Plant Cell Physiol.

43(9): 10171026 (2002) JSPP 2002

Physiological Functions of the WaterWater Cycle (Mehler Reaction) and the Cyclic Electron Flow around PSI in Rice Leaves
Amane Makino 1, 4, Chikahiro Miyake 2 and Akiho Yokota 3
1 2

Graduate School of Agricultural Sciences, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan Graduate School of Bioresource and Bioenvironmental Sciences, Kyusyu University, Hokozaki, Higashi-ku, Fukuoka, 812-8581 Japan 3 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, 630-0101 Japan

Changes in chlorophyll fluorescence, P700+-absorbance and gas exchange during the induction phase and steady state of photosynthesis were simultaneously examined in rice (Oryza sativa L.), including the rbcS antisense plants. The quantum yield of photosystem II (FPSII) increased more rapidly than CO2 assimilation in 20% O2. This rapid increase in FPSII resulted from the electron flux through the waterwater cycle (WWC) because of its dependency on O2. The electron flux of WWC reached a maximum just after illumination, and rapidly generated non-photochemical quenching (NPQ). With increasing CO2 assimilation, the electron flux of WWC and NPQ decreased. In 2% O2, WWC scarcely operated and FPSI was always higher than FPSII. This suggested that cyclic electron flow around PSI resulted in the formation of NPQ, which remained at higher levels in 2% O2. The electron flux of WWC in the rbcS antisense plants was lower, but these plants always showed a higher NPQ. This was also caused by the operation of the cyclic electron flow around PSI because of a higher ratio of FPSI/FPSII, irrespective of O2 concentration. The results indicate that WWC functions as a starter of photosynthesis by generating DpH across thylakoid membranes for NPQ formation, supplying ATP for carbon assimilation. However, WWC does not act to maintain a high NPQ, and FPSII is down-regulated by DpH generated via the cyclic electron flow around PSI. Keywords: Chlorophyll fluorescence Cyclic electron flow (PSI) Gas exchange (leaf) Oryza sativa L. Ribulose1,5-bisophosphate carboxylase/oxygenaseWaterwater cycle (Mehler reaction).
Abbreviations: IRGA, infrared gas analyzer; Ja, rate of alternative electron flow; Jf, rate of electron flow calculated from Chl fluorescence; Jg, rate of electron flow calculated from gas exchange; MDAR, monodehydroxylascorbate radical reductase; NPQ, nonphotochemical quenching; PPFD, photosynthetic photon flux density; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; DpH, pH gradient across the thylakoid membrane; FPSII, quantum yield of PSII photochemistry; P700, primary electron donor of PSI

Introduction
There are several electron transport pathways in photosyn4

thesis in addition to the linear electron flow from water to NADP+ used for CO2 assimilation and photorespiration. Additional electron pathways in chloroplasts, including the Mehler reaction (Mehler 1957), the cyclic electron flow around PSI (Heber et al. 1978), the cyclic electron flow within PSII (Miyake and Yokota 2001, Miyake et al. 2002) and nitrate assimilation have been referred to as alternative electron flows. The Mehler reaction represents the photoreduction of O2 at PSI. This photoreduction produces superoxide radicals (O2 ), which are disproportionate to H2O2 and O2 with the aid of superoxide dismutase. The H2O2 is rapidly detoxified to water by the ascorbate peroxidase pathway. Since the electron flow from water in PSII to water in PSI occurs in this process, it has been termed the waterwater cycle (Asada 1999). The waterwater cycle not only scavenges O2 and H2O2, but also generates a pH gradient (DpH) across the thylakoid membranes when little electron transport acceptors are available in PSI. This DpH enhances non-radiative dissipation of light energy as observed by non-photochemical quenching (NPQ). Therefore, the waterwater cycle is considered to function to dissipate the energy of excess photons (Asada 1999, Asada 2000, Foyer and Noctor 2000, Osmond et al. 1997, Osmond and Grace 1995). Quantitative analysis of the waterwater cycle in vivo is important for the understanding of the implications of this cycle. Asada et al. (1974) estimated that the maximum rate of O2 photoreduction was about 7.5 mmol O2 (mol Chl)1 s1 (30 mmol (mg Chl)1 h1) in washed thylakoids, which corresponds to a 510% rate of total electron transport. In addition, this O2-reduction rate reached a maximum around 2.0 kPa O2 (Heber and French 1968, Takahashi and Asada 1982). Significant photoreduction of O2 was also found in intact cells by mass-spectrometric measurements (Furbank et al. 1982). In intact leaves, while simultaneous measurements of gas exchange and Chl fluorescence have not always provided an evidence for the electron flow to O2 (Genty et al. 1989, Cornic and Briantais 1991, Edwards and Baker 1993, Ruuska et al. 2000a, Cheng et al. 2001), Laisk and Loreto (1996) and Miyake and Yokota (2000) have shown that the electron flux in PSII clearly exceeds the total electron flux required for CO2 assimilation and photorespiration under high light conditions. In addition, Miyake and Yokota (2000) have reported that since

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Corresponding author: E-mail, makino@biochem.tohoku.ac.jp; Fax: +81-22-717-8765. 1017

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Table 1 The amounts of total leaf-N, Chl and Rubisco and the specific activity of Rubisco per mol of enzyme protein in the wildtype and rbcS antisense (Anti-rbcS) rice plants Leaf-N (mmol m2) Wild type Anti-rbcS
a b

Chl (mmol m2) 0.6500.024 0.8750.033

Rubisco (g m2) 2.920.07 (27) b 1.680.03 (14)

Rubisco specific activity (mol mol1 s1) c I=0d I = 900 2.50.5 1.40.2 12.10.4 11.60.3

1247 a 1426

Means SE (n = 3 4). Values in parentheses show the percentage of Rubisco-N to total leaf-N contents. c Specific activity is expressed per mol of Rubisco protein assuming a mol mass of 520 kDa. Enzyme assays were carried out at 25C after incubation for more than 5 min with 15 mM MgCl2 and 20 mM NaHCO3 at pH 8.0. d I = 0 indicates the samples kept in dark and I = 900 indicates the samples illuminated at 900 mmol m2 s1. The samples in the dark were collected from the leaves after 5 h in darkness, and the illuminated samples were collected from the leaves showing the steady-state rate of photosynthesis with the gas-exchange system (Nakano et al. 2000).

this extra electron flow strongly depends on O2 concentration, it can be regarded as the waterwater cycle. According to them, the maximum rate of this electron flow and the Km for O2 were calculated to be about 15 mmol O2 (mol Chl)1 s1 and 8 kPa, respectively. Since Fd and MDAR can produce O2 at a maximum rate of 75 mmol O2 (mol Chl)1 s1 (Furbank and Badger 1983, Miyake et al. 1998) and since their Kms for O2 were similar to that estimated by Miyake and Yokota (2000), the potential capacity of the waterwater cycle under ambient conditions is expected to be higher than previously estimated (for a review, see Asada 2000). Thus, the waterwater cycle can be a major electron sink in photosynthesis. The physiological functions of the cyclic electron flow around PSI in C3 plants still remain uncertain. This electron flow also produces ATP without NADP+ reduction. Heber et al. (1978) pointed out that since both this cyclic electron flow and CO2 assimilation are sensitive to antimycin A, the cyclic electron flow contributes to ATP synthesis for carbon assimilation. Similar observations were found for barley protoplasts by Furbank and Horton (1987). On the other hand, a linear relationship between the quantum yields of PSI and PSII in carbon assimilation was reported by Genty et al. (1990). This suggests that the cyclic electron flow occurs little in photosynthesis. In addition, recent studies on the bioenergetics of photosynthesis (H+/e = 3 in the presence of a Q-cycle and H+/ATP = 4 of ATP synthesis) have suggested that the cyclic electron flow is not necessarily essential for C3 photosynthesis (Kobayashi and Heber 1995). However, Endo et al. (1999) have reported that the cyclic electron flow plays an important role in the regulation of PSII quantum yield, namely it protects PSII from photoinhibition because of its generation of DpH across the thylakoid membranes for NPQ formation. Similar conclusions have been described with beans under conditions of low-CO2 (Cornic et al. 2000) and with barley and cucumber under conditions of low-temperature stress (Clarke and Johnson 2001, Kim et al. 2001). In the formation of DpH, since the cyclic electron flow around PSI does not produce any harmful radical species, it is emphasized that the cyclic electron transport is more

important than the waterwater cycle (Cornic et al. 2000, Clarke and Johnson 2001). The purpose of this study was to investigate the physiological functions of the waterwater cycle and the cyclic electron transport around PSI during the induction phase and steady state of photosynthesis in rice leaves. We simultaneously measured Chl fluorescence, P700+ absorbance and gas exchange in rice, including the transgenic plants with decreased amounts of Rubisco, and analyzed alternative electron flows under different O2 concentrations. These transgenic plants have low capacity for CO2 assimilation without any decrease in electron transport capacity (Makino et al. 1997). This means that these plants have a small electron transport sink. In fact, these plants showed a high NPQ because of a high DpH across the thylakoid membranes. Thus, these transgenic plants may be very useful material for the analysis of alternative electron flows. In addition, since the waterwater cycle strongly depends on O2 concentration (Miyake and Yokota 2000), simultaneous analyses of Chl fluorescence, P700+ absorbance and gas exchange were able to quantitatively distinguish among the respective alternative electron flows.

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Results
Characteristics of the wild-type and rbcS antisense plants Table 1 shows leaf N, Chl and Rubisco contents and Rubisco specific activity in rice leaves used for this study. Rubisco content in the rbcS antisense rice was about 60% of the wild-type level. The ratio of Rubisco to leaf N in the antisense rice was about 50% of that in the wild-type rice because of a small increase in leaf N content in the antisense rice. Rubisco specific activity per mol of enzyme protein did not differ between the plants. However, the specific activity from the leaves in darkness was much lower than that from the illuminated leaves in both plants, and it was only 20% in the wild type and 12% in the antisense plants, respectively. This indicates that rice plants belong to a species with extensive dark inhibition of Rubisco (Makino et al. 1994b).

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Fig. 1 Left panel: FPSII and CO2 assimilation rate (A) (top), electron transport rates from Chl fluorescence (Jf), gas exchange (Jg) and alternative electron flow (Ja) (middle) and NPQ (bottom) during photosynthetic induction in the wild-type rice. Gas exchange and Chl fluorescence were simultaneously measured at 20.2% O2 from darkness to an irradiance of 900 mmol m2 s1. O2 concentration was reduced to 2% after 30 min, and irradiance was decreased to 200 mmol m2 s1 after 40 min. Right panel: Rate of CO2 assimilation (top) and electron transport rate from gas exchange (Jg) and FPSI (bottom) during photosynthesis in the wild-type rice. Gas exchange and P700+-absorbance were simultaneously measured at 20.2% O2 from darkness to an irradiance of 900 mmol m2 s1. O2 concentration was reduced to 2% after 30 min.

FPSII increases more rapidly than CO2 assimilation during photosynthetic induction in 20% O2 The responses of FPSII calculated from Chl fluorescence and CO2 assimilation during photosynthetic induction in 20% O2 were examined in the leaf of the wild-type rice plants. The responses of FPSI calculated from the change in the P700+absorbance and CO2 assimilation were also examined. Before both experiments, the leaves were first adapted to darkness for more than 60 min, and then exposed to a sudden increase in PPFD of 900 mmol m2 s1. Typical examples with similar CO2assimilation rates are shown in Fig. 1. FPSII increased more rapidly than CO2 assimilation during the first 10 min, then increased gradually more slowly, and finally it reached the steady-state rate at the same time as CO2 assimilation (top in left panel). Since these parameters were simultaneously recorded, it was possible to directly compare the electron transport rate through PSII (Jf) calculated from the FPSII with the

electron transport rate required to sustain CO2 assimilation and photorespiration (Jg) from the gas exchange data. The difference between Jf and Jg was regarded as the rate of alternative electron transport (Ja). As shown in Fig. 1, Ja increased rapidly just after illumination and Jg increased gradually with decreasing Ja after Ja reached its maximum (middle in left panel). This rapid increase in Ja was similar to a response of NPQ just after illumination. If most of Ja is accounted for by the electron flux in the waterwater cycle (Miyake and Yokota 2000), this means that the electron flow to oxygen and oxidized ascorbic acid during the first 10-min induction phase generated a high DpH across the thylakoid membranes. After that, CO2 assimilation (Jg) started when Ja and NPQ decreased, and then reached the steady-state rate. When the O2 concentration was decreased to 2%, CO2 assimilation increased, whereas FPSII decreased slightly. This increase in CO2 assimilation was caused by a suppression of oxygenation of RuBP (pho-

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in 20% O2. Since the steady-state level of P700+ was not affected by O2 concentration, these results additionally suggest that the electron flux through PSI was greater in 2% O2 and that the cyclic electron transport did occur. FPSII and CO2 assimilation increase in parallel with each other during photosynthetic induction in 2% O2 The responses of PSII, PSI and CO2 assimilation in the wild-type leaf during photosynthetic induction were next examined under conditions of 2% O2. Typical examples are also shown in Fig. 3. No difference in the response between FPSII and CO2 assimilation was found during photosynthetic induction in 2% O2. As a result, Ja was almost zero, although a small Ja was detected during the first 10-min induction. This suggests little electron flow to oxygen under the conditions of 2% O2. NPQ increased during the first phase of the induction. However, the rate of the NPQ formation was appreciably slower than that in 20% O2, although it remained at higher levels. When the O2 concentration was increased to 20%, CO2 assimilation decreased and FPSII increased slightly. Since Jf increased a little and Jg decreased, Ja appeared in 20% O2. NPQ decreased in 20% O2. The response of FPSI was also similar to that of Jg, but FPSI was always greater than FPSII in 2% O2 (right panel). When the O2 concentration was increased to 20%, FPSI decreased and reached levels close to those of FPSII. These results thus indicate that the cyclic electron flow around PSI also occurred in 2% O2 just after illumination throughout the induction phase. The rbcS antisense plants show a higher NPQ because of a higher ratio of FPSI to FPSII Fig. 4 shows the responses of FPSII, FPSI and CO2 assimilation in the rbcS antisense rice during photosynthetic induction in 20% O2. FPSII increased more rapidly than CO2 assimilation, then increased gradually, and finally reached the steady-state rate at the same time as CO2 assimilation. During the first 5-min induction phase, most of Jf was derived from Ja. When the O2 concentration was decreased to 2%, Ja decreased to zero. These responses were similar to those found for the wild-type rice. In the antisense rice, however, NPQ remained at higher levels throughout the induction phase. FPSI also remained at higher levels (about 0.45) than FPSII (about 0.25), irrespective of O2 concentrations. This suggests that the cyclic electron flow around PSI occurs in the antisense rice even in 20% O2 as well as in 2% O2. Since this cyclic electron flow generates DpH across the thylakoid membranes, it probably resulted in a higher NPQ. We summarize the photosynthetic characteristics during the steady-state phase of the wild-type and rbcS antisense rice in 2% and 20% O2 (Table 2). In the wild-type rice, Ja was strongly suppressed and NPQ was enhanced at 2% O2. Whereas the ratio of FPSI/FPSII was almost 1 in 20% O2, this ratio increased to 1.66 in 2% O2. These results suggest that a higher NPQ in 2% O2 is caused by the cyclic electron flow

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Fig. 2 Transient changes in P700+ absorbance by a 50-ms saturating light pulse during the steady-state phase of photosynthesis where the rates of CO2 assimilation were 21 mmol m2 s1 in 20.2% O2 (bold line) and 30 mmol m2 s1 in 2% O2 (light line), respectively, in the wild-type rice. The arrow indicates the time when the saturating light pulse was fired.

torespiration) and a stimulation of carboxylation. Interestingly, Ja was strongly suppressed in 2% O2. When the PPFD was reduced to 200 mmol m2 s1, CO2 assimilation decreased quickly and FPSII increased. At this time, Jf and Jg decreased and Ja was down to almost zero. FPSI also increased more rapidly than CO2 assimilation during the induction phase in 20% O2 (right panel of Fig. 1). This response of FPSI was similar to that of FPSII, but the increase in FPSI during the first induction phase was slightly faster than that of FPSII. When FPSI reached the steady-state level, it was close to FPSII levels (0.40.5). However, when the O2 concentration was decreased to 2%, FPSI increased substantially (~0.7), whereas FPSII slightly decreased (~0.37). This suggests that the rate of the electron flow through PSI was enhanced in 2% O2 and was faster than that through PSII in 2% O2. The faster rate of electron flow through PSI than through PSII suggests the operation of the cyclic electron transport around PSI. To examine the difference in the transfer rate of electrons from plastocyanin to P700 between 2% and 20% O2 concentrations, the transient changes in P700+-absorbance caused by a 50-ms saturating light pulse during the steady-state of photosynthesis were monitored on a millisecond time scale with a transient recording system as described in Materials and Methods (Fig. 2). The height of the peak in response to a saturating pulse was higher in 2% O2, and the reduction rate of P700+ after the saturating pulse was clearly faster in 2% O2 than that

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Fig. 3 Left panel: FPSII and CO2 assimilation rate (A) (top), electron transport rates from Chl fluorescence (Jf), gas exchange (Jg) and alternative electron flow (Ja) (middle) and NPQ (bottom) during photosynthetic induction in the wild-type rice. Gas exchange and Chl fluorescence were simultaneously measured at 2% O2 from darkness to an irradiance of 900 mmol m2 s1. O2 concentration was increased to 20.2% after 45 min, and irradiance was decreased to 200 mmol m2 s1 after 60 min. Right panel: Rate of CO2 assimilation (top) and electron transport rate from gas exchange (Jg) and FPSI (bottom) during photosynthesis in the wild-type rice. Gas exchange and P700+-absorbance were simultaneously measured at 2% O2 from darkness to an irradiance of 900 mmol m2 s1. O2 concentration was enhanced to 20.2% after 37 min.

around PSI. In the antisense rice, CO2 assimilation and FPSII were lower than in the wild-type rice, irrespective of O2 concentration. Ja also decreased and was suppressed in 2% O2. However, FPSI was similar to that in the wild-type rice, which resulted in the higher ratio of FPSI to FPSII. NPQ was also enhanced and increased more in 2% O2. These results also suggest that a high NPQ in the antisense plants is caused by the operation of the cyclic electron flow around PSI.

Discussion
The waterwater cycle is a starter of photosynthesis FPSII increased more rapidly than CO2 assimilation during the early phase of photosynthetic induction under normal O2 concentrations (Fig. 1). A similar response during photosynthetic induction was found previously in tobacco by Ruuska et al. (2000b). Regarding this different response between FPSII

and CO2 assimilation, they discussed several candidates of extra electron sinks as possible explanations. One is the oxaloacetatemalate shuttle which can transport reducing equivalents from chloroplasts to cytosol (Backhausen et al. 1998). The other is nitrate reduction or photoreduction of O2 which can also consume the extra electron (waterwater cycle). In algae, O2 uptake and O2 evolution were simultaneously observed during a lag in CO2 fixation just after illumination (Radmer and Kok 1976). Probably, this is similar to our present observation. Our results clearly indicate that the rapid increase in FPSII strongly depends on O2 concentration. Whereas a more rapid response of FPSII was found in 20% O2 (Fig. 1), there was no difference in induction rate between FPSII and CO2 assimilation in 2% O2 (Fig. 3). Therefore, extra electron flow in 20% O2 was regarded as the waterwater cycle. This cycle was first induced when the leaf was illuminated. In addition, NPQ increased more rapidly just after illumination in 20% O2 than in 2% O2.

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Fig. 4 Left panel: FPSII and CO2 assimilation rate (A) (top), electron transport rates from Chl fluorescence (Jf), gas exchange (Jg) and alternative electron flow (Ja) (middle) and NPQ (bottom) during photosynthetic induction in the rbcS antisense rice. Gas exchange and Chl fluorescence were simultaneously measured at 20.2% O2 from darkness to an irradiance of 900 mmol m2 s1. O2 concentration was reduced to 2% after 32 min, and irradiance was decreased to 200 mmol m2 s1 after 50 min. Right panel: Rate of CO2 assimilation (top) and electron transport rate from gas exchange (Jg) and FPSI (bottom) during photosynthesis in the wild-type rice. Gas exchange and P700+-absorbance were simultaneously measured at 20.2% O2 from darkness to an irradiance of 900 mmol m2 s1. O2 concentration was reduced to 2% after 31 min.

Thus, our results indicate that the waterwater cycle first generates DpH across the thylakoid membranes and leads to such a rapid formation of NPQ. When the electron-flow rate of the waterwater cycle reached a maximum, it was 100 mmol e m2 s1 (Fig. 1), corresponding to 75 mmol O2 (mol Chl)1 s1. This is comparable to the maximum rate of O2 production mediated by Fd or MDAR in vitro (Furbank and Badger 1983, Miyake et al. 1998). The potential capacity of the waterwater cycle, thus, is considerably greater than previously estimated. Generally, stomatal opening and several enzymes of the Calvin cycle need a few min to be activated after illumination (Usuda 1985, Kirschbaum and Pearcy 1988, Seemann et al. 1988, Mott et al. 1997, Hammond et al. 1998). Therefore, a rapid formation of NPQ would be required to prevent photoinhibition. Furthermore, according to Seemann et al. (1988) and Portis (1992), an increase in photosynthesis after illumination can be limited by the rate at which Rubisco activity

increases in vivo. In this regulation, Rubisco activase releases RuBP or a nocturnal tight-binding inhibitor, 2-carboxy-Darabinitol-1-phosphate (CA1P), from inactivated Rubisco (Andrews et al. 1995, Salvucci and Ogren 1996). This process requires ATP hydrolysis by Rubisco activase (Portis 1990). Therefore, although the energy costs for activation of Rubisco by Rubisco activase have not been established, it is possible that the waterwater cycle functions as an ATP supplier for Rubisco activase during the early phase of photosynthetic induction. In fact, rice produces large amounts of CA1P in darkness (Table 1). In the present experiments, the plants were adapted to darkness more than 60 min before measurements. However, when the plants were exposed to a low light flux (100 mmol quanta m2 s1) for 60 min before measurements, Rubisco was not inhibited and there was no difference between FPSII and CO2 assimilation during photosynthetic induction by an actinic light of 900 mmol quanta m2 s1. In addition, a

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Table 2 Summary of the photosynthetic characteristics during the steady-state phase in the wild-type and rbcS antisense rice (Anti-rbcS) under 2 and 20.2% O2 concentrations A Ci (mmol m2 s1) (Pa) a Wild type (20%) b Wild type (2%) Anti-rbcS (20%) Anti-rbcS ( 2%) 23.20.9 c 32.41.1 18.41.6 25.12.5 291 281 321 311 FPSI 0.470.01 0.650.02 0.460.01 0.560.06 FPSII 0.430.02 0.390.02 0.230.03 0.210.03 Jg Jf Ja PSI/PSII (mmol m2 1 s ) (mmol m2 s1) (mmol m2 s1) NPQ 1.09 1.66 1.95 2.64 129 6 148 5 98 9 11211 179 8 160 8 12915 11619 5113 1214 30 6 4 8 1.190.16 1.750.26 2.490.11 2.730.14

Measurements were made at a PPFD of 900 mmol quanta m2 s1, a leaf temperature of 23C and an external CO2 pressure of 37.5 Pa. a Ci shows the intercellular CO2 partial pressure. b Values in parentheses show the concentration of O2 (%). c Means SE (n = 36).

rapid formation of NPQ was not also found at this time (data not shown). With increasing CO2 assimilation, the electron flux of the waterwater cycle decreased as deduced from a decrease in Ja, and then photosynthesis reached the steady-state rate (Fig. 1). During the latter phase of the induction, NPQ also decreased with the decrease of the electron flux of the waterwater cycle. Therefore, we propose that the waterwater cycle functions as a starter of photosynthesis during the induction phase. Cyclic electron flow around PSI is a starter of photosynthesis when the waterwater cycle is suppressed During the photosynthetic induction in 2% O2, the electron flux of the waterwater cycle was scarcely found (Fig. 3). Nevertheless, NPQ was formed and CO2 assimilation increased smoothly. In 2% O2, FPSI was higher than PSII. This suggests the operation of the cyclic electron flow around PSI. The cyclic electron flow may have generated DpH across the thylakoid membranes and formed NPQ. However, the rate of NPQ formation was slower in 2% O2 than in 20% O2. On the other hand, during the latter phase of the induction, NPQ remained at higher levels in 2% O2. This may have been caused by the absence of a decline in the cyclic electron flow during this phase. By contrast, the electron flow rate of the waterwater cycle in 20% O2 declined gradually during the latter phase of induction (see Fig. 1). Clarke and Johnson (2001) examined PSII and PSI photochemistry in barley leaves in 2% and 21% O2. They found that while the rate of electron transport through PSI was apparently independent of O2 concentration, FPSII was substantially reduced in 2% O2. Their results are a little different from ours, but also suggest the existence of the cyclic electron flow in barley in 2% O2. Jot et al. (2001) used the ndhB-inactivated tobacco mutant with less activity of the cyclic electron transport around PSI, and found that both CO2 assimilation and FPSII in this mutant are strongly inhibited under the conditions of 2% O2 concentration. In addition, Jot et al. (2002) observed an increased activity of the cyclic electron transport around PSI in tobacco leaf discs under anaerobic conditions. Thus, we conclude that when the waterwater cycle is suppressed, the cyclic electron flow can also be a main starter

of photosynthesis. In 20% O2, the increase in FPSI during the first induction phase was slightly faster than that of FPSII (Fig. 1). This suggests the possibility that the cyclic electron flow around PSI also functions during the first induction phase in 20% O2. On the other hand, as shown in Fig. 3, a slight Ja was found during the first induction phase in 2% O2. If this is the waterwater cycle, the waterwater cycle might also be essential as a starter of photosynthesis, even under O2-limited conditions. Otherwise, it is possible that this is the cyclic electron flow within PSII (Miyake and Yokota 2001, Miyake et al. 2002). Further work aimed at resolving these problems is in progress. High NPQ of the rbcS antisense rice with reduced Rubisco is maintained not by the waterwater cycle but by the cyclic electron flow around PSI Our rbcS antisense rice plants with decreased Rubisco have low capacity for CO2 assimilation without any decrease in electron transport capacity (Makino et al. 1997). Although such characteristics suggest that these plants should be sensitive to photoinhibition, our antisense plants grew well even under high light conditions. This is because they had a higher NPQ (Table 2). Similarly, high NPQ has been frequently observed in several transgenic plants with suppressed photosynthesis such as tobacco plants with reduced Rubisco (Quick et al. 1991, Ruuska et al. 2000b) and phosphoribulokinase (Habash et al. 1996), and potato and tobacco plants with reduced fructosebisphosphatase (Bilger et al. 1995). In such plants, Bilger et al. (1995) and Ruuska et al. (2000b) found that high NPQ is associated with enhancement of violaxanthin de-epoxidation among xanthophyll cycle pigments. In their plants, the low capacity of CO2 assimilation did not affect total carotenoid and xanthophyll cycle pigment content per unit of Chl content (Bilger et al. 1995, Ruuska et al. 2000b). Quite similar phenomena were also found in our antisense rice (data not shown). Since such leaf pigments per Chl generally increase when the plants are grown under conditions of higher irradiance (Demmig-Adams and Adams 1996, Demmig-Adams et al. 1998) or N deficiency (Logan et al. 1999, Verhoeven et al. 1997), the capacity for photoprotection in our transgenic rice plants may be suffi-

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Waterwater cycle and cyclic electron flow in rice (Makino et al. 1997), and their R2 segregants with about 50% wildtype Rubisco were selected (AS-71; Makino et al. 2000). All plants were grown hydroponically in an environmentally controlled growth chamber (Makino et al. 1994a). The chamber was maintained with a 14-h photoperiod, 25/20C day/night temperature, 60% relative humidity and a photosynthetic photon flux density (PPFD) of 1,000 mmol quanta m2 s1 during the photoperiod. The basal nutrient solution was as previously described by Makino et al. (1988) except that 2 mM NH4NO3 was used instead of 1 mM NH4NO3. The measurements were done on uppermost, fully expanded leaves of 70- to 80day-old plants. Chl fluorescence, P700+-absorbance and gas exchange measurements Chl fluorescence and gas exchange, and P700+-absorbance and gas exchange, respectively were measured simultaneously (the three parameters were not measured simultaneously). The measurements of the leaf attached to the plant were done over a leaf area of 1.75 cm2. The basal system of gas exchange was used as previously detailed by Miyake and Yokota (2000). Differences in the partial pressures of CO2 and H2O entering and exiting the chamber were measured with an IRGA (LI-6262; Li-Cor, Lincoln, NE, U.S.A.). The absolute pressure of CO2 was measured with another IRGA (LI-6252, Li-Cor) and controlled at 37.50.5 Pa in the chamber by mixing 1% (v/v) CO2 in 99% (v/v) N2, 2%(v/v) O2 in 98% (v/v) N2 and 20.2% (v/v) O2 in 79.8% (v/ v) N2 with a mass-flow controller (Kofloc model GB-3C, Kojima, Kyoto, Japan). The partial pressure of H2O entering chamber was strictly maintained at 17.5 kPa with a dew point generator (LI-610, LiCor). Leaf temperature was maintained at 231C. Irradiance was provided by a halogen lamp (KL-1500; Walz, Effeitrich) and adjusted to a PPFD of 900 or 200 mmol quanta m2 s1 at the position of the leaf in the chamber. It entered the chamber at an angle of 90 through glassfiber optics linked to a pulse-amplitude-modulated (PAM) Chl fluorometer (PAM-101; Walz). Chl fluorescence was measured with the PAM Chl fluorometer through the same fiber-optic probe positioned in the chamber. The steady-state fluorescence yield (Fs) was monitored continuously and a 900-ms pulse of saturating light was supplied at intervals of 6090 s to determine maximum variable fluorescence (Fm). The quantum efficiency of PSII (FPSII) is defined as (Fm Fs)/Fm or DF/Fm as proposed by Genty et al. (1989). Non-photochemical quenching (NPQ) was calculated as (Fm Fm)/Fm according to Bilger and Bjrkman (1994). The absorbance of P700+ was measured with the same PAM fluorometer equipped with an ED 830-nm emitter detector unit (Walz) as described by Klughammer and Schreiber (1994). The change in the absorbance of P700+ was continuously monitored and a 1-s pulse of saturating light was supplied at intervals of 6090 s. After cessation of actinic illumination, far-red light was illuminated for 6090 s, and then a 1-s saturating light pulse was irradiated to determine the total P700+. The quantum efficiency of PSI was calculated according to Klughammer and Schreiber (1994). For the evaluation of the rate of both reduction and oxidation of P700 during the steady-state phase of photosynthesis, the transient changes in P700+ absorbance by the saturating light pulse were also monitored on a millisecond time scale with a transient recording system (Mac Lab 200; ADI Instruments, NSW, Australia). For this analysis, a 50-ms saturating light pulse was applied to the leaf in the chamber from a Xenon discharge lamp with sharp on/ off characteristics (XST 103, Walz). Before the above measurements were taken all plants were adapted to darkness for more than 60 min. Calculations of electron transport rates The rate of electron transport required to account for the photo-

ciently high to maintain PSII functionality in spite of a decrease in CO2 assimilation under conditions of the present growth-light. The electron flow rate of the waterwater cycle was lower in the rbcS antisense plants with 50% wild-type Rubisco (Table 2). In rbcS antisense tobacco, the relationship between electron transport rates from Chl fluorescence and those from gas exchange has been found to be similar to those in wild-type tobacco, indicating that the low capacity for CO2 assimilation does not enhance extra electron transport to O2 (Ruuska et al. 2000a). The electron flow rate of the waterwater cycle during the early phase of photosynthetic induction in 20% O2 was also lower in the antisense rice (Fig. 4). Nevertheless, NPQ increased up to a level similar to that in the wild-type rice and remained at higher levels. This may have been caused by the higher rate of the cyclic electron flow around PSI. Higher ratios of FPSI to FPSII were observed in the rbcS antisense plants, irrespective of O2 concentration (Table 2 and Fig. 4). Therefore, we conclude that in the rbcS antisense plants, the water water cycle is not important in maintaining DpH across the thylakoid membranes and the cyclic electron flow around PSI acts to maintain a higher NPQ to prevent photoinhibition. However, since a rapid formation of NPQ just after illumination was not observed in 2% O2 (data not shown), the waterwater cycle also may function as a main starter of photosynthesis in the antisense plants

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Conclusions
The waterwater cycle functions as a starter of photosynthesis and leads to the rapid formation of DpH across the thylakoid membranes just after illumination. This response may be essential for not only protection of PSII just after a sudden illumination but also for activation of Calvin cycle enzymes including Rubisco activase during photosynthetic induction. In addition, the potential capacity for the electron flow through the waterwater cycle is considerably greater than previously estimated. It is comparable to the maximum rate of O2 production at the site of Fd or MDAR in vitro (75 mmol O2 (mol Chl)1 s1). However, this electron flow does not act to maintain a high NPQ and to regulate the quantum yield of PSII. These regulations are performed by the cyclic electron flow around PSI. For example, a high NPQ of the rbcS antisense plants is maintained by the cyclic electron flow. Since the cyclic electron flow does not produce any toxic active oxygen species, it may be more important for the long-term regulation of the quantum yield of PSII and NPQ.

Materials and Methods

Plant materials Rice (Oryza sativa L. cv. Notohikari) and transgenic rice plants with reduced amounts of Rubisco were used. The transgenic plants were obtained by transformation with the rice rbcS antisense gene

Waterwater cycle and cyclic electron flow in rice synthetic carbon reduction and photorespiration (Jg) was calculated from gas exchange data according to the following equation (von Caemmerer and Farquhar 1981): Jg = (A + Rd)(4Cc + 8G*)/(Cc G*) (1)

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where A is the net rate of CO2 exchange, Rd is non-photorespiratory respiration and Cc is the CO2 partial pressure in the chloroplast stroma deduced from the assumption that CO2 transfer conductance between the intercellular air spaces and the chloroplast stroma is 0.5 mol CO2 m2 s1 (von Caemmerer and Evans 1991 for rice, Makino et al. 1994a). G* is the partial pressure of CO2 in the chloroplast at which photorespiratory CO2 evolution equals the rate of carboxylation, G* = 0.5 Vo Kc O/Vc Ko (2)

where Vc and Vo denote the maximum Rubisco activity of carboxylation and oxygenation, Kc and Ko are the Michaelis-Menten constants for CO2 and O2 (Makino et al. 1994a), and O is the partial pressure of O2 in the chloroplast but assumed to be the same as in the external air. The respective Vc and Vo values used here are 17.5 and 5.7 mol (mol Rubisco)1 s1, and the respective Kc and Ko values are 24 Pa and 28 kPa (Makino et al. 1994a, Makino et al. 1997). The rate of electron transport through PSII (Jf) was calculated from the Chl fluorescence data according to the following equation (Genty et al. 1989): Jf = a I DF/Fm (3)

where DF/Fm is the quantum yield of PSII, I is the incident PPFD and a is the fraction of absorbed light distributed to PSII. The value of a was calculated from the assumption that Jg is equal to Jf at external CO2 and O2 partial pressures of 37.5 Pa and 2 kPa, respectively and a PPFD of 200 mmol quanta m2 s1. Values of 0.460.04 (n = 4) were obtained with rice leaves. The rate of alternative electron transport (Ja) was calculated from the equation Ja = Jf Jg (4)

Biochemical assays The amounts of Rubisco, Chl and total leaf N were determined by the method of Makino and Osmond (1991). Rubisco activity was measured at 25C according to Nakano et al. (2000).

Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (Nos. 11460029 and 14360036 to A.M.) and by a Research for the Future from the Japan Society for the Promotion of Science (JSPS-00L01604 to C.M. and A.Y.). We thank two anonymous reviewers for invaluable and constructive comments. A.M. deeply appreciates C.M. for allowing him to be the first author of this paper.

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(Received April 19, 2002; Accepted July 4, 2002)