases were found in most plants of common oat and triticale. The peptidase detected in wheat had an estimated molecular weight of 80 kDa, the same as one of the two enzymes in triticale [a hybrid of wheat and rye (Secale cereale L.)]. However, we did not test any accession of rye, so no comparison of the second peptidase (and its potential conservation) with rye germplasm is possible.
Other authors have purified and characterized peptidases in forage species, but the molecular weights they reported were usually much less than the values we obtained. For example, a 23- and 30-kDa peptidase in alfalfa, and a 28-kDa peptidase in barley have been reported (Finley et al., 1980; Miller and Huffaker, 1981; Nieri et al., 1998) compared with our observations of 112 and 77 kDa. We consistently failed to find peptidases of less than 50 kDa, and the weight of the enzymes we found were usually higher than values in the literature. We hypothesize that the intensive extraction and purification processes used in many studies may reduce peptidase stability or induce proteolysis of enzyme fragments not needed to hydrolyze proteins. The mild extraction methodology used in this study—without acid or salt precipitation—may conserve the integrity of peptidases, leading to molecular weight values greater than estimated by other authors.
The analysis of many reports suggests that serine- and cysteine-peptidases are the most commonly found peptidases in extracts of forage and grain plants (Finley et al., 1980; McKersie, 1981; Miller and Huffaker, 1981; Morris et al., 1996). The main peptidases in extracts of the six evaluated grass and legume species in our study belonged to the serine class. In alfalfa, our findings are in good agreement with the results from McKersie (1981) but in contrast with those of Nieri et al. (1998), who described a metallo-peptidase responsible of 90% of the proteolytic activity. However, the last authors evaluated leaves from dark-induced senescent plants, which may induce program cell death and expression of peptidases not present in fully active leaves (Buchanan-Wollaston, 1997; Herrmann and Feller, 1998).
An explanation for the increased peptidase activity observed in perennial ryegrass when incubated with metallo-peptidase inhibitors may be that the metal-chelating properties of these compounds can induce changes in the tertiary structure of the peptidases, which could enhance its activity. A similar response was observed in alfalfa by Nieri et al. (1998) with the nonspecific inhibitor pCMB.
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CONCLUSIONS
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We observed a wide variation in proteolytic activity measured by incubation of fresh plant tissue over a protein-containing gel. Our findings support the idea that a reduction in protease activity in forages is feasible by selection of appropriate germplasms. The wide variation in proteolysis observed among the evaluated accessions should encourage plant breeding efforts to look for and select genetic lines with reduced protein degradation rates via lower peptidase content or greater peptidase inhibitor concentrations. The existence of one or two relevant peptidases in each species and the fact that these main peptidases belong to the serine class may allow the expansion of research in technologies that could reduce plant protein degradation during ensiling and during early stages of ruminal digestion.
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ACKNOWLEDGMENTS
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The authors thank IGER (United Kingdom), SARDI (Australia), ANASAC (Chile), ECSA (Chile), INIA (Chile), and SG2000 (Chile) for providing the seed stock used. We are also very grateful to Luis Barrales and Ivan Peña for their advice in statistical analysis and Eduardo Leiva for his assistance in laboratory work.
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NOTES
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Financial support for this study was provided by Fondecyt (Chile) grants No. 1000267 and 1030918.
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