RESEARCH PAPER |
Can the cold tolerance of C4 photosynthesis in Miscanthusxgiganteus relative to Zea mays be explained by differences in activities and thermal properties of Rubisco?
1Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
2Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
3Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
4US Department of Agriculture, Photosynthesis Research Unit, Agricultural Research Service, Urbana, IL 61801, USA
To whom correspondence should be addressed. E-mail: slong{at}uiuc.edu
Received 13 November 2007; Revised 6 February 2008 Accepted 19 February 2008
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Abstract |
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The previous investigations show that the amount and activity of Rubisco appears the major limitation to effective C4 photosynthesis at low temperatures. The chilling-tolerant and bioenergy feedstock species Miscanthusxgiganteus (M.xgiganteus) is exceptionally productive among C4 grasses in cold climates. It is able to develop photosynthetically active leaves at temperatures 6 °C below the minimum for maize, and achieves a productivity even at 52° N that exceeds that of the most productive C3 crops at this latitude. This study investigates whether this unusual low temperature tolerance can be attributed to differences in the amount or kinetic properties of Rubisco relative to maize. An efficient protocol was developed to purify large amounts of functional Rubisco from C4 leaves. The maximum carboxylation activities (Vmax), activation states, catalytic rates per active site (Kcat) and activation energies (Ea) of purified Rubisco and Rubisco in crude leaf extracts were determined for M.xgiganteus grown at 14 °C and 25 °C, and maize grown at 25 °C. The sequences of M.xgiganteus Rubisco small subunit mRNA are highly conserved, and 91% identical to those of maize. Although there were a few differences between the species in the translated protein sequences, there were no significant differences in the catalytic properties (Vmax, Kcat, and Ea) for purified Rubisco, nor was there any effect of growth temperature in M.xgiganteus on these kinetic properties. Extracted activities were close to the observed rates of CO2 assimilation by the leaves in vivo. On a leaf area basis the extracted activities and activation state of Rubisco did not differ significantly, either between the two species or between growth temperatures. The activation state of Rubisco in leaf extracts showed no significant difference between warm and cold-grown M.xgiganteus. In total, these results suggest that the ability of M.xgiganteus to be productive and maintain photosynthetically competent leaves at low temperature does not result from low temperature acclimation or adaptation of the catalytic properties of Rubisco.
Key words: Activation energy, activation state, C4 photosynthesis, chilling tolerance, maize, Miscanthus, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)
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Introduction |
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Although C4 photosynthesis has a higher potential efficiency of light use than C3 photosynthesis (Long, 1983, 1999; Sage, 1999), the productivity of many C4 species including some crop species of the Andropogoneae such as maize (Zea mays), sorghum (Sorghum bicolor), and sugarcane (Saccharum officinarum) is limited by poor photosynthetic performance at low temperatures. An exception is the biofuel crop Miscanthusxgiganteus (M.xgiganteus), a rhizomatous C4 perennial grass of Andropogoneae, which is not only able to survive at low temperatures but is highly productive in cool temperate climates (Beale and Long, 1995, 1996; Bullard et al., 1995; Heaton et al., 2004). At 52° N in the UK it produced as much as 30 tonnes of dry matter per hectare per year, exceeding the most productive C3 crops and showed no loss of maximum photosynthetic efficiency in the field in contrast to Z. mays at the same latitude (Beale and Long, 1995; Beale et al. 1996). When Z. mays is grown at 14 °C, the maximum photosynthetic rate of CO2 uptake (Asat) is decreased by more than 90% relative to leaves grown at 25 °C, when both are measured at a common temperature. In contrast, growth at 14 °C causes no reduction in the photosynthetic capacity of leaves of M.xgiganteus, and only a small reduction is observed in leaves grown at 10 °C (Naidu and Long, 2004; Farage et al., 2006).
The molecular mechanism by which M.xgiganteus and other chilling-tolerant C4 species maintain high photosynthetic rates at low temperature remains unclear. In saturating light and chilling temperatures (<15 °C), CO2 assimilation for chilling-intolerant species is severely reduced and, in turn, utilization of observed excitation energy leads to photoinhibition and photo-oxidation (Long, 1983; Long et al., 1994). M.xgiganteus avoids this damage both by maintaining a high rate of CO2 uptake (Beale et al., 1996) and by increased non-photochemical quenching that correlates with a large increase in xanthophyll content (Farage et al., 2006). Chilling-induced decreases in C4 photosynthesis have been correlated with decreases in carboxylation efficiency via PEP carboxylase (PEPc) (Kingston-Smith et al., 1997; Chinthapalli et al., 2003), capacity for PEP regeneration via pyruvate orthophosphate dikinase (PPDK) (Du et al., 1999), Rubisco activity (Kingston-Smith et al., 1997; Du et al., 1999; Pittermann and Sage, 2000, 2001; Chinthapalli et al., 2003) or a combination of the above. Recent studies have focused on Rubisco as exerting most or all control over the rate of light-saturated C4 photosynthesis at low temperature. Using antisense Rubisco small subunit transgenic Flaveria bidentis (a C4 dicot) grown at warm temperatures, the amount of Rubisco was shown to control the rate of C4 photosynthesis at low temperatures (Kubien et al., 2003). In addition, in vivo photosynthetic rates below 20 °C showed the same pattern of decline as maximum activities of Rubisco in crude extracts from the chilling-tolerant species Bouteloua gracilis, Muhlenbergia glomerata, and Amaranthus retroflexus (Kubien and Sage, 2004; Pittermann and Sage, 2000; Sage, 2002). This has led Sage and McKown (2006) to propose that C4 plants with small spatial capacity for Rubisco due to their Kranz anatomy will be inherently limited by Rubisco at low temperatures.
Increase in the amount of Rubisco in response to low temperature has not been commonly observed in C4 species (Pittermann and Sage, 2000, 2001; Naidu et al., 2003) Similarly, previous analyses of M.xgiganteus have shown that it maintains a similar Rubisco content when grown at 14 °C as at 25 °C, while amounts of PPDK are elevated greatly (Naidu et al., 2003). Given the key role of Rubisco at low temperature in C4 photosynthesis, if the amount of Rubisco is not increased does its Rubisco have catalytic properties such as a lower activation energy or a higher Kcat that would allow higher rates of photosynthesis at low temperatures even if the amount of Rubisco is unchanged? Previous surveys of C3 plants from different habitats showed that, on average, the Rubisco of C3 plants from cool habitats had a higher Kcat than those from warm habitats, when measured at chilling temperatures (Seemann et al., 1984; Sage, 2002). In addition, the Kcat of purified Rubisco was higher for the C3 plant spinach when it was grown at low temperature versus high temperatures (Yamori et al., 2006). Since multiple genes are only known for the small subunit of Rubisco (RbcS), this implies expression of different members of the subfamily at low temperature. Most previous analyses on the activities of Rubisco from C4 species were limited to crude leaf extracts that may contain species-specific inhibitors affecting the apparent properties of Rubisco in vitro. Therefore, a protocol was developed in this study to obtain significant quantities of purified and functional Rubisco. The maximum carboxylation activities (Vmax), activation states, Kcat, and Ea of purified Rubisco and Rubisco in crude leaf extracts from M.xgiganteus grown at 14 °C and 25 °C were compared with those of Zea mays grown at 25 °C.
This study tests the hypotheses that: (i) the putative translated protein sequence from RbcS cDNA of M.xgiganteus would show significant differences from those of Z. mays, which may explain cold tolerance; (ii) the catalytic properties of Rubisco formed at low temperature in M.xgiganteus results in a higher catalytic rate per active site (Kcat) than Z. mays; (iii) Rubisco from M.xgiganteus grown at low temperature has lower activation energy (Ea) than that of Z. mays.
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Materials and methods |
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Plant material
Miscanthusxgiganteus and Zea mays FR1064 (a commercial inbred line, Illinois Foundation Seeds, Tolono, IL, USA) were grown in soil-less potting media (Sunshine Mix LC1, Sun Gro Horticulture Inc., Bellevue, WA, USA), well watered and fertilized once a week according to Naidu and Long (2004). Plants were grown at 25/20 °C (warm grown) day/night temperature and 14/10 h day/night cycle under 500 µmol photons m–2 s–1 and a water–air vapour pressure deficit of <1 kPa in controlled-environment chambers utilizing a mixture of high-pressure sodium and mercury lamps to deliver a balanced light spectrum (E15, Conviron, Winnipeg, Canada) (Naidu and Long, 2004). In addition, M.xgiganteus was grown at 14/12 °C (cold grown) day/night temperature, all other conditions as above (Naidu and Long, 2004). Maize grew only very slowly at 14/12 °C and produced chlorotic leaves of insufficient area for analysis. Leaves which had just completed expansion, as judged by ligule emergence, were collected after 3 h of illumination, washed in cold distilled water and the main vein removed. For assays based on crude extracts, leaf discs (1.1 cm diameter) were cut from mature leaves after 3 h of illumination and immediately frozen into, and then stored in, liquid nitrogen.
Cloning and sequencing of Rubisco small subunit (RbcS) cDNA of M.xgiganteus
Total RNA was extracted from fully-expanded M.xgiganteus leaves grown at 25 °C using the RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's recommended protocols. The first-strand DNA was synthesized from total RNA (2.4 µg) by reverse transcription (Superscript III RT; Invitrogen, Carlsbad, CA, USA) using a poly-T primer according to the manufacturer's recommended protocol. The forward and reverse primers for cloning the RbcS gene of M.xgiganteus were based on the sequence of RbcS cDNA of Saccharum officinarum (GenBank accession no. M86717
[GenBank]
), 5'-GCGGCCACGGGAGAACGGT-3' and 5'-TGATAGCCGGAGCTTGCA-3', respectively. For PCR, initial denaturation at 94 °C for 2 min was followed by 34 cycles of denaturation (94 °C for 30 s), annealing (63 °C for 30 s), and extension (68 °C for 75 s), and then a final extension at 68 °C for 4 min. HiFiTaq DNA Polymerase (Invitrogen) and 2 µl of cDNA from the original RT-PCR reaction were used for PCR. Resulting PCR fragments were cloned into the pCR4-TOPO vector (Invitrogen). The constructs were transformed into E. coli DH5
(Invitrogen). Plasmid DNAs were isolated from positive colonies and sequenced using Prism BigDye Terminators v 3.0 (Applied Biosystems, Foster City, CA, USA) followed by electrophoresis on ABI 377 sequencers (Applied Biosystems).
Purification of Rubisco
A protocol using a bundle sheath enrichment procedure with two-step blending (2SB) was developed based on Edwards and Black (1971), in order to minimize contamination by mesophyll cells relative to the traditional protocol that uses a single blending or grinding step for purification of Rubisco from C3 leaves. The first blending (up to five times) was applied to break open mesophyll cells, leaving the bundle sheath largely intact. The subsequent wash step removed the broken mesophyll cell contents. The second longer blending is then applied to break open the bundle sheath cells and extract the Rubisco. All procedures were performed at 4 °C. Leaves were cut into pieces in an ice-cold blending buffer containing 50 mM Tricine–NaOH (pH 8.0), 10 mM NaHCO3, 2 mM EDTA, 10 mM DTT, 10 mM MgCl2, 3 mM mercaptobenzothiazole, 1.5% insoluble PVPP, 1 mM PMSF, 1 mM benzamidine, and 10 µM leupeptin, with three 5 s bursts in a Waring blender. The brei was filtered through four layers of cheesecloth and one layer of Miracloth (Calbiochem, CA, USA). The filtered solution enriched in mesophyll cell extracts was discarded. The remaining leaf tissue collected by cheesecloth and Miracloth filtration was washed twice more with ice-cold blending buffer before being re-blended for ten 30 s cycles with 1 min intervals on ice. The solution was then filtered through four layers of cheesecloth and one layer of Miracloth, and then centrifuged at 13 700 g (rotor SLA-3000, Sorvall, Newtown, CT, USA) for 40 min. Ammonium sulphate was added to the supernatant to 10% of complete saturation and then centrifuged at 10 800 g for 30 min (rotor SLA-3000, Sorvall). The supernatant was then subjected to 35% and then 55% saturation as above. The protein precipitated by the 35% saturation step was resuspended in Column Buffer A (20 mM Tricine–NaOH, 10 mM NaHCO3, and 10 mM MgCl2, pH 8.0), desalted through a Sephadex G-25 column, and resolved by chromatography on a 30 ml ion-exchange Q-Sepharose column (GE Healthcare, Piscataway, NJ, USA). The Rubisco was eluted with Column Buffer A, which was progressively mixed with increasing amounts of Column Buffer B, which was the same as Buffer A except for the addition of NaCl to 1 M. The resulting NaCl gradient was from 0 to 500 mM over 200 ml, with a flow rate of 1 ml min–1 and the fraction volume of 3 ml. Fractions showing maximum absorbance at 280 nm contained the bulk of the Rubisco, and were desalted and concentrated with Microcon YM-100 (Millipore, Bedford, MA, USA). Success in purifying Rubisco was evaluated by 12.5% SDS-PAGE.
Preparation of crude leaf extracts for Rubisco assays
This method followed the procedure by Sharkey et al. (1991) with modifications as described below. One protease inhibitor cocktail tablet (EDTA-free, Roche Applied Science, Indianapolis, IN, USA) per 10 ml of extraction buffer was added to inhibit protease activities. Leaf discs were rapidly ground in extraction buffer on ice using a pre-chilled Tenbroeck tissue homogenizer. The extract was then centrifuged at 15 000 g for 15 s and the supernatant incubated with 20 mM MgCl2 and 10 mM NaHCO3 at room temperature for 10 min to activate Rubisco fully (Sharkey et al., 1991).
In vitro assay of Rubisco activity
The Rubisco activity assay was adapted from Kallis et al. (2000) with the following modifications. Except where noted below, all enzymes and chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). NADH oxidation was monitored in a dual-beam UV/VIS spectrophotometer at a wavelength of 340 nm with temperature controlled cuvettes (Cary I and Temperature Control Accessory, Varian, Inc., Tempe, AZ, USA). The measurement temperatures were 6, 8, 10, 12, 15, 18, 20, 25, and 30 °C. The 1 ml assay buffer (preincubated to the assay temperatures) contained 100 mM Tricine–NaOH (pH 8.0), 20 mM KCl, 10 mM MgCl2, 2 mM ATP, 2 mM DTT, 0.3 mM NADH, 10 mM NaHCO3, 5 µg (or 20 µg at temperatures lower than 18 °C) of fully activated purified Rubisco or 50 µl aliquot of the crude leaf extract (fully activated), 2 mM phosphoenolpyruvate, 10 units ml–1 pyruvate kinase, 20 units ml–1 glycerate 3-phosphate kinase, 10 units ml–1 glyceraldehyde 3-phosphate dehydrogenase, 100 units ml–1 triose phosphate isomerase, and 10 units ml–1 glycerol phosphate dehydrogenase. The reaction was initiated by the addition of 4 mM RuBP.
The Rubisco activation assays were performed as described in Crafts-Brandner and Salvucci (2002). Leaves were illuminated in a temperature-controlled leaf chamber integrated into an open gas-exchange system (LI-6400, Li-Cor Inc, Lincoln, NB, USA). Once a steady-state rate of CO2-uptake was obtained, the illuminated leaf disc was freeze-clamped at liquid N2 temperature and stored briefly at –80 °C. The Rubisco extraction and activity assay was as described above. The initial activity of Rubisco was assayed immediately after extraction, and then incubated with 50 mM MgCl2 and 50 mM NaHCO3 to obtain total activity. The activation state of Rubisco was described as the ratio of the initial to total enzyme activities.
Calculations
The Rubisco activity of each sample was determined from the absorbance per minute and extinction coefficient of NADH (6221 µl µmol–1 cm–1), accounting for the stoichiometry of the reactions linking each enzyme from NADH to CO2. One mol RuBP is consumed for every 4 mol of NADH and 1 mol of CO2 is consumed for every 1 mol of RuBP. The maximum enzyme rate of reaction was reported as Vmax in CO2 equivalents. To calculate the activation energy (Ea), Log10 Vmax was linearly regressed against the inverse of the measuring temperature (K) x1000 to give an Arrhenius plot (OriginPro 7.5, Northampton, MA, USA). Separate regressions were conducted for high (18–30 °C) and low (6–18 °C) temperature ranges where a break was apparent. Ea was calculated as the product of the slope, 2.3 (to convert from log10 to ln) and the ideal gas constant (R=8.3143 J mol–1 K) to yield Ea in units of kJ mol–1. Where two fitted lines appeared to provide a better description of the relationship, two were accepted if they accounted for significantly more variation than a single line. Statistical differences were determined using analysis of variance (OriginPro 7.5) with a threshold significance of P < 0.10. This threshold, rather than 0.05, was chosen, in order to minimize the occurrence of possible Type II errors, at this small sample size.
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Results |
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Purification of Rubisco from M.xgiganteus
The protocol efficiently released the Rubisco from bundle sheath cells and minimized contamination with mesophyll proteins such as PEPc and PPDK (Fig. 1; data for warm-grown M.xgiganteus are shown as an example). After the first round of initial blending, the majority of bundle sheath was found by light microscopy to remain intact (data not shown). The subsequent wash steps removed the broken mesophyll cells and their contents. The second round of blending ruptured the bundle sheath cell wall and released the Rubisco. By comparison with the traditional protocol, the two-step blending (2SB) protocol substantially reduced contamination with mesophyll proteins PEPc and PPDK (98–100 kDa bands in Fig. 1, lanes 1 and 2). The Rubisco was further enriched by 10–35% saturation of ammonium sulphate precipitation (Fig. 1; lanes 3–5). Upon separation on the Q-Sepharose column, the peak fractions containing highly purified Rubisco were eluted at [NaCl] of 0.31 and 0.34 M (data not shown). The Rubisco from M.xgiganteus grown at 14 °C and from Z. mays grown at 25 °C exhibited similar characteristics of ammonium sulphate precipitation and column elution as those from M.xgiganteus grown at 25 °C (data not shown).
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Cloning and characterization of RbcS cDNAs from M.xgiganteus
Two full-length RbcS cDNAs of M.xgiganteus were identified: RbcSMg1 (GenBank accession no. EU219922) and RbcSMg2 (GenBank accession no. EU219923). The cDNA sequences were highly conserved, with 98% identity between each other (Fig. 2B). When compared to the Z. mays RbcS cDNAs (GenBank accession nos AY103730 [GenBank] , D00170 [GenBank] , and Y00322 [GenBank] ), M.xgiganteus RbcS cDNAs showed 91% identity to those of Z. mays (Fig. 2B). The translated putative protein sequences of M.xgiganteus RbcS were 89% identical to those of Z. mays (Fig. 2A). The amino acids with significant differences on a hydrophobicity index are highlighted in dark grey (Fig. 2A) and summarized in Table 1. The larger the index the more hydrophobic the amino acid (Kyte and Doolittle, 1982). Amino acid residues highlighted in light grey in Fig. 2A are those previously reported to affect either catalytic rate, holoenzyme assembly, or thermal stability of Rubisco (Fitchen et al., 1990; Spreitzer, 1993; Spreitzer et al., 2001), and were conserved between M.xgiganteus and Z. mays (Fig. 2A).
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Temperature dependency of maximum RuBP carboxylation rates of purified Rubisco
The in vitro maximum RuBP carboxylation rates (Vmax) catalysed by purified Rubisco progressively increased with temperature up to 30 °C (Fig. 3A). The Vmax of Rubisco purified from warm- and cold-grown M.xgiganteus showed no significant difference in specific activities across the entire range of measuring temperatures from 6 °C to 30 °C, and were similar to those of Rubisco from Z. mays (Fig. 3A). The Kcat at 25 °C or 15 °C was also similar for Rubisco purified from warm- and cold-grown M.xgiganteus and for Rubisco from Z. mays (Table 2). The Arrhenius plots of Vmax showed no significant difference either between warm- and cold-grown M.xgiganteus or between warm-grown M.xgiganteus and Z. mays across all measuring temperatures (Fig. 3B). No apparent break-points in the Arrhenius plot were observed at temperatures below 18 °C (Fig. 3B). A shift in the slopes of the fitted lines at high (18–30 °C) and low (6–18 °C) temperature was observed (Table 2). Therefore, the apparent activation energy (Ea) of the Rubisco was calculated separately for these two regions of the plot. The Ea at high measuring temperatures was about 1.5-fold of that below 18 °C for Rubisco from M.xgiganteus, and 1.6-fold for Rubisco from Z. mays. No significant differences in the Ea in either temperature range was observed between Rubisco from warm- and cold-grown M.xgiganteus (Table 2). The Ea values were not significantly different between Rubisco from M.xgiganteus and from Z. mays.
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Temperature dependency and activation state of Rubisco activity in crude extracts
The Vmax of the Rubisco in crude leaf extracts increased with temperature from 6 °C to 30 °C (Fig. 4A). The in vitro activity of Rubisco in leaf extracts was similar to the observed CO2 assimilation rates in vivo. For example, the Vmax of Rubisco from M.xgiganteus were 18.1±0.8 and 25.8±1.4 µmol m–2 s–1 at 25 °C and 30 °C, respectively, close to the CO2 assimilation rates (21.6±1.8 and 27.8±2.2 µmol m–2 s–1 measured under 500 µmol photon m–2 s–1 light) of M.xgiganteus leaves grown under 25 °C and those previously reported (Naidu et al., 2003). No significant difference in the Vmax of Rubisco in leaf extracts between warm-, and cold-grown M.xgiganteus and warm-grown Z. mays were observed on a leaf area basis or when compared at any given measurement temperature (Fig. 4A). As was found for purified Rubisco, the Arrhenius plot of Vmax based on crude extracts showed no significant difference for M.xgiganteus grown at either 14 °C or 25 °C, or between M.xgiganteus and Z. mays (Fig. 4B). No apparent break-points in the Arrhenius plot were observed at temperatures below 18 °C (Fig. 4B). Due to the shift in the slopes of the lines fitted at high (18–30 °C) and low (6–18 °C) temperature ranges (data not shown), the apparent activation energy (Ea) of the extracted Rubisco was calculated separately for these two regions. The Ea at high measuring temperatures was about 1.8-fold of that below 18 °C for Rubisco from M.xgiganteus, and 1.7-fold for Rubisco from Z. mays (Table 2). No significant differences were found in the Ea of the extracted Rubisco between the crude extracts of Z. mays and of warm- or cold-grown M.xgiganteus in either measurement temperature range (Table 2). Purified Rubisco from M.xgiganteus and Z. mays exhibited higher Ea than the Rubisco in crude extracts above 18 °C, but the differences were not significant below 18 °C (Table 2).
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Although the Vmax of Rubisco in crude extracts declined significantly with decrease in temperature below 20 °C (Fig. 4A), the Rubisco activation states remained high (Fig. 5). As temperatures decreased, the Rubisco activation state declined only slightly (Fig. 5). The extent of decrease in activation state of Rubisco was similar between cold-grown and warm-grown M.xgiganteus.
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Discussion |
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Previous studies have indicated Rubisco activity at low temperature as the major limitation to C4 photosynthesis. Here, however, it is shown that Miscanthusxgiganteus, a species known to maintain high rates of photosynthesis and productivity at low temperatures, does not produce Rubisco with thermal catalytic properties different from that of the cold-intolerant species, maize. Differences have been investigated at the levels of gene sequence, activation energy, and activation state.
Although the Rubisco small subunit (RBCS) does not form part of the Rubisco active sites, RBCS plays an important role in holoenzyme assembly, is essential for maximal activity, and has a pervasive influence on enzymes kinetics (Spreitzer, 1993; Spretizer and Salvucci, 2002). In addition, the RBCS polypeptide is encoded by a gene family, in contrast to the plastid encoded large sub-unit. As a result RbcS sequences vary more than those of the large subunit (rbcL) (Stein et al., 1990; Spreitzer, 1993). Therefore, changes in the population of RbcS mRNAs transcribed in response to environmental stimuli might be a mechanism for molecular adaptation or acclimation to low temperature as appears evident for spinach (Yamori et al., 2006). Although the translated protein sequences of RbcS showed some minor differences between M.xgiganteus and Z. mays, none of the residues reported to affect either catalytic efficiency or holoenzyme assembly/stability of Rubisco differed (Fig. 2). This is consistent with the biochemical analysis of catalytic properties which failed to show any significant or even indication of differences in Rubisco between M.xgiganteus and Z. mays (Figs 3–5
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In order to avoid the effects of incomplete extraction (Rogers et al., 2001) and potential inhibitors in crude leaf extracts that might complicate comparative activity assays, Rubisco was purified and its thermal properties measured. The purified Rubisco exhibited a similar temperature dependency of RuBP carboxylation rate as Rubisco in the crude leaf extracts. The only noticeable difference between purified Rubisco and Rubisco in crude extract was the Ea. When assayed in the low temperature range (6–18 °C) the Ea showed no significant difference, while Ea was higher for purified Rubisco than in the crude extract at high temperatures (18–30 °C). This suggests that the contents of the crude extract such as enzymes, metabolites, or inhibitors, may change the apparent activity of the enzyme at this temperature range. However, the fact that the extracted activity of Rubisco in vitro was 17.9±0.8 µmol m–2 s–1 at 25 °C and was close to the observed rate of photosynthesis in vivo suggests that any effect in crude leaf extracts was small (Naidu and Long, 2004).
No difference in activation energy (Ea) or Kcat between warm and cold-grown M.xgiganteus or between M.xgiganteus and Z. mays were observed, suggesting that there is no acclimation of Rubisco in M.xgiganteus to low temperature, nor adaptation relative to the Rubisco of Z. mays. These results suggest that the ability of M.xgiganteus effectively to assimilate CO2 at 14 °C and below in contrast to Z. mays, and to develop photosynthetically functional leaves at lower temperatures than Z. mays is not the result of any apparent difference in the catalytic properties of its Rubisco. Although acclimation of Rubisco to low temperature has been shown in the C3 plant spinach (Yamori et al., 2006), the present results suggest that this is not a universal phenomenon in plants able to acclimate to lower temperature.
What does facilitate the sustained CO2 uptake in leaves formed and photosynthesizing at low temperature in M.xgiganteus? It is assumed that two enzymes, Rubisco and PPDK, dominate metabolic control of the Vpr, the CO2-saturated photosynthetic rate (Naidu and Long, 2004). However, no significant differences in activation energy, Vmax and Kcat of Rubisco were detected between warm and cold-grown M.xgiganteus, or warm-grown Z. mays. Possibly, differences in CO2 assimilation may have resulted from variations in Rubisco or PPDK content. It has been reported that, in contrast to a significant decline in Rubisco content in Z. mays grown at 14 °C, Rubisco content in leaves of M.xgiganteus grown at 14 °C remained constant relative to comparable leaves of M.xgiganteus grown at 25 °C while PPDK content increased (Naidu et al., 2003).
Overall the results fail to support the hypothesis that Rubisco is the key limitation of C4 photosynthesis and success of C4 plants in cool climates. Since this study only concerns one species, this does not exclude the likelihood that Rubisco is a key limitation in other C4 taxa. However, the results do indicate that Rubisco is not a universal limitation to the productivity of C4 plants at low temperature.
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Acknowledgements |
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This project is supported by National Science Foundation (grant No. 0446018). The authors thank Long laboratory members for their advice at all stages of this work.
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Footnotes |
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* Present address: School of Molecular and Cell Biology, University of Illinois, 505 S Goodwin Ave, 393 Morrill Hall, Urbana, IL 61801, USA.
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