Annals of Botany 89: 731-740, 2002
© 2002 Annals of Botany Company
An Assessment of the Ability of the Stay-green Phenotype in Lolium Species to Provide an Improved Protein Supply for Ruminants
1Department of Animal Science and Microbiology, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK and 2Department of Plant Breeding, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK
* For correspondence. Fax +44 (0)1970 828357, e-mail alison.kingston-smith{at}bbsrc.ac.uk
Received: 4 February 2002; Returned for revision: 21 February 2002; Accepted: 8 March 2002.
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ABSTRACT |
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The stay-green phenotype results from a naturally occurring mutation in which senescent leaves retain their chlorophyll and the associated apoprotein, LHCPII. Protection of this protein pool could deliver grass with enhanced protein content and could decrease the extent of protein degradation by plant proteases in the rumen. This would enhance the efficiency of protein utilization in livestock to the benefit of the environment. Field plots of stay-green and wild-type Lolium perenne were defoliated at intervals to simulate grazing. There were variations in foliar protein content and proteolysis throughout the year, but no significant differences between genotypes when material was analysed fresh or after it was cut and dried to simulate hay-making, which possibly induced senescence. In a subsequent experiment with stay-green and wild-type L. temulentum, increased protein retention and decreased protein degradability were observed in stay-green leaves that were allowed to senescence naturally and extensively on the plant. That there is no difference between the two L. perenne genotypes suggests that as a field crop in grazed pastures the stay-green genotype would not confer a nutritional advantage in terms of protein degradability. It is possible that grazing promotes a high proportion of non-senescent to senescent leaf material within the sward and thus any advantage conferred by the stay-green phenotype would be effectively masked by an abundance of mature foliage. It is suggested that the stay-green trait would be of benefit in areas where agricultural practice permits extensive natural senescence to occur.
Key words: Lolium perenne, Lolium temulentum, stay-green, forage, protein, protease, Rubisco, LHCPII.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As part of the developmental process, leaves of plants mature and then enter a period of senescence in which protein and carbohydrate are remobilized for transport to other organs. Typically, senescence is accompanied by yellowing as chlorophyll is degraded (Matile et al., 1999). A naturally occurring mutant of Festuca pratensis in which the chlorophyll is not degraded during senescence has been designated the stay-green phenotype. This phenotype owes its characteristics to a mutation in the sid gene that prevents operation of the macrocycle-opening reaction in chlorophyll catabolism, catalysed by the enzyme phaeophorbide a oxygenase (Vincenti et al., 1995). Interestingly, retention of chlorophyll in the leaves of the stay-green mutant is associated with increased persistence of several chlorophyll binding proteins (Thomas, 1987; Thomas et al., 1992), although retention of these proteins is not associated with increased photosynthetic performance (Hauck et al., 1997; Kingston-Smith et al., 1997). Through conventional breeding, the sid gene has been transferred to forage and amenity grasses to produce stay-green varieties of ryegrass (Lolium perenne) and darnel (Lolium temulentum) (Thorogood, 1996; Thomas et al., 1997, 1999; Wilman et al., 2001).
The aim of this study was to determine whether stay-green grasses provide a nutritional advantage to ruminant animals grazing at pasture. This possibility arises because of the apparent increased protein stability in the stay-green lines. Obviously, an adequate protein supply to the ruminant animal is crucial for growth and development. However, excessive protein degradation in the rumen, surplus to the demands of the rumen microbial population, results in deamination of amino acids and excretion of ammonia (Beever and Siddons, 1986; Wetherall et al., 1995; Dewhurst et al., 1996). This is a wasteful process causing a net loss of nitrogen to the animal and pollution of the environment. If protein breakdown in the rumen can be attenuated (either through slowed rate or decreased extent) so that more of the ingested protein passes through the rumen intact, to be degraded by acid hydrolysis in the abomasum (the true stomach), there may be a nutritional advantage to the animal, decreasing the need for dietary protein supplements, thereby limiting N pollution to the environment.
The conventional view that proteolysis in the rumen is due to rumen micro-organisms has recently been questioned (Theodorou et al., 1996; Kingston-Smith and Theodorou, 2000). The potential for plant enzymes to participate in degradation of plant protein in the rumen has been largely overlooked previously: our recent data indicate that proteolysis in the rumen is mediated by plant proteases at least during the initial stages of digestion (Theodorou et al., 1996; Zhu et al., 1999; Kingston-Smith and Theodorou, 2000; Beha et al., 2002). Because rumen conditions constitute a stressful environment for plant cells, ingested plant material may undergo a form of cell death, during which protein is degraded by endogenous plant proteases (Theodorou et al., 1996; Kingston-Smith and Theodorou, 2000; Beha et al., 2002). In planta, retention of chlorophyll in the stay-green phenotype means that the apoproteins associated with it in vivo are not degraded to the same extent as in the wild-type during natural or induced senescence (Thomas, 1977, 1982a; Hilditch et al., 1989). Such prolonged association could result in a smaller proportion of the protein pool being degraded and so enable more protein to persist through the rumen. By avoiding rumen proteolysis, more protein would be available for direct use by the animal. Alternatively, the protease activities of the stay-green leaves may differ from those present in the wild-type leaves and this, in itself, may confer a nutritional advantage during their degradation in the rumen.
An in vitro system was used to determine the rate of proteolysis in the absence of rumen micro-organisms in stay-green and wild-type genotypes of L. perenne and L. temulentum. First, a simulated grazing regime was imposed on replicated, paired experimental lines to investigate seasonal changes in protein stability and nutritional quality of fresh grass and hay from stay-green and wild-type genotypes of L. perenne. Secondly, proteolysis in undefoliated stay-green and wild-type genotypes of L. temulentum were studied at various growth stages under controlled environment conditions.
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MATERIALS AND METHODS |
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Plant material
The sid mutant, originally characterized in Festuca pratensis (Thomas, 1982a, b), was transferred to perennial ryegrass (L. perenne L.) using Festuca–Lolium intergeneric crossing procedures (Humphreys and Thorogood, 1993). A homozygous sid mutant donor genotype of L. perenne (provided by D. Thorogood) was crossed into a high water soluble carbohydrate (WSC) accumulating line of perennial ryegrass and a segregating F2 population was produced. From the F2 population stay-green and yellowing phenotypes were selected and polycrossed. Further selection based on progeny tests was made within the yellowing polycross progeny for homozygous wild-type plants, and within stay-green polycross progeny for confirmed homozygous sid mutant genotypes. Seed from selected lines (stay-green IGER ref. 94/4 and 94/6; wild-type IGER ref. 94/21 and 94/22) was sown in 1996 in 2 m2 plots, twice replicated in randomized blocks on Cae Banadl (Plas Gogerddan, Ceredigion, UK). This material was used for comparison of the response of stay-green and wild-type plants (four plots each of stay-green and wild-type) to simulated grazing. Plots were cut approximately monthly between April 1998 and March 1999 and were supplied with 50 g fertilizer per plot (21 : 8 : 11, N : P : K) initially in April 1998 and then after each cut between May and August 1998. After cuts in September and October, fertilizer was supplied at a ratio of 9 : 24 : 24, N : P : K. Except for the removal of experimental material, plots were not cut (or fertilized) between November 1998 and March 1999 in accordance with common agricultural practice.
Two cuts were taken for hay from replica, randomized field plots of stay-green and wild-type L. perenne in June and September 1999, by cutting about 5 cm above the soil surface. Samples were bulked and dried to 90 % dry matter over 3 d in a growth cabinet (300 µmol m–2 s–1 irradiance, 20 °C, 8 h light period) with regular turning. This simulated hay-making.
Plants of L. temulentum were grown from seed in trays of John Innes no. 3 compost under controlled environment conditions (300 µmol m–2 s–1 irradiance, 20 °C, 8 h light period) for 8 weeks. Mature leaves (determined by ligule formation on leaves) were harvested and used fresh or made into hay as described above. Some plants were allowed to grow for a further 4 weeks until senescent material was abundant on the plant.
Field sampling procedure
Within a single plot, about 5 g f. wt of grass was removed by cutting about 2·5 cm above the soil at each of three random positions in each plot. This material was then combined into one sample. Hence, the harvested plant material contained a range of leaf ages from senescent to immature. The samples collected from the eight plots were placed in a pre-cooled box which was then transported to the laboratory. Harvesting and transport to the laboratory took a maximum of 20 min (15 min were required for harvesting).
Determination of nutritional quality
Samples (2 g) of material from each plot were oven-dried for 2 d at 80 °C before being ground to a fine powder in a ball mill. The nutritional quality (nitrogen, carbohydrate and fibre content) of the dried powder was analysed chemically as described in Leco Instruments UK (1992), Arthur (1977), MAFF/ADAS (1986) and Goering and van Soest (1970).
In vitro determination of rate of plant-mediated proteolysis
In the laboratory, leaf blades were surface sterilized by immersion in 200 ml 80 % (v/v) ethanol for 2 min, rinsed three times with cold water, blotted dry and cut into 1–2 cm lengths. Then 200 mg f. wt of grass was weighed into a microfuge tube and frozen in liquid nitrogen for compositional analysis of the standing crop.
For each sample from the eight plots, about 250 mg of cut grass was accurately weighed into perforated plastic tubes, in triplicate. Under a stream of anaerobic gas (80 % N2, 10 % CO2, 10 % H2), four of these tubes were placed in 100 ml Duran bottles (Schott) containing 100 ml anaerobic phosphate buffer (50 mol m–3 Na2HPO4, 50 mol m–3 KH2PO4, 1 mol m–3 dithiothreitol, 0·01 % (v/v) methylene blue, pH 6·8) pre-warmed to 39 °C (Beha et al., 2002). The buffer had been made up to exclude oxygen (determined by loss of colour of the indicator dye) by using freshly boiled (and cooled) water and gassing with the anaerobic gas mix for 4 h. After 0, 2, 6 and 24 h of incubation of plant tissue in buffer at 39 °C, three replicate perforated tubes were removed from the buffer and the tissue placed in clean microfuge tubes. These were frozen in liquid nitrogen for subsequent determination of foliar protein content. This procedure was repeated concurrently for each plot sample.
Biochemical measurements
Leaf tissue was ground to a fine powder in liquid nitrogen and 0·5 ml extraction buffer (50 mol m–3 Na2HPO4, 50 mol m–3 KH2PO4, pH 6·8, 1 mol m–3 EDTA, 5 mol m–3 dithiothreitol, 1 mol m–3 PMSF, 1 mmol m–3 E-64, 0·1 % (v/v) Triton X-100). The extracts were centrifuged for 5 min at 10 000 g and the supernatant recovered. The pellet was put through one freeze/thaw cycle and then re-extracted with extraction buffer (as above). The supernatant was recovered. The protein content of the extracts was determined following Bradford (1976) with suitable controls to negate interference of the detergent with the dye-binding reaction. The chlorophyll content of the extracts was determined according to Lichtenthaler and Wellburn (1983) by placing a 50 µl aliquot of the crude (uncentrifuged) extract in 950 µl of 80 % acetone and measuring absorbance at 663, 646 and 470 nm. Protein extracts were assessed qualitatively with denaturing PAGE (Laemmli, 1970).
Protease activity
Protein was extracted from fresh grass, senescent grass, or hay in 50 mol m–3 Na2HPO4, 50 mol m–3 KH2PO4, pH 6·8, 1 mol m–3 EDTA, 5 mol m–3 dithiothreitol, 0·1 % (v/v) Triton X-100. Protease activity of the extracts was determined by a modification of the gel clearing assay of Zavaleta-Mancera et al. (1999). Briefly, tissue extracts were loaded onto filter paper discs which were applied to the surface of a Petri dish containing a solidified mixture of agarose and gelatin buffered at either pH 5·6 (previously determined to be the optimum for senescence-induced protease activity in L. perenne; A. H. Kingston-Smith, unpubl. res.) or pH 6·8 (rumen pH). After incubation at 39 °C for 24 h, the zones of clearing were determined by amido black staining and densitometry (GS 710 scanning densitometer equipped with Quantity One imaging software; BioRad UK Ltd, Hemel Hempstead, UK) where the result of multiplying the area of clearing (mm2) by 1/OD was obtained in arbitrary clearance units. In this way it was possible to assess proteolysis within the gel even in the absence of substantial radial diffusion.
Protease isozymes were revealed by semi-denaturing PAGE by a method modified from Morris et al. (1996) as described in Beha et al. (2002). Gels were cast to contain bovine serum albumin as a substrate. After electrophoresis, gels were incubated overnight and then stained with Coomassie blue to reveal clear bands of protease activity on a blue background.
Statistical analysis
For field trial data three replicate measurements were performed on each of the four lines representing either the stay-green or wild-type genotype. ANOVA showed no significant differences among the four lines within each genotype. For simplicity of presentation, data have therefore been compiled to allow genotype comparison. For the experiment in which hay was prepared, the results of a factorial design were compared by ANOVA using the GENSTAT package. Unless otherwise indicated, results presented are means and associated standard errors, and the significance of differences between genotypes was determined by Student’s t-test. Regression and first order rate equations were fitted to data by FigP software (Biosoft, UK Ltd, Cambridge, UK). Calculation of the time required for the leaf protein pool to decrease by a factor of two (g) was based on equations used to calculate microbial doubling time. Microbial growth is described by the expression:
ln Z – ln Z0 = µ (t – t0)
where Z and Z0 are the number of bacterial cells (or in this case protein) at times t and t0, respectively, and µ is the growth rate constant. If the constant g is substituted for (t – t0) then: µ = (ln 2/g) and µ can be substituted by the first order rate constant, derived experimentally, to give the value of g in hours.
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RESULTS |
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Seasonality in field-grown stay-green and wild-type L. perenne
A comparison was made of the composition of leaves taken from stay-green and wild-type L. perenne grown in field plots and subjected to regular defoliation. Total leaf nitrogen increased during the sampling period and, with the exception of measurements made after February 1999, was similar in stay-green and wild-type L. perenne (Fig. 1). Over the same period, the readily extractable protein and chlorophyll contents of the field-grown grass fluctuated but stay-green plants did not have greater protein contents than wild-type plants at any time of year (Fig. 1). There was a trend towards decreased protein in early autumn. In September 1998, the chlorophyll content of stay-green leaves exceeded that of the wild-type but there was no corresponding increase in leaf protein (Fig. 1). WSC in leaves of stay-green plants tended to decrease during the latter phase of the sampling period, from August 1998 to April 1999 (Fig. 1), compared with that in the wild-type.
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Both LHCPII content (Fig. 2) and protease profiles (Fig. 3) were generally similar for stay-green and wild-type plants at a given time of the year. The band density of LHCPII decreased over the autumn cuts (Fig. 2). Activity of two main protease isoforms, at 67 and 52 kDa, were clearly resolved on stained gels. Densitometry confirmed that protease activity varied with season (Fig. 3). Both proteases showed peaks of activity during spring and summer, with activity decreasing during the autumn and winter (Fig. 3). The ratio of 67 kDa protease activity to 52 kDa protease activity changed with season (compare Fig. 3A and B).
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When L. perenne leaf material was subjected to rumen-like conditions in the in vitro degradation assay, chlorophyll degradation was minimal, and largely similar between stay-green and wild-type plants (Table 1). In contrast, protein breakdown was extensive but there were no significant differences in terms of extent of degradation of the protein (percentage of the original protein pool remaining after incubation for 24 h) between corresponding stay-green and wild-type plants (Table 1). There was a trend towards more extensive protein degradation with progressive harvest dates that was not seen for chlorophyll (Table 1). Protein degradation proceeded according to first order rate kinetics which allowed calculation of the theoretical value g, the time required for the protein pool to decrease by a factor of two. No significant differences in the values of g were observed for corresponding stay-green and wild-type leaves of L. perenne (Fig. 4). Linear regression showed a weak trend towards an increased rate of protein degradation as the season progressed (Fig. 4), indicating that protein was less stable in March 1999 than in May 1998.
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Performance of the L. perenne wild-type and stay-green phenotypes as hay
Field plots of L. perenne were cut for hay in June and September and compared with the relevant fresh material (Table 2). Nutritional quality parameters varied across harvests but were largely unchanged by the preservation, with the exception of WSC content (Table 2). Except for neutral detergent fibre (NDF), there were no consistent differences in nutritional parameters, including nitrogen content, between corresponding stay-green and wild-type genotypes for each harvest (Table 2).
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When hay from the wild-type and stay-green L. perenne phenotypes was incubated anaerobically at 39 °C, rates of protein loss were rapid and did not differ (Fig. 5). Protease activities per unit of protein were not significantly altered in extracts from hay compared with those from fresh grass when measured at pH 5·6 (299 and 428 units mg–1 protein, respectively) or pH 6·8 (285 and 442 units mg–1 protein) due to the statistical variation (coefficient of variation = 83 %). There were no significant differences due to genotype (mean 388 and 339 units mg–1 protein for stay-green and wild-type leaves, respectively).
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Comparison of senescent material from stay-green and wild-type plants of L. temulentum
As observed previously with L. perenne, there was no advantage to the stay-green line of L. temulentum in terms of protein conservation when examined as fresh material or after conservation as hay (data not shown). The progress of senescence in L. temulentum was monitored by changes in leaf protein and chlorophyll content. When leaves were allowed to senesce naturally (without being cut from the plant) over the 40 d following emergence of the fourth leaves (DAE), chlorophyll content in wild-type plants decreased from 0·26 to 0·15 mg g–1 f. wt, consistent with the onset and progression of senescence. In stay-green plants, however, the chlorophyll content fell only slightly, from 0·29 to 0·26 mg g–1 f. wt. Over the same period there was a significant divergence, at around 25 DAE, in protein content (Fig. 6A), resulting in an almost three-fold greater protein content in stay-green compared with wild-type L. temulentum. This was reflected in a greater loss of ribulose 1,5 biophosphate carboxylase/oxygenase (Rubisco) (Fig. 6B) and, to a lesser extent, loss of LHCPII (Fig. 6C) in leaves of wild-type plants compared with stay-green plants. In contrast, the protease profile was unchanged between leaves of stay-green and wild-type plants during the course of natural senescence (Fig. 6D). When leaves were harvested late into senescence (27 DAE), the initial difference in protein content between senescent leaves of stay-green and wild-type plants persisted during in vitro analysis and was significant at incubation times over 2 h (Fig. 7A). The protease isoforms present during in vitro incubation of senescent material were not altered in activity or number and there was no difference between genotypes (Fig. 7B).
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DISCUSSION |
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In theory, the stay-green phenotype has the potential to increase the protein supply to the ruminant in a form that will be utilized more efficiently, thereby reducing the need to supply additional concentrated forms of protein. We have shown previously (Zhu et al., 1999; Kingston-Smith and Theodorou, 2000; Beha et al., 2002) that protein degradation in the rumen can be addressed from a plant perspective; proteolysis occurs in the presence and absence of rumen micro-organisms, at least in the first few hours after ingestion. We believe that a significant proportion of the cells in freshly ingested, excised leaves undergo a cell death response in the rumen that contains elements of, but is not identical to, the induced senescence pathway (Kingston-Smith and Theodorou, 2000; Beha et al., 2002). Thus, because stay-green leaves undergoing normal senescence retain LHCPII, stay-green leaves in the rumen may lose a smaller proportion of protein if proteolysis is associated with normal senescence. Here we have tested the hypothesis that the stay-green mutation will preserve protein (1) in a standing crop similar to that used for grazing; (2) in hay making; and (3) during incubation of fresh and dried leaves under rumen-like conditions.
Protein remobilization is known to be affected by changing environmental conditions and nutritional limitations (Volenec et al., 1996; Fischer et al., 1998; Louahlia et al., 1999; Thornton et al., 2002). Therefore, the observed changes in protein and protease activity during the year of the field trial probably reflect changing demands of the plant for mobilization of assimilated nitrogen. Interestingly, protease activities were high at the end of the experiment, in contrast to May of the preceding year. This could explain why a high rate of protein degradation (low g-value) was achieved in the March 1999 samples as opposed to degradation rates returning to the relatively lower values seen in samples from May 1998. This effect could have been induced by lack of fertilizer after August 1998, leading to increased remobilization of internal nitrogen reserves and seasonal variation in parameters of quality and yield; without sufficient nutrients in the soil a good quality crop cannot be produced, hence the standard farm practice of a spring application of fertilizer. It has been suggested that due to the increased demand for carbon and nitrogen to form protein (at the expense of carbohydrate) in the stay-green lines, the phenotype might represent a low carbohydrate line. The observation that stay-green grass contains less carbohydrate than wild-type is also interesting from a practical stand-point; such grass may be of use as forage for horses, which can suffer serious conditions such as laminitis when grazing forage containing high concentrations of water soluble carbohydrates (Longland et al., 1999).
From the field trial evaluation of stay-green and wild-type L. perenne there was little evidence of an advantage of the trait in terms of protein supply or rate of protein degradation either when the crop was harvested fresh or conserved as hay. While senescence is a useful model for what happens when fresh forage enters the rumen (for a review, see Kingston-Smith and Theodorou, 2000), it does not fully explain the plant responses in the rumen (Beha et al., 2002). Despite significant differences in protein contents of stay-green and wild-type leaves after 24 h incubation in vitro under rumen-like conditions (Table 1), the lack of extensive degradation of chlorophyll indicates that degradation of protein and chlorophyll are not tightly linked processes under these conditions. This confirms previous observations (Beha et al., 2002) that in vitro degradation of chlorophyll was not as extensive as that which occurred during natural senescence. This might relate to the necessity for oxygen in the normal path of chlorophyll disassembly (Matile et al., 1999). While this would explain why more chlorophyll was lost in fresh forage during in vitro incubation (anaerobic conditions), it would not explain why differences in chlorophyll content in stay-green and wild-type plants were not observed when the grass was made into hay. It must be concluded that although senescence can be induced in attached leaves by drought conditions (Thomas and Stoddart, 1980; Khanna-Chopra et al., 1999; Yang et al., 2000), normal senescence cannot proceed in excised leaves very deficient in water. Interestingly, the data suggest that plant proteases may survive the prolonged drying of the hay-making process and can be re-activated on subsequent exposure to water.
When leaves were allowed to senesce naturally on the plant, the divergence of leaf protein content in stay-green and wild-type L. temulentum was mainly due to retention of Rubisco in stay-green plants (Fig. 6). This is interesting as both L. perenne and L. temulentum are believed to be type-C stay-green, in which initiation and rate of senescence are normal except for the retention of chlorophyll and LHCPII (Thomas and Howarth, 2000). This result contrasts with previous observations of the stay-green phenotype in Festuca pratensis in which disappearance of Rubisco was unconnected to genotype (Thomas, 1977). However, genotype-related differences in protein retention of senescent leaves were maintained during in vitro incubation, suggesting that the mechanism of protein degradation under rumen-like conditions was unchanged by the stay-green phenotype. Previously, protease activities were shown to be little changed during natural senescence in stay-green or wild-type Festuca pratensis (Thomas, 1982b). During incubation under rumen-like conditions, the stay-green trait resulted in improved protein content of senescent material compared with that of wild-type plants (Fig. 7), despite extractable protease activities being similar in the two genotypes. This indicates that increased protein persistence in senescent stay-green leaves subjected to rumen-like conditions was due to a simple substrate : enzyme relationship.
An advantage of the stay-green plants in terms of increased protein availability was seen in leaves from undefoliated plants after extended, natural senescence, but not when grass was subjected to regular cutting to simulate grazing. This indicates that an advantage from the stay-green phenotype would be gained in field situations in which a standing crop of largely senescent leaf material is used as the fodder source. For instance, stay-green could be exploited to maximize the nutritional quality of dead and dried sorghum leaves which are used as fodder for livestock in the semi-arid tropics, or to decrease the environmental impact of methods currently used to prevent protein losses during preparation of standing hay in Australia (Dove et al., 1999; Gatford et al., 1999; Leury et al., 1999; Siever-Kelly et al., 1999). One scenario resulting in a lack of an advantage of the stay-green phenotype during repeated defoliation of a sward (through grazing or cutting) could be if the sward contained mainly mature leaf blades, and only a relatively small percentage of senescent leaves. This could occur because grass leaf growth originates from a basal meristem, which together with production of tillers allows regrowth of laminae after defoliation (Tallowin et al., 1995). Hence, after defoliation, excised leaves will be replaced by high protein content juvenile leaves, which mature but may themselves be removed (grazing or cutting) before they begin to senesce; thus, the ratio of senescent to non-senescent leaves would favour non-senescent leaves. In the field trial, any advantage of retention of LHCPII in stay-green leaves in terms of protein content could therefore have been masked by an abundance of high-protein, mature, non-senescent leaf material. In addition, retention of LHCPII was of minor significance compared with rates of disappearance of Rubisco. Although stability of LHCPII contributes to the rate of protein degradation in the rumen, our data demonstrate the need to address stability of Rubisco to make a major impact on protein quality in forage.
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ACKNOWLEDGEMENTS |
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The authors thank Mr M. Farrell for help in the field, Mrs D. Jones and the Analytical Chemistry Group for analyses of nutritional quality, Professor H. Thomas and Dr H. Ougham for supplying stay-green Lolium temulentum and many useful discussions, and Mr M. S. Dhanoa for assistance with statistical analyses. This work was funded by the BBSRC and a LINK Sustainable Livestock Production programme involving the Ministry of Agriculture, Fisheries and Food, Milk Development Council, the Meat and Livestock Commission and Germinal Holdings.
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LITERATURE CITED |
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