Journal of Experimental Botany, Vol. 51, No. 347, pp. 1089-1097,
June 2000
© 2000 Oxford University Press
Respiratory costs and rate of protein turnover in the roots of a fast-growing (Dactylis glomerata L.) and a slow-growing (Festuca ovina L.) grass species
1 Plant Ecophysiology, Faculty of Biology, Utrecht University, PO Box 80084, NL-3508 TB Utrecht, The Netherlands
2 Institut für Botanik, University of Münster, Schlossgarten 3, D-48149 Münster, Germany
3 Plant Sciences, Faculty of Agriculture, The University of Western Australia, Nedlands WA 6907, Australia
Received 23 July 1999; Accepted 4 February 2000
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Abstract |
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Protein turnover is generally regarded as one of the most important maintenance processes in plants in terms of energy requirements. In this study, the contribution of protein turnover to the respiratory costs for maintenance in the roots of two grass species, the fast-growing Dactylis glomerata L. and the slow-growing Festuca ovina L., is evaluated. Plants were grown under controlled-environment conditions in a nutrient solution to which
was added at a relative addition rate of 0.2 and 0.1 mol N mol-1 N already present in the plant d-1 for D. glomerata and F. ovina, respectively, so as to obtain a steady exponential growth rate close to the plants' maximum relative growth rate. Pulse-chase labelling with 14C-leucine was used to determine the rate of protein turnover in the grass roots. The rate of turnover of the total protein pool did not differ significantly between the two species. The protein degradation constant in D. glomerata and F. ovina was 0.156 and 0.116 g protein g-1 protein d-1, respectively, which corresponds with a total protein half-life of 4 d and 6 d. Assuming specific respiratory costs for protein turnover of 148 mmol ATP g-1 protein, the estimated respiratory costs for protein turnover in the roots were 2.8 and 2.4 mmol ATP g-1 root DM d-1 in D. glomerata and F. ovina, respectively. Both the fast- and the slow-growing grass spent between 22–30% of their daily ATP production for maintenance on protein turnover, which corresponds to 11–15% of the total root ATP production per day. Note that the data presented in this abstract are based on the assumption that 50% recycling of the 14C-labelled leucine took place in the roots of both grass species. Key words: Dactylis glomerata L., Festuca ovina L., maintenance, protein turnover, relative growth rate, specific respiratory costs.
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The fraction of daily fixed carbon that is used in root respiration is larger in inherently slow-growing herbaceous species than that in their fast-growing counterparts, when the plants are grown with free access to nutrients (Poorter et al., 1990
; Van der Werf et al., 1992b; Lambers et al., 1996). This is not due to the slow-growing species exhibiting higher rates of respiration per unit mass and time, since the rate of root respiration is often somewhat lower in slow-growing species than in fast-growing ones. However, this difference in root respiration is not as large as might be expected from the large differences in rate of ion uptake and maximum relative growth rate (RGR, the increase in dry mass per unit mass and time; Poorter et al., 1991; Van der Werf et al.,1994; Atkin et al., 1996; Scheurwater et al., 1998). Clearly some aspect of root respiratory metabolism is less efficient in the slow-growing plant species.
Root respiration provides metabolic energy for growth and maintenance of root biomass and for ion transport (Veen, 1980; Van der Werf et al., 1988; Bouma et al., 1996; Mata et al., 1996; Scheurwater et al., 1998). Scheurwater et al., using a linear regression approach to determine specific respiratory costs in a fast- and a slow-growing grass species, found 3-fold higher specific costs for ion transport, expressed as mol O2 per net mol taken up, in the slow-growing grass (Scheurwater et al., 1998). These higher costs are at least partly due to a higher efflux of N per net taken up in slow-growing grass species than in fast-growing ones (Scheurwater et al., 1999).
Specific costs for growth and for maintenance of biomass, however, are only slightly lower in the slow-growing Festuca ovina, compared with those in the fast-growing Dactylis glomerata (Scheurwater et al., 1998). The specific respiratory costs for growth have been calculated for the roots of a range of fast- and slow-growing herbaceous species (Poorter et al., 1991), using information on the chemical composition of the roots and the oxygen consumption associated with constructing specific compounds (cf. Penning de Vries et al., 1974). The specific costs for growth increased with increasing RGR and varied from 5.5–8.0 mmol O2 g-1 root DM (Poorter et al., 1991).
Several authors have analysed the underlying processes that determine the differences in specific costs for ion transport and growth between fast- and slow-growing grass species (Poorter et al., 1991; Scheurwater et al., 1999). In contrast, no study has yet investigated the underlying processes that determine respiratory maintenance costs in roots of fast- and slow-growing grasses. Respiratory costs for maintenance of root biomass differ only slightly between fast- and slow-growing grass species (Scheurwater et al., 1998); however, this does not necessarily mean that the costs of the underlying processes are also similar. Protein turnover and the maintenance of ion gradients are generally regarded as the two most important maintenance processes in terms of energy requirements (Penning de Vries, 1975; Bouma et al., 1994). Therefore, in this study, the rate of protein turnover was measured and the respiratory costs estimated for protein turnover in the roots of a fast- and a slow-growing grass species in order to evaluate the contribution of one of the major maintenance processes to the respiratory costs for maintenance.
Protein turnover is ‘the flow of amino acids from existing protein into newly synthesized protein’ (Hatfield and Vierstra, 1997). Thus, both biosynthetic and breakdown processes affect the rate of protein turnover. Both protein synthesis and protein degradation require respiratory energy (De Visser et al., 1992; Vierstra, 1993). Protein turnover has several important functions in regulating the plant's metabolism. Together with protein synthesis, degradation is essential to maintain appropriate enzyme levels and to modulate these levels based on internal and external signals (Hatfield and Vierstra, 1997). For instance, rate-limiting enzymes, like nitrate reductase, have fast degradation rates (Vierstra, 1993). Furthermore, protein degradation is important in allowing a plant to cope with changing environmental conditions. When nutrients become limiting, the rate of protein turnover is accelerated by increasing the rate of degradation relative to synthesis, which generates a pool of free amino acids from less essential proteins that can be used to assemble more essential ones (Hatfield and Vierstra, 1997). This has been shown for the total soluble protein pool of Lemna minor (Davies, 1979). Acceleration of protein degradation is also important in response to changing light conditions. In a canopy, leaves that are initially exposed to high irradiance at the top of the leaf canopy during early growth, are progressively shaded during later development as shoot growth progresses. To improve nitrogen-use efficiency at the whole plant level, nitrogen must be mobilized in these shaded leaves, through acceleration of protein turnover, and reallocated to leaves in the more favourable light environment (Pons et al., 1993). For soybean, it has been shown that, after the start of the shading treatment, leaf nitrogen content per unit area gradually declined over a period of 12 d, indicating export of nitrogen from the leaf (Pons and Pearcy, 1994).
Other factors also influence protein turnover rates. It has been shown that the rate of protein turnover increases with increasing temperature in wheat roots (Ferguson et al., 1990). The rate of protein turnover also depends on the developmental stage of a plant or plant organ. For example, the rate of protein turnover of individual proteins, such as Rubisco (Mae et al., 1983) and of the total protein pool in Lolium perenne leaves (Barneix et al., 1988), increase with plant age. In contrast, the rate of protein turnover in bean leaves is higher in expanding than in fully-grown leaves (Bouma et al., 1994).
Protein degradation also has a housekeeping function. Abnormal proteins that appear in cells due to biosynthetic errors, spontaneous denaturation and free radical-induced damage (Vierstra, 1993), have to be degraded before they accumulate to toxic levels. In most situations the balance between free radical formation and detoxification is tightly controlled (Foyer et al., 1997; Millar and Day, 1997). Starvation and stressful conditions such as high and low temperatures, dehydration and high light intensities can accelerate damage by free radicals (Foyer et al., 1997; Hatfield and Vierstra, 1997). Metabolic imbalance can lead to an increase in the steady-state concentration of free radicals, which eventually can lead to protein damage (Foyer et al., 1997). Since the metabolic rates in the roots of fast-growing species are higher than those in their slow-growing counterparts, the degree of metabolic imbalance might be greater in fast–growing species. If this were the case, then interspecific differences in free radical-induced damage might be coupled to interspecific differences in the rate of protein degradation.
Apart from possible differences in the rate of protein turnover, the protein concentration of the roots is a factor that can cause differences in costs for protein turnover between species. A positive correlation has been found between the protein concentration in roots and the RGR (Poorter et al., 1990). If the rates of protein turnover are similar in fast- and slow-growing grass species, a higher protein concentration will result in higher costs for protein turnover.
To investigate whether the rate of protein turnover and the respiratory costs for protein turnover differ between the roots of the fast-growing Dactylis glomerata and the slow-growing Festuca ovina, the rate of protein degradation of the total protein pool was determined using pulse-chase labelling with 14C-leucine (cf Van der Werf et al., 1992a). To calculate the respiratory costs associated with protein turnover, information about the protein concentration of the roots, the specific respiratory costs for protein turnover (the amount of ATP per protein mass) and the rate of protein turnover was used. The costs for protein turnover are compared with the previously determined respiratory costs for maintenance respiration.
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Materials and methods |
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Plant growth
Two monocotyledonous species were selected that differ in maximum relative growth rate (RGR): Dactylis glomerata L. and Festuca ovina L., which have RGR values of 228 and 102 mg g-1 d-1, respectively (Scheurwater et al., 1998
). Seeds of D. glomerata were commercially obtained from Van Engelen Zaden bv (Vlijmen, the Netherlands) and seeds of F. ovina were collected in a heathland area in the Netherlands at the ‘Uddelse Heide’.
Plants were grown under the following controlled-environment conditions: PAR: 500 µmol m-2 s-1 (using high-pressure mercury lamps, HPI-T 400W, Philips BV, Eindhoven, the Netherlands); temperature: 20 °C; relative humidity: 70%; light period: 14 h. Seeds were germinated in the growth room in Petri dishes on moistened filter paper. Subsequently, the seedlings were transferred to sand moistened with half-strength of the following nutrient solution: 795 µM KNO3, 602.5 µM Ca(NO3)2, 270 µM MgSO4, 190 µM KH2PO4, 41 µM Fe-EDTA, 20 µM H3BO3, 2 µM MnSO4, 0.85 µM ZnSO4, 0.25 µM Na2MoO4, and 0.15 µM CuSO4 (Poorter and Remkes, 1990). After 7–11 d of establishment, the seedlings were transferred to 32 l tanks containing an aerated full-strength nutrient solution, as described above, minus nitrate. To compensate for KNO3 and Ca(NO3)2, 397.5 µM K2SO4 and 602.5 µM CaSO4 were added to the medium. Using the method of Ingestad (Ingestad, 1981), KNO3 was added exponentially to the nutrient solution once a day (cf Van der Werf et al., 1992a), at a rate of 0.2 and 0.1 d-1 for the inherently fast-growing D. glomerata and slow-growing F. ovina, respectively. The pH of the nutrient solution was adjusted regularly to 5.8 and the nutrient solution was changed once a week. When grown like this, the values for RGR (g g-1 d-1) are the same as those of the relative addition rate (RAR) of (mol N mol-1 N d-1) (Fig. 1 in Van der Werf et al., 1993). Therefore, it is possible to be certain of the actual RGR of the plants and of that of the proteins (Van der Werf et al., 1992a), which are needed in this study's calculations of the rate of protein turnover.
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), in the roots of Dactylis glomerata (A) and Festuca ovina (B). Time zero represents the end of the 48 h labelling period.After the plants had reached 60–80 mg FM, they were transferred to the growth room in the isotope laboratory, which had similar growth conditions as described above except for PAR, which was 320 µmol m-2 s-1 (using the same high pressure mercury lamps as mentioned above) and a temperature of 22 °C.
Experimental design
It is important to compare plants at a similar mass, as preliminary experiments showed that the rate of protein turnover increases with age (data not shown). Therefore, the plants of both species that were used for the turnover rate experiments had an initial root fresh mass of 40 mg (at the beginning of the labelling period). The root mass at the last harvest was 0.5 and 1 g for F. ovina and D. glomerata, respectively.
To determine the rate of protein turnover in the roots, a similar experimental design to that of Van der Werf et al. (1992a) was used. Plants were transferred from the 32 l tank to a 14 ml tube, which contained the same nutrient solution as described above and was aerated continuously. Each tube contained three plants. The RAR was maintained at 0.2 and 0.1 d-1 for D. glomerata and F. ovina, respectively. Uniformly labelled L-[14C]-leucine (324.6 Ci mol-1) was added to the nutrient solution in the tubes to radioactively label the root proteins. During a 48 h pulse period, each tube received 187.5 nCi four times, at intervals of 12 h. After this pulse period, the plants were transferred back to the 32 l tank and were kept there for 14–62 h (chase period) to reduce the specific radioactivity of leucine in the roots. After this chase period plants were harvested at regular intervals of 2–6 d over a period of 14 and 21 d for the fast-growing D. glomerata and the slow-growing F. ovina, respectively. Three plants per harvest were used to determine the protein content and the protein-bound radioactivity of each individual root separately.
Protein content and protein-bound radioactivity
During the measurement period, after pulse-chase labelling with 14C-leucine, protein extraction, protein precipitation, determination of the protein contents of the two fractions (see below), and radioactivity determinations were performed according to Van der Werf et al. (Van der Werf et al., 1992a).
According to their solubility, proteins were subdivided into two fractions. After a two-step extraction in 30 mM potassium phosphate buffer (KPi-buffer, pH 7.0) at 40 °C and centrifugation (15 min, 12 500 g, 20 °C), the combined supernatants were denoted fraction 1. The remaining pellet was resuspended in 0.2 M NaOH and heated at 65 °C. After centrifugation (see above), the supernatant of fraction 2 contained proteins soluble in NaOH. Van der Werf et al. (Van der Werf et al., 1992a) checked whether the residue after extraction in KPi-buffer and NaOH still contained protein by extraction with 0.1 M NaOH containing 1% (w/v) SDS. This resulted in very low amounts of protein.
Proteins in fractions 1 and 2 were precipitated with trichloric acid (TCA, final concentration 5% (w/v) for 24 h at 4 °C) to remove radioactivity not incorporated into proteins. After centrifugation as described above, the protein pellet was washed with acid ethanol (0.1 M HCl : ethanol 1 : 11, v/v) and centrifuged again. Solutions obtained after dissolving the pellets in 0.1 M NaOH were denoted protein fractions 1 and 2. Another experiment performed by Van der Werf et al. (Van der Werf et al., 1992a) indicated that 14C-labelled carbon from leucine was not rapidly incorporated into other carbon skeletons. After hydrolysis of precipitated protein and amino acid separation by HPLC, radioactivity was absent in all amino acids, except leucine. Therefore, the radioactivity in non-protein compounds was presumably relatively low compared with that in 14C-leucine-labelled proteins.
Radioactivity was determined by liquid scintillation counting (Packard 2200 AC). In a glass vial, 5 ml Lumagel (Lumac, the Netherlands) was added to a 0–50 µl sample of the protein fractions. Decay rates (dpm) were obtained from counting rates (cpm) by using an external standard and a quench calibration curve. From these decay rates and the protein content of the fractions, the measured specific radioactivity (MSRA, dpm µg-1 protein) of each protein fraction can be calculated.
Absolute protein concentration
The method used to determine the protein content of fractions 1 and 2 (Bradford, 1976) does not give information on absolute protein content. Consequently, the data of fractions 1 and 2 cannot be combined to calculate the absolute protein concentration of the roots. Therefore, the organic N concentration in non-labelled roots was determined and the protein concentration subsequently calculated from this value (see below). D. glomerata and F. ovina plants were grown for 4–6 weeks, under the same environmental conditions and with the same RARs as mentioned above. During this period, plants were harvested three times to determine the total N and concentration. The fresh mass of the roots was within the mass range mentioned for the 14C-labelled roots.
The total N concentration of freeze-dried (Unitop 600SL and Freezemobile 12SL, The Virtis Company, Inc. Gardiner, New York, USA) roots was determined with a C-H-N analyser (Carlo-Erba, model 1106, Milan, Italy) using combustion gas chromatography (Pella and Colombo, 1973). was determined in water extracts of the freeze-dried root samples, using a modified salicylic acid method (Cataldo et al., 1975) that enabled determination of the concentration in 10 µl of water extract.
The organic N concentration of the non-labelled roots, taken as the upper estimate of the protein-N concentration, was calculated by subtracting the concentration from the total N concentration. Subsequently, the root protein concentration was calculated by multiplying the organic N concentration (mmol ‘protein’ N g-1 root DM) by 87.5 g protein mol-1 protein N (cf. Beevers, 1976, and Lehninger, 1982, as cited in De Visser et al., 1992).
Statistics
Significance of linear regressions was tested using the SAS statistical package (SAS, 1988). An analysis of covariance was used to test whether differences between the slopes of regression lines were significant (Sokal and Rohlf, 1981).
Calculations
The relative growth rates of the extracted proteins in the two fractions (RGRpr1 and RGRpr2) were calculated as the slope of the natural logarithm of the total amount of protein in the fraction versus time (Hunt, 1982).
The measured specific radioactivity (MSRA) decreases with time due to protein turnover (replacement of labelled proteins by newly synthesized non-labelled ones) and due to newly synthesized proteins that are produced in excess of the proteins synthesized to replace degraded proteins, thus enabling growth. To obtain rates of protein turnover the MSRA must be corrected for the proteins that are synthesized to enable root growth. At each harvest time (t), the corrected specific radioactivity (SRAt, dpm µg-1 protein) was calculated, using the following equation (cf. Van der Werf et al., 1992a):
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) these costs are 11.8–20.8 mol ATP mol-1 peptide bonds, which equal 107–189 mmol ATP g-1 protein (assuming an average of 1 mol of peptide bonds per 110 g protein, De Visser et al., 1992). |
Results and discussion |
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Costs for protein turnover in roots
Do fast- and slow-growing grass species differ in their costs for root protein turnover? The costs for protein turnover (i.e. the amount of ATP required per root mass and time) in the roots of the fast-growing Dactylis glomerata and the slow-growing Festuca ovina were estimated, using the protein concentration in the roots, the specific respiratory costs for protein turnover (the amount of ATP per protein mass) and the rate of protein turnover (cf. equation 4).
Firstly, systematic differences in root protein concentration could cause differences in the costs for root protein turnover between fast- and slow-growing grass species. Poorter et al., investigating 24 herbaceous species, found a significant positive correlation between root organic N concentration and RGR (Poorter et al., 1990). If the rate of protein turnover were similar for fast- and slow-growing species, a higher root protein concentration will result in higher costs for protein turnover (cf. equation 4). The protein concentration in the roots, calculated from the root organic N concentration, was 123 and 140 mg protein g-1 root DM in D. glomerata and F. ovina, respectively (Table 1), and did not change with time during the experiment (data not shown). The organic N concentration in the roots of eight grass species that differ in RGR, varied from 1.3–1.8 mmol N g-1 root DM, corresponding with 114–158 mg protein g-1 root DM (I Scheurwater, M Koren, H Lambers, and OK Atkin, unpublished results). No significant correlation was found between the root organic N concentration and the RGR. Thus, differences in the costs for protein turnover between the roots of fast- and slow-growing grass species are not expected to be due to differences in protein concentration.
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Secondly, the roots of fast- and slow-growing species might differ in specific respiratory costs for protein turnover. However, given there is no evidence to support this contention, it was assumed that biochemical costs for this process are the same for all plant species. Similar assumptions have been made in previous studies (De Visser et al., 1992
; Bouma et al., 1994; Van der Werf et al., 1994). The third factor that might cause differences in the respiratory costs for root protein turnover between fast- and slow-growing grass species is the rate of protein turnover. To assess this the rate of protein degradation was determined in roots labelled with 14C-leucine, assuming that this rate equals the rate of protein turnover in a growing root.
Protein degradation constant and rate of protein turnover in roots
To determine the rate of root protein turnover, the plants were labelled for 48 h. After this labelling, there is a period during which the 14C-leucine is incorporated into new proteins while 14C-labelled proteins are degraded simultaneously. Therefore, the protein-bound radioactivity first increases, but after the specific radioactivity of the free 14C-leucine in the roots has been sufficiently reduced (chase period), the measured specific radioactivity (MSRA, radioactivity per protein mass) declines with time due to the replacement of labelled proteins by newly synthesized non-labelled proteins (protein turnover). Proteins were extracted by a two-step procedure, which resulted in protein fractions 1 and 2, extracted in potassium phosphate buffer and NaOH, respectively. In D. glomerata roots, the MSRA immediately declined after the 14 h chase period (Fig. 1A
). In F. ovina roots, however, the decline in MSRA started much later (Fig. 1B), suggesting that the incorporation of 14C-labelled leucine into proteins was slower in the roots of the slow-growing F. ovina than that in the fast-growing D. glomerata roots. This could be due to a slower rate of protein synthesis, to a bigger size of the pool of free amino acids and/or to a longer mean residence time of leucine in the free amino acid pool before it is incorporated into protein, in F. ovina roots compared with that in the roots of D. glomerata.
To correct the MSRA for newly synthesized proteins that are used for growth in excess of those used for protein turnover (cf. equation 1), the relative growth rate of the proteins (RGRpr) must be determined. The RGRpr of the two protein fractions was calculated as the slope of the average values of the natural logarithm of the protein content in the fraction versus time. The ratio of the protein content in fraction 1 to that in fraction 2 was on average 1.6 and 0.9 in D. glomerata and F. ovina, respectively. In the roots of D. glomerata the RGRpr of fractions 1 and 2 was 0.218 and 0.157 g protein g-1 protein d-1, respectively. RGRpr1 and RGRpr2 were 0.120 and 0.094 g protein g-1 protein dd-1, respectively, in F. ovina roots. In both species RGRpr1 and RGRpr2 were not significantly different. The RGRpr values obtained from the pooled protein fractions were 0.192 and 0.107 d-1 in the roots of D. glomerata and F. ovina, respectively (Fig. 2), and equalled the relative addition rate of . This indicates that the change in light intensity from 500 to 320 µmol m-2 s-1 upon transfer of the plants to the isotope laboratory did not affect RGRpr as measured during the harvesting period. The straight lines in Fig. 2 show that protein turnover was measured while plants were growing exponentially in steady state. After determining RGRpr, the corrected specific radioactivity (SRA) was calculated according to equation 1. For F. ovina, data obtained from day 5.6 onwards were used because the protein-bound radioactivity only started to decline after that day (see above). The absolute value of the slope of the natural logarithm of SRA versus time, as depicted in Fig. 3, represents the protein degradation constant (kd, cf. equation 2). In both species, an analysis of covariance did not reveal a significant difference between the degradation constants of fractions 1 and 2. So, on average, proteins of both fractions were degraded at the same rate. This was also found by Van der Werf et al., who used the same method to determine the protein degradation constant in roots (Van der Werf et al., 1992a).
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) and Festuca ovina (
). Time zero represents the end of the 48 h labelling period. The slope of the regression line equals the relative growth rate of the proteins (g protein g-1 protein d-1). Both linear regressions are significant (P<0.001).
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The degradation constant of the pooled protein fractions in D. glomerata roots was 0.078 g protein g-1 protein d-1 (0.062 and 0.094 d-1 in fractions 1 and 2, respectively; Fig. 3A
). In the roots of F. ovina, a degradation constant of 0.058 g protein g-1 protein d-1 was obtained for the pooled protein fractions (0.058 d-1 in both fractions; Fig. 3B). The protein degradation constant of the pooled protein fractions in the roots of the two grass species (Fig. 3C) was slightly higher in D. glomerata (0.078 d-1) compared with that in F. ovina (0.058 d-1). This corresponds with a protein half-life of 9 d in the roots of D. glomerata and 12 d in F. ovina roots (cf. equation 3). No significant difference between the slopes of the regression lines (Fig. 3C) was found, however, suggesting that the root proteins of both species are degraded at the same rate. This was also found for leaves of grass species differing in RGR. The leaves of a fast- and a slow-growing population of Lolium perenne did not differ significantly in their rate of protein turnover (Barneix et al., 1988). In the present study, the pooled degradation constant for the roots of the two species, as obtained from the analysis of covariance, was 0.066 d-1. This value is identical to the protein degradation constant that Van der Werf et al. (Van der Werf et al., 1992a) obtained for the roots of D. glomerata at a developmental stage similar to that of the plants used in these experiments. Taken together, the rate of protein turnover does not differ significantly between the roots of the fast-growing D. glomerata and the slow-growing F. ovina in the present experiment.
The protein degradation constant determined in a pulse-chase experiment is a minimum estimate, because the labelled amino acids from degraded proteins can be re-utilized for protein synthesis (Davies and Humphrey, 1978; Davies, 1982; Eising and Süselbeck, 1991), thus reducing the apparent rate of degradation. Davies and Humphrey (Davies and Humphrey, 1978) measured the extent of recycling for a number of amino acids in Lemna minor and determined a value for leucine recycling of 50%. In roots this value can be lower, as 14C-leucine, originating from degraded proteins, can be transported to the shoot (Van der Werf et al., 1992a). Furthermore, Van der Werf et al. (Van der Werf et al., 1992a) showed that in D. glomerata roots, all radioactivity was confined to leucine, suggesting that recycling of the radioactive label only occurs via 14C-leucine. If at most 50% recycling of 14C-leucine takes place in the roots of both species, the maximum estimates of the protein degradation constants are 0.156 and 0.116 d-1 in D. glomerata and F. ovina, respectively (Table 1).
Contribution of protein turnover to the respiratory costs for maintenance in roots
Do the fast-growing D. glomerata and the slow-growing F. ovina differ in respiratory costs for root protein turnover? De Visser et al. (De Visser et al., 1992) calculated 11.8–20.8 mol ATP mol-1 peptide bonds as specific costs for protein turnover, which equals 107–189 mmol ATP g-1 protein. In this study's calculations, the average estimate of these specific respiratory costs for protein turnover (148 mmol ATP g-1 protein, De Visser et al., 1992), the protein concentration of the roots (Table 1), and the maximum estimates of the protein degradation constant (rate of protein turnover, Table 1) of both species (cf. equation 4) was used. This implies that 50% recycling of 14C-leucine was assumed to take place in the roots of both species. The respiratory costs for root protein turnover as calculated from these primary data and taking into account the assumptions mentioned above, were very similar in the fast- and the slow-growing species: 2.8 mmol ATP g-1 root DM d-1 in the roots of D. glomerata and 2.4 mmol ATP g-1 root DM d-1 in F. ovina roots.
What portion of root maintenance respiration can be ascribed to protein turnover? Scheurwater et al. (Scheurwater et al., 1998) calculated the specific respiratory costs for the maintenance of root biomass, which were 2.2±0.18 and 1.8±0.23 mmol O2 g-1 root DM d-1 for the fast-growing D. glomerata and the slow-growing F. ovina, respectively. These maintenance costs, which equal the rate of maintenance respiration in the roots, accounted for 50% of the total rate of root respiration in both D. glomerata and F. ovina (Scheurwater et al., 1998). Using maximum and minimum estimates of the contribution of the non-phosphorylating alternative respiratory pathway to the total rate of root respiration (44% and 3% for D. glomerata and 37% and 1% for F. ovina; Lambers et al., 1997), the rate of root respiration for maintenance processes can be calculated on an ATP-basis (cf. Scheurwater et al., 1998). The rate of root maintenance respiration ranges from 9–13 mmol ATP g-1 root DM d-1 in D. glomerata and from 8–11 mmol ATP g-1 root DM d-1 in F. ovina. Thus, the respiratory costs for maintenance of root biomass may be slightly higher in the faster-growing D. glomerata. As the respiratory costs for protein turnover were 2.8 and 2.4 mmol ATP g-1 root DM d-1 in the roots of D. glomerata and F. ovina, respectively, the percentage of maintenance respiration that can be ascribed to protein turnover is 22–30% in both species. Maintenance respiration accounted for approximately 50% of the total rate of root respiration in both D. glomerata and F. ovina (Scheurwater et al., 1998), thus 11–15% of the total ATP production in roots per day is spent on protein turnover in the roots of the fast- and the slow-growing grass species. These data only pertain to the steady-state situation in which the measurements were carried out. In ageing roots the percentage of respiration that can be ascribed to maintenance increases (Van der Werf et al., 1988). Therefore, the percentage of total root ATP production spent on protein turnover will increase as well, irrespective of whether protein turnover remains constant or whether it increases. Van der Werf et al. (Van der Werf et al., 1992a), using lower specific respiratory costs for protein turnover and a higher root protein concentration than in this study, calculated that 24–48% of the ATP production for maintenance in roots of D. glomerata is used for protein turnover in these roots, which corresponds with 4–7% of the total ATP production per day. In Lolium perenne leaves, costs for protein turnover accounted for 27–36% of leaf dark respiration (Barneix et al., 1988), and the protein turnover costs in expanding bean leaves amounted to 17–35% of total leaf dark respiration (Bouma et al., 1994).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Assuming 50% recycling of 14C-leucine in both species (see below) and assuming the specific respiratory costs for protein turnover to be 148 mmol ATP g-1 protein, the respiratory costs for protein turnover were only slightly higher in the roots of the fast-growing D. glomerata compared with those in the slow-growing F. ovina roots. As this was also the case for the respiratory costs for maintenance of root biomass (Scheurwater et al., 1998
), both the fast- and the slow-growing grass spent 22–30% of their daily ATP production for maintenance on protein turnover. This corresponds with 11–15% of the total root ATP production per day in both grass species. The rate of protein degradation, as determined via pulse-chase labelling with 14C-leucine, was not significantly different between the roots of the fast- and the slow-growing grass, provided the species did not differ in the extent of recycling of 14C-labelled leucine. Assuming 50% recycling in both species, the protein degradation constant was 0.156 and 0.116 d-1, corresponding with a total protein half-life of 4 d and 6 d, in the roots of the fast-growing D. glomerata and the slow-growing F. ovina, respectively.
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Acknowledgments |
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The authors thank Freya Dictus and Rob Welschen for technical assistance and Jolanda Schuurmans for her help in the isotope laboratory. Owen Atkin provided valuable comments on previous versions of this manuscript. We thank Ad Borstlap, Frank Millenaar and Hendrik Poorter for helpful suggestions. Seeds of Festuca ovina were kindly provided by Hendrik Poorter, Utrecht University, the Netherlands. This study was financially supported by the Life Sciences Foundation (SLW), which is subsidized by the Netherlands Organization for Scientific Research (NWO).
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Notes |
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4 To whom correspondence should be addressed. Fax: +31 30 2518366. E-mail:F.I.Scheurwater{at}bio.uu.nl
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