Differences between Maize and Rice in N-use Efficiency for Photosynthesis and Protein Allocation

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1 Plant Cell Physiol. 44(9): (2003) JSPP 2003 Short Communication Differences between Maize and Rice in N-use Efficiency for Photosynthesis and Protein Allocation Amane Makino 1, Hiroe Sakuma, Emi Sudo and Tadahiko Mae Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Japan The N-use efficiency for photosynthesis was higher in a C 4 plant, maize, than in a C 3 plant, rice, including rbcs antisense rice with optimal ribulose-1,5-bisphosphate carboxylase (Rubisco) content for CO 2 -saturated photosynthesis, even when photosynthesis was measured under saturating CO 2 conditions. The N cost for the C 4 cycle enzymes in maize was not large, and the lower amount of Rubisco allowed a greater N investment in the thylakoid components. This greater content of the thylakoid components as well as the CO 2 concentrating mechanism may support higher N-use efficiency for photosynthesis in maize. Keywords: Gas exchange (leaf) N-use efficiency Oryza sativa L. Protein allocation Ribulose-1,5-bisphosphate carboxylase/oxygenase Zea mays L. Abbreviations: CF, coupling factor; LHC, light-harvesting Chl protein complex; ME, malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate orthophosphate dikinase; PPFD, photosynthetic photon flux density; PS, photosystem; RuBP, ribulose-1,5- bisphosphate; Rubisco, RuBP carboxylase/oxygenase. Generally, C 4 plants show a greater N-use efficiency for photosynthesis than C 3 plants (Brown 1978, Schmitt and Edwards 1981, Sage and Pearcy 1987, Sage and Pearcy 2000). This has been considered to be because C 4 plants have a mechanism for eliminating photorespiration by increasing the concentration of CO 2 around ribulose-1,5-bisphosphate carboxylase (Rubisco). Since the K m values for CO 2 and O 2 of Rubisco from higher plants are close to atmospheric CO 2 and O 2 levels, respectively, Rubisco in C 3 plants fixes CO 2 at about 25% of its potential activity (Farquhar et al. 1980), but operates close to its potential activity in C 4 plants (Usuda 1984, Usuda et al. 1984, Sage et al. 1987). In addition, the specific activity of Rubisco in C 4 plants is about two times higher than that in C 3 plants (Seemann et al. 1984, Sage 2002). Thus, a lower amount of Rubisco may be sufficient to achieve higher rates of photosynthesis in C 4 plants. In fact, although the amount of Rubisco in C 3 plants accounts for 20 30% of leaf-n content (Evans and Seemann 1989, Makino 2003, Kumar et al. 2002), it is esti- ; mated to be only 5 9% in C 4 plants (Schmitt and Edwards 1981, Sage et al. 1987). This means that C 4 plants can save substantial N. However, C 4 plants must invest N in the C 4 cycle enzymes. Sage and Pearcy (2000) described that the N cost of the C 4 cycle enzymes is relatively low. For example, in a C 4 plant, Amaranthus retroflexus, phosphoenolpyruvate carboxylase (PEPC) accounts for 2 5% of total leaf N, which combined with Rubisco accounts for less than 14% of total leaf N (Sage et al. 1987). However, there is no available data on the N cost of other C 4 cycle enzymes. Thus, it is not known whether the N saving because of a lower amount of Rubisco is offset by the N cost for total C 4 cycle enzymes. Moreover, higher rates of photosynthesis require higher rates of ribulose-1,5-bisphosphate (RuBP) regeneration as well as CO 2 assimilation. Nevertheless, it is also not known whether C 4 plants have greater capacity of RuBP regeneration. In this study, we compared the light-saturated rates of photosynthesis per unit of leaf-n content in young, expanded leaves of a C 4 plant, maize (Zea mays L. cv. Honeybantam), and a C 3 plant, rice (Oryza sativa L. cv. Notohikari), including rbcs antisense rice. We also used spinach (Spinacia oleracea L. cv. Nobel) and bean (Phaseolus vulgaris L. cv. Tendergreen). All plants were grown hydroponically with different N concentrations. Photosynthetic measurements were made at ambient normal CO 2 and saturating CO 2 levels. Since CO 2 - saturated photosynthesis is limited by RuBP regeneration (Farquhar et al. 1980), we also compared the difference in RuBP regeneration capacity. In addition, we examined the differences in N allocation to proteins of the photosynthetic apparatus between maize and rice. The rates of light-saturated photosynthesis measured at atmospheric partial pressure of CO 2 (36 Pa) were linearly correlated with leaf-n content in both maize and several C 3 plants including rice (Fig. 1). At the same N content, although rice had slightly higher rates of photosynthesis than spinach and bean, the photosynthetic rate in maize was about two times higher than that in rice. When the photosynthetic rates were measured under saturating CO 2 conditions (100 Pa CO 2 ), the rate in maize was still about 1.6-fold higher than that in rice (Fig. 2). Higher rates of photosynthesis in C 4 plants have been generally considered to be due to the suppression of photores- 1 Corresponding author: , makino@biochem.tohoku.ac.jp; Fax,

2 Allocation of protein N in maize and rice 953 Fig. 1 Rate of photosynthesis at 36 Pa CO 2 versus total leaf N content in young leaves of maize (open squares), rice (closed circles), spinach (open triangles) and bean (closed triangles). Measurements were made at a PPFD of 1,800 mol quanta m 2 s 1, a leaf temperature of 25 C, and a leaf-to-air vapor pressure difference of 1.0 to 1.2 kpa. All plants were grown hydroponically with different N levels. Data for rice were taken from Makino et al. (1997). Fig. 2 Rate of photosynthesis at 100 Pa CO 2 versus total leaf N content in young leaves of maize (open squares), rbcs antisense rice (open triangles) and wild-type (non-transgenic) rice (closed circles). Measurements were made at a PPFD of 1,800 mol quanta m 2 s 1, a leaf temperature of 25 C and a leaf-to-air vapor pressure difference of 1.0 to 1.2 kpa. Data for rice including rbcs antisense rice were taken from Makino et al. (1997). piration because of the contribution of the CO 2 concentrating pathway to Rubisco (Brown 1978, Schmitt and Edwards 1981, Sage et al. 1987). However, CO 2 -saturated photosynthesis in rice was not comparable to that in maize. Thus, photosynthetic N-use efficiency is higher in maize than in rice even under conditions where photorespiration is eliminated. This means that another factor(s) besides the CO 2 concentration mechanism also contributes to higher N-use efficiency of photosynthesis in maize. Since C 3 photosynthesis under conditions of saturating CO 2 is strongly limited by RuBP regeneration capacity determined by electron-transport activity (Von Caemmerer and Farquhar 1981) or the re-availability of P i during starch and sucrose synthesis (Sharkey 1985), the amount of Rubisco in C 3 plants is thought to be excessive for CO 2 -saturated photosynthesis (for a review, see Makino and Mae 1999). Therefore, it is possible that an excess investment of N in Rubisco led to lower N-use efficiency of photosynthesis in rice than in maize under conditions of saturating CO 2. Since we previously selected rbcs antisense rice with 65% wild-type Rubisco as a plant set with optimal Rubisco content for CO 2 -saturated photosynthesis (Makino et al. 1997), the data on the photosynthetic rate obtained with those transgenic plants are included in Fig. 2. CO 2 -saturated photosynthesis in maize was still higher than that in transgenic rice for the same N content. This means that even if photorespiration is eliminated and if Rubisco content is optimized for saturating CO 2 conditions, the photosynthetic N- use efficiency is still higher in maize. In addition, since CO 2 - saturated photosynthesis in C 3 plants is limited by RuBPregeneration capacity, this indicates that RuBP regeneration capacity is also greater in maize than in rice. Fig. 3 shows differences in the partitioning of protein-n in a leaf between maize and rice. The N partitioning into soluble protein was significantly lower in maize (33%) than in rice (50%). This was due to a smaller amount of Rubisco in maize (8.5%). The N costs of PEPC and pyruvate orthophosphate dikinase (PPDK) were only 2.8 and 1.5%, respectively. Although the standard error of the ratio of N investment in PEPC protein was relatively large ( %), this was not due to scatter of the data. The ratio of PEPC to leaf N strongly depended on N treatment and increased with increasing leaf-n content. It was % in the 1.0 mm N treatment, % in the 4.0 mm N treatment and % in the 12.0 mm N treatment. A similar response to N treatment has been previously reported by Sugiyama et al. (1984) with maize. However, the ratios of other photosynthetic components to leaf N were relatively constant irrespective of N treatment (data not shown). Sage et al. (1987) deduced the N costs of Rubisco and PEPC in a C 4 plant, A. retroflexus, from their measured activities and the mean specific activities, respectively, and reported that the ratio of N invested in Rubisco ranged from 5 to 9% and that in PEPC ranged from 2 to 5%. These are close to our values obtained with maize. Although the N costs of other C 4 cycle enzymes such as NADP-malic enzyme (NADP-ME), adenylate kinase and cytosolic carbonic anhydrase are not known, the fact that the N investment of Rubisco in rice (27%) was close to that of total soluble protein in maize (33%) suggests that N saving due to the lower amount of Rubisco in C 4 plants is not offset by the N cost for the C 4 cycle enzymes. In addition, although it is possible that the N cost for the photorespiratory enzymes is smaller in maize than in rice, the fact that

3 954 Allocation of protein N in maize and rice Fig. 3 Allocation of protein-n in young leaves of maize and rice. Rubisco, PEPC and PPDK were determined spectrophotometrically by formamide extraction of Coomassie Brilliant Blue R-250-stained bands on SDS-PAGE. For the CF 1 /CF 0 complex, and subunits of CF 1 were also determined by SDS-PAGE and the amount of CF 1 /CF 0 protein complex was estimated by multiplication by 1.65 (Blankenship 2002). For the Cyt b6/f complex, the Cyt f content was determined spectrophotometrically from the difference between hydroquinone-reduced and the ferricyanideoxidized spectra (Evans and Terashima 1987) and the amount of Cyt b 6 /f protein complex was estimated by multiplication by 3.25 (Cramer et al. 1996). The N content of proteins was assumed to be 16% of the mass of proteins. The N contents of PSI, PSII and LHC II complexes were assumed to be 37, 83.5 and 25.5 mol N mol 1 Chl, respectively (Evans and Seemann 1989). In rice, since the Chl a/b ratio was , the ratios of Chl in PSI, PSII and LHC II were assumed to be 0.308, and 0.566, respectively (Leong and Anderson 1984). Ninety percent of Chl b was assumed to be bound to LHC II complex including its minor proteins. In maize, the amounts of PSI, PSII and LHC II were estimated on the assumption that the ratio of PSII to LHC II is the same as that in rice. The Chl a/b ratio in maize was Maize was grown with 1.0, 4.0 and 12.0 mm N, and rice was grown with 0.5, 2.0 and 8.0 mm N. The results are the mean and the values in parentheses show standard error (P <0.05, n = 8 25). The average contents of total leaf N were 90 and 124 mmol N m 2 for maize and rice, respectively. the investment of N in other soluble proteins did not differ (20% for maize and 23% for rice) also shows that this is probably not substantial. By contrast, the N partitioning into insoluble fraction was appreciably greater in maize (53%) than in rice (37%). This fraction includes mainly thylakoid-membrane proteins associated with light harvesting, electron transport and photophosphorylation. The total amounts of PSI, PSII, LHC II, CF 1 /CF 0 and Cyt b/f accounted for 34% of leaf N in maize and 24% of leaf N in rice, respectively. Thus, the greater content of the thylakoid components in maize may contribute to higher capacity of RuBP regeneration. However, some of these components in C 4 plants are responsible for the energy supply of the C 4 cycle. The total energy required to fix one molecule of CO 2 into carbohydrate in C 4 plants is theoretically estimated to be three ATP and two NADPH molecules for RuBP regeneration plus at least an additional two ATP molecules for the C 4 cycles, giving a total of more than five ATP and two NADPH molecules for fixation of one molecule of CO 2 (Kanai and Edwards 1999). Thus, the ratio of energy cost for the C 4 cycle to RuBP regeneration is about 2 : 3. However, since C 4 plants do not require additional reduction power to drive the C 4 cycle, the ratio of the N cost for the thylakoid components between them is not necessarily 2 : 3. In maize, although oxaloacetate is reduced to malate by NADPH in the mesophyll chloroplasts, NADPH produced by ME reaction in the C 4 cycle is reused for the Calvin cycle in the bundle sheath chloroplasts. In addition, a significant cyclic electron flow around PSI is suggested to be driven in the bundle sheath chloroplasts (Asada et al. 1993, Kubicki et al. 1996), and the shuttle pathway of PGA and triose-phosphate operates between the mesophyll and bundle sheath chloroplasts (Hatch 1988). Thus, because of the complexity of the C 4 photosynthesis, it is very difficult to distinguish the N cost of the thylakoid components between the C 4 cycle and RuBP regeneration in the Calvin cycle. In summary, the N saving due to the lower amount of Rubisco in maize is not offset by the N cost for the C 4 cycle enzymes, and allows a greater N investment in the thylakoid components. Since C 4 plants have the higher specific activity form of Rubisco associated with a low affinity for CO 2 (Seemann et al. 1984, Sage 2002, Yoeh et al. 1980), a lower amount of Rubisco is sufficient to achieve high rates of photosynthesis. A greater N investment in the thylakoid components as well as the CO 2 concentrating mechanism may support higher N use efficiency for photosynthesis in C 4 plants. One approach to improving N use efficiency in crops is to introduce C 4 characteristics into C 3 crops by genetic manipulation. The recombinant DNA technology to express high levels of C 4 cycle enzymes at the target locations in C 3 crops is becoming well established (for a review, see Matsuoka et al.

4 Allocation of protein N in maize and rice ). For example, the introduction of the intact maize gene of the C 4 enzymes such as PEPC and PPDK led to high level expression of C 4 enzymes in rice leaves, their expression levels being comparable to those in the C 4 plants (Ku et al. 1999, Fukayama et al. 2001). In addition, maize NADP-ME located in the bundle sheath cells could be expressed at high levels in the mesophyll cells in rice under the control of the rice Cab promoter (Tsuchida et al. 2001). Such results may lead to the eventual successful introduction of the complete C 4 cycle into the C 3 plants. To improve N-use efficiency of photosynthesis in C 3 crops, however, we must create Kranz leaf anatomy to obtain an efficient CO 2 concentrating mechanism and transport systems between the mesophyll and bundle sheath cells (Kanai and Edwards 1999). In addition, our results strongly indicate that we must establish optimal protein allocation as shown in Fig. 3, including a lower amount of Rubisco of a type different from that of C 3 plants. All plants, excluding spinach, were grown hydroponically in growth chambers at a day/night temperature of 25/20 C, a photosynthetic photon flux density (PPFD) of 1,000 mol quanta m 2 s 1 and a 14-h photoperiod. Spinach was grown at a day/night temperature of 19/14 C, a PPFD of 900 mol quanta m 2 s 1 and a 9-h photoperiod. The hydroponic solution used has been previously described by Makino and Osmond (1991) and was continuously aerated. All measurements were made on young, expanded leaves of 3- to 10-week-old plants. One to two weeks before measurements, plants were supplied with a hydroponic solution containing different N concentrations from 0.5 to 12 mm N as previously described (Makino and Osmond 1991). These nutrient solutions were changed every 5 d for maize and every 7 d for other plants. For maize, to maintain the ph of the solution during the 5-day culture, 5 mm MES was added. Gas exchange was determined with an open gas-exchange system using a temperature-controlled chamber equipped with a fan (Makino et al. 1988). A fresh, young leaf blade was homogenized with a chilled pestle and mortar in 50 mm Naphosphate buffer (ph 7.0) containing 2 mm iodoacetic acid, 120 mm 2-mercaptoethanol and 5% (v/v) glycerol. Total leaf-n was determined from part of the homogenate before centrifugation (Makino et al. 1988). Total soluble-n and soluble protein- N were determined from the supernatant of the homogenate after centrifugation. Soluble protein-n was measured using trichloroacetic acid precipitate of the supernatant. Insoluble-N was calculated by subtracting soluble-n from total leaf-n. Trichloroacetic acid soluble-n was calculated by subtracting soluble protein-n from soluble-n. N content was determined with Nessler s reagent after Kjeldahl digestion. The remaining homogenate was used for the determinations of Chl, Rubisco, PEPC, PPDK and CF 1. Chl content was determined from part of the homogenate before centrifugation by the method of Arnon (1949). Rubisco, PEPC and PPDK contents were determined using the homogenate after the addition of Triton X-100 (0.1%, final concentration). After this homogenate was centrifuged at 10,000 g for 3 min, the supernatant was treated with lithium dodecylsulfate (1.0%, w/v) at 100 C for 90 s and analyzed by SDS-PAGE. Rubisco, PEPC and PPDK were determined spectrophotometrically by formamide extraction of the respective Coomassie-Brilliant-Blue-R-250-stained bands corresponding to 99 kda for PEPC (Uedan and Sugiyama 1976), 94 kda for PPDK (Sugiyama 1973), and 52 and 15 kda for Rubisco. Whereas the calibration curve for the Rubisco determination was made with the purified Rubisco, those for PEPC and PPDK were made with BSA because of a large difference in dye-efficiency among proteins (Makino et al. 1986, Fukayama et al. 2001). For the determination of CF 1 content, a portion of this homogenate was filtered through four layers of cheesecloth and centrifuged. After washing the pellet with 50 mm Tris/HCl buffer (ph 7.2), it was treated with lithium dodecylsulfate (2.5%, w/v) at 100 C for 90 s and analyzed by SDS-PAGE. CF 1 was determined spectrophotometrically at 595 nm by formamide extraction of the respective dye-stained bands corresponding to 55 and 52 kda for the CF 1 - and -subunits (Blankenship 2002). Bovine serum albumin was used as the standard. 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