Masahiro Ikeuchi, Nozomu Uebayashi, Fumihiko Sato and Tsuyoshi Endo* Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan *Corresponding author: E-mail, [email protected]; Fax, +81-75-753-6398. (Received November 22, 2013; Accepted May 7, 2014)
The PsbS protein plays an important role in dissipating excess light energy as heat in photosystem II (PSII). However, the physiological importance of PsbS under naturally fluctuating light has not been quantitatively estimated. Here we investigated energy allocation in PSII in PsbSsuppressed rice transformants (psbS) under both naturally fluctuating and constant light conditions. Under constant light, PsbS was essential for inducing the rapid formation of light-inducible thermal dissipation (NPQ), which consequently suppressed the rapid formation of basal intrinsic decay (f,D), while the quantum yield of electron transport (II) did not change. In the steady state phase, the difference between the wild type (WT) and psbS was minimized. Under regularly fluctuating light, the reduced PsbS resulted in higher II upon the transition from high light to low light and in lower II upon the transition from low light to high light, indicating that II was, to some extent, controlled by PsbS. Under naturally fluctuating light in a greenhouse, rapid changes in II were compensated by NPQ in the WT, but by f,D in psbS. As a consequence, a significantly lower NPQ integrated NPQ over a whole day) and higher f,D were found in psbS. Furthermore, thermal dissipation associated with photoinhibtion was enhanced in psbS. These results suggest that PsbS plays an important role in photoprotective process at the induction phase of photosynthesis as well as under field conditions. The physiological relevance of PsbS as a photoprotection mechanism and the identities of NPQ and f,D are discussed. Keywords: Energy allocation in PSII Naturally fluctuating light Non-photochemical quenching (NPQ) PsbS Thermal dissipation of absorbed light energy. Abbreviations: pH, transthylakoid pH gradient; psbS, PsbS-suppressed rice transformants; Fm (Fm0 , Fm00 ), the maximum fluorescence level achieved with a saturating pulse in the absence of NPQ (or in the presence of NPQ, or after a brief dark treatment during daytime); Fo (Fo0 ), the minimum fluorescence level achieved under a measuring light in the dark (or in the presence of NPQ); Fs, steady-state fluorescence level
in the light; fast (fast), the quantum yield (or daily integrated flux) of the fast-relaxing portion of light-inducible dissipation in the Kasajima model; f,D (f,D), the quantum yield (or daily integrated flux) of basal dissipation in the Hendrickson model; NPQ (NPQ), the quantum yield (or daily integrated flux) of light-inducible dissipation in the Hendrickson model; slow (slow), the quantum yield (or daily integrated flux) of the slowly-relaxing portion of light-inducible dissipation in the Kasajima model; II (II), the quantum rate (or daily integrated flux) of absorbed light energy in PSII allocated to electron transport; LHC, light-harvesting chlorophyll-protein complex; NPQ, non-photochemical quenching; PAM, pulse amplitude modulation; PAR, photosynthetically active radiation; PQ, plastoquinone; qE, energy quenching; qI, photoinhibitory quenching; qP, photochemical quenching; qT, state transition quenching; RH, relative humidity; SE, standard error; PAR, integrated PAR over an entire day; TD, thermal dissipation; VDE, violaxanthin deepoxidase; WT, wild type rice.
Introduction Under natural conditions, changes in light intensity follow diurnal and seasonal cycles and irradiance from the sun can be blocked by clouds, trees and buildings. Moreover, in a threedimensional canopy, leaves are exposed to fluctuating light (Zhu et al. 2004). Plants have evolved an efficient light-harvesting system in response to these fluctuating light conditions. The allocation of absorbed light energy in photosystem II (PSII) is highly controlled and some of the excess light energy is dissipated as heat, which can be monitored as non-photochemical quenching (NPQ) of chlorophyll fluorescence (Horton et al. 1996, Niyogi 1999, Maxwell and Johnson 2000). NPQ is the sum of at least three different mechanisms, i.e. qE (energy quenching), qT (state transition) and qI (photoinhibitory quenching), which are defined on the basis of their relaxation kinetics (Quick and Stitt 1989, Walters and Horton 1991). A dominant part of thermal dissipation that is associated with NPQ (NPQ-TD), qE-associated thermal dissipation (qE-TD),
Plant Cell Physiol. 55(7): 1286–1295 (2014) doi:10.1093/pcp/pcu069, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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Special Focus Issue – Regular Paper
Physiological Functions of PsbS-dependent and PsbS-independent NPQ under Naturally Fluctuating Light Conditions
Role of PsbS under naturally fluctuating light
these parameters were calculated from the chlorophyll fluorescence determined during the induction period. Thus, it is not yet clear how the mechanism of energy allocation changes under prolonged or fluctuating light illumination. The aim of this study was to estimate the physiological importance of PsbS under naturally fluctuating light conditions. We first obtained data on energy allocation in PSII, using the model proposed by Hendrickson et al. (2004), in PsbS-suppressed rice transformants under constant or regularly fluctuating light to understand the basic changes in energy allocation in the presence and absence of PsbS. Based on the data obtained above, we then analyzed the diurnal changes in energy allocation in plants cultivated in a semi-open greenhouse to reveal the mechanism of energy allocation under naturally fluctuating light conditions. As a result, drastic changes in energy allocation in PSII were induced depending on the amount of PsbS and the duration of illumination. Thus, we could confirm that PsbS protein plays an important role in the photoprotective process and its content is nearly optimized to adapt to natural conditions.
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protects reaction centers of PSII from photoinhibition. As shown previously, qE-type energy dissipation is fast-forming and rapidly reversible in the dark and is induced by a proton gradient across the thylakoid membrane (pH) caused by excess light (Krause and Weis 1991, Horton and Ruban 1992, Horton et al. 1996, Mu¨ller et al. 2001). PSII subunit S (PsbS) protein and violaxanthin de-epoxidase (VDE), which converts violaxanthin to zeaxanthin, are associated with this quenching process and are protonated at a low pH in the thylakoid lumen (Demmig-Adams 1990, Horton et al. 1996). The de-epoxidation state of xanthophyll pigments may regulate the affinity of LHCII for protons (Rees et al. 1992, Horton et al. 2000). The PsbS protein has been shown to enhance the dynamics of thylakoid membranes by reducing the ordering and increasing the fluidity of protein organization, which accelerates the reorganization and aggregation of LHCII (Goral et al. 2002, Kiss et al. 2008, Goral et al. 2012). However, a qE-type quenching process is an intrinsic property of LHCII, and thus can be induced independently of VDE or PsbS protein (Rees et al. 1989, Noctor et al. 1991, Johnson and Ruban 2010, Johnson and Ruban 2011). Therefore, PsbS facilitates reorganization of LHCII, which results in acceleration of qE induction. The most slowly relaxing component of NPQ, qI, supposedly reflects photoinhibitory quenching. However, there is no clear relationship between the extent of qI and photodamage to the PSII reaction center itself (Walters and Horton 1993). A recent study has shown that the thioredoxin-like/b-propeller protein SOQ1 could prevent the formation of a slowly reversible NPQ, and there might be other uncharacterized quenching pathways (Brooks et al. 2013). Additionally, some part of the NPQ induced by pH across thylakoid membranes, which is the same mechanism as in qE-type energy dissipation, had slow induction and relaxation kinetics, and thus was monitored as qI. This slow kinetics was associated with zeaxanthin, trapped protons and aggregated LHCII (Ruban et al. 1993, Ruban and Horton 1995, Nilkens et al. 2010). To maximize photosynthetic productivity under naturally fluctuating light, each component of NPQ-TD should be identified and the photoprotective process should be optimized. A modeling analysis has demonstrated that slowly-reversible qI-type energy dissipation prevented rapid recovery of the CO2 assimilation rate and reduced canopy photosynthesis (Zhu et al. 2004). Most studies on PsbS protein have been conducted under artificial and continuous light illumination, and no clear phenotype with respect to growth has been reported. However, a mutant of Arabidopsis thaliana npq4 that lacks PsbS protein showed a significant decrease in plant growth under field conditions and when grown under fluctuating light in a growth chamber (Ku¨lheim et al. 2002, Ku¨lheim and Jansson 2005). We previously showed, using the light energy allocation model in PSII proposed by Hendrickson et al. (2004), that the suppression of PsbS protein in rice resulted in a decrease in light-inducible dissipation (NPQ) and a consequent increase in basal intrinsic decay (f,D), whereas the allocation of energy to electron transport (II) did not change over a wide range of light intensities (Ishida et al. 2011). However,
Results Properties of "psbS We used a transgenic line of rice in which both of the two psbS genes had been silenced by RNAi (psbS; Ishida et al. 2011). At the T2 generation, quantitative reverse transcription-PCR (RTPCR) analysis revealed that the amounts of both gene transcripts were significantly reduced in this transgenic line, and no PsbS protein was detected by an immunoblot analysis (Ishida et al. 2011). The T4 generation of the transformant used in the present study also had no detectable PsbS protein by an immunoblot analysis (data not shown). The chlorophyll contents estimated by a SPAD chlorophyll meter in the 9-week-old wild type (WT) and psbS cultivated in a semi-open greenhouse were 42.6 ± 0.7 and 43.2 ± 0.7, respectively (means ± SE, n = 5). The aboveground dry weights of a plant grown in the greenhouse after the maturation of seeds in the WT and psbS were 48.9 ± 2.6 g and 41.1 ± 3.4 g, respectively, (means ± SE, n = 6) in the 2013 season.
Induction kinetics of (II, (NPQ and (f,D under constant light illumination We first investigated the time course of changes in II, NPQ and f,D under exposure to constant light (Figs. 1A, B). The parameter II represents the effective quantum yield of electron transport through PSII (Genty et al. 1989). The parameters NPQ and f,D represent the quantum yield of lightinducible energy dissipation and that of the intrinsic decay of excited chlorophylls, respectively (Hendrickson et al. 2004). There was no significant difference in Fv/Fm between the two genotypes in the dark-adapted state before light illumination (WT: 0.836 ± 0.001, psbS: 0.837 ± 0.001, means ± SE, n = 5).
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At 420 mmol m2 s1, NPQ in the WT plants was highest 2 min after the onset of actinic illumination (2 min L) and then gradually decreased to the steady state level at 60 min after the onset of light exposure (60 min L; Fig. 1A). In contrast, NPQ in psbS increased gradually and reached the steady state, at which the yield was almost identical to that in the WT. On the other hand, f,D was highest in both genotypes at 2 min L during the induction phase, and f,D in psbS was about
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two-fold greater than that in the WT. Subsequently, f,D gradually decreased after a rapid decrease at 4 min after the onset of illumination (4 min L) in both genotypes. The difference between them was minimized and the yield nearly returned to the darkadapted value in both genotypes at 60 min L. The quantum yield II showed an almost identical pattern regardless of the PsbS level; it was lowest at 2 min L, and then slowly increased to reach a steady state level at 60 min L after a
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Fig. 1 Changes in chlorophyll fluorescence parameters during induction from dark-adapted states. The quantum yields of light-inducible dissipation (NPQ), basal and constitutive dissipation (f,D) and electron transport (II) according to Hendrickson et al. (2004), and photochemical quenching (qP) were determined in both the wild type (WT) and the transgenic T4 plant in which the PsbS level was reduced (psbS). Leaves were dark-adapted for over 1 h before the measurement of Fo and Fm, and then actinic light of 420 (A) or 1150 mmol m2 s1 (B) was applied. After illumination for 60 min, the actinic light was turned off and leaves were kept in the dark for 12 min. Means ± SE (n = 5).
Role of PsbS under naturally fluctuating light
low light, II and f,D were at the same level in both genotypes at 60 min L (Fig. 2A). However, under high light, II in psbS was reduced at 60 min L, and this reduction was compensated by an increase in f,D (Fig. 2B). This reduced II and increased f,D may be associated with photoinhibition under high light, as discussed below.
Energy allocation in PSII under regularly fluctuating light To obtain insight into the energy allocation in PSII under fluctuating light, we conducted a series of experiments using low-, moderate- and high-light fluctuation after photosynthesis had reached a steady state (Fig. 3). Light-treated plants were subjected to light fluctuation where the light intensity was changed every 5 min, in the order 420, 125, 1150 and 125 mmol m2 s1 as one cycle. In this light fluctuation treatment, NPQ in the WT was much greater than that in psbS throughout light exposure. On the other hand, while f,D remained at the dark-treated level in the WT plant, a large increase was seen with an increase in light intensity in psbS. The changes in II under fluctuating light in psbS showed a characteristic pattern. Under moderate and high light (420 and 1150 mmol m2 s1), there was a 10–20% reduction in psbS, as was found in the induction phase upon the transition from dark to light shown in Figs. 1A, B. During light fluctuation treatment, the value of qP in psbS was consistently lower than that in the WT, and the difference between the two genotypes significantly increased under moderate and high light. This indicates that a higher reduction state of the PQ pool in psbS could reduce II under this condition. However, under exposure to low light (125 mmol m2 s1) just after moderate light illumination, there was no significant difference in II between the two genotypes. Furthermore, under low light just after high-light treatment (dark grey bar after the white bar in Fig. 3), II in psbS was higher 30 seconds after the light shift and gradually settled to a level comparable to that in the WT. This pattern in II could be observed even after the light fluctuation treatment was repeated several times.
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rapid increase between 2 to 4 min L. At 2 min L, II in psbS and the WT were nearly indistinguishable. Subsequently, II in the WT was slightly higher than that in the psbS, but the difference between the two genotypes was minimized at the steady state (60 min L). With regard to photochemical quenching (qP), the WT showed a significantly higher qP value than psbS at 2 min L, but the difference between the two genotypes became smaller at 60 min L (Fig. 1A). After the actinic illumination was turned off, there was no significant difference in these parameters between the two genotypes. To evaluate photoinhibition, we used the Fv00 /Fm00 value after a brief dark treatment instead of Fv/Fm, which is determined after an overnight dark treatment. The identical yield of II in the dark (i.e. Fv00 /Fm00 ) indicates that photoinhibition did not occur under this low-light illumination. At 1150 mmol m2 s1, the levels of NPQ in the WT and psbS at 2 min L were approximately 20% and 60% higher than the peak observed at 420 mmol m2 s1, respectively (Fig. 1B). However, under this high-light level, NPQ in the WT was not highest at 2 min L, and gradually increased to form a gentle peak around 10 min L. By contrast, in psbS, it gradually increased to a steady state level at 60 min L. The highest f,D in the first 2 min was about 20–30% higher than that at 420 mmol m2 s1 in both genotypes. After reaching the maximum at 2 min L, the yield in the WT decreased and returned to the dark-adapted value as in the pattern at 420 mmol m2 s1, while psbS showed an approximately 15% higher yield (P < 0.05) than the WT plant. The difference in f,D between the two genotypes was greater than that at 420 mmol m2 s1. The yield of II at 1150 mmol m2 s1 was approximately 40% lower than that at 420 mmol m2 s1 in both genotypes. At 2 min L during the induction phase, there was no significant difference between the two genotypes, after which psbS showed about 10% lower II than in the WT throughout the illumination period. Additionally, qP in psbS was significantly lower than that in the WT throughout light illumination, suggesting that the plastoquinone (PQ) pool remained more reduced than that in the WT. The lower II in the dark (i.e. Fv00 /Fm00 ) in both genotypes after high light than after low light indicates that photoinhibition occurred under high-light illumination, and the extent of the reduction in Fv00 /Fm00 in psbS after high light was much greater than that in the WT. It has also been reported that continuous illumination with high light causes a greater rate of photoinhibition in npq4, an Arabidopsis mutant in which the psbS gene is inactive, than in the WT (Li et al. 2002, Sarvikas et al. 2006). Rough estimates of the energy allocation in PSII, calculated from the same data set as for Figs. 1A, B, are shown in Fig. 2. Under both low and high light, at 2 min L during the induction phase, II was almost the same regardless of the amount of PsbS, and the lower level of NPQ in psbS than in the WT was compensated by an increase in f,D (left panels of Fig. 2). In contrast, at 60 min L during the steady state phase, the absence of PsbS had no effect on NPQ (right panels of Fig. 2). Under
Effect of the PsbS level on energy allocation in PSII under naturally fluctuating light Typical diurnal changes in the quantum yields for electron transport and dissipations (Sept 13, 2012) as well as PAR are shown in Fig. 4. In the WT plant, f,D remained constant regardless of the changes in light intensity. In contrast, NPQ rapidly tracked light fluctuation and showed a pattern identical to the light intensity. This rapid fluctuation pattern of NPQ was also observed in field-grown WT rice (Ishida et al. 2014). On the other hand, NPQ in psbS showed a smaller fluctuation while f,D responded more sensitively to changes in light intensity compared to the WT. This indicates that different dissipation mechanisms may respond to rapidly fluctuating light in the presence and absence of PsbS. These dissipation patterns
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were consistent with those in regularly fluctuating light treatment, as shown in Fig. 3.
Efficiency of the daily utilization of absorbed light energy in PSII We estimated the total daily allocation of absorbed light energy in PSII by calculating the integrated flux of de-excitation processes over an entire day (Fig. 5). In this analysis, we divided NPQ into slow and fast, according to Kasajima et al. (2009) with a minor modification: 00 slow ¼ 1=Fm 20 min dark 1=Fm x Fs fast ¼ NPQ slow In this model, it has been proposed that NPQ-TD associated with photoinhibition (qI-type NPQ) is mainly reflected in slow and NPQ-TD induced by pH (qE-type NPQ) is included in fast (Kasajima et al. 2009). On a high-light day (Oct 3, 2012, integrated PAR, 12.8 molm2), slow in psbS was twice as high as that in the WT plant (Fig. 5A). This is in agreement with a previous report that PsbS-suppressed Arabidopsis is more susceptible to photoinhibition than the wild type plant (Li et al. 2002). In contrast, fast in psbS was approximately 40% lower than
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that in the WT. Overall, NPQ (= slow + fast) in the WT was about 30% higher than that in psbS. The decrease in NPQ in psbS was nearly compensated by an increase in f,D. As a result, the lower level of PsbS had little impact on II under naturally fluctuating light. On a low-light day (Sept 15, 2012, integrated PAR, 8.4 mol m2), there was little difference in slow between psbS and the WT (Fig. 5B). This suggests that photoinhibition had little effect on the daily energy allocation under low-light conditions. fast in psbS was significantly lower than that in the WT. Additionally, the difference in f,D between the WT and psbS on a low-light day was smaller than that on a highlight day. As a result, II in psbS was at the same level as in the WT. Overall, the energy allocation under naturally fluctuating light was close to that in the induction phase as shown in Fig. 2, in that total NPQ (=slow + fast) and f,D in psbS were lower and higher, respectively, than those in the WT.
Relationship between DII in the PsbS-suppressed plant and integrated PAR Data in Fig. 5 indicate that the deficiency in PsbS did not result in any changes in integrated daily efficiency of electron
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Fig. 2 Rough estimates of energy allocation in PSII depending on the phase of photosynthesis and the light condition calculated from the data set as for Fig. 1 in the WT and psbS plants. Induction and the steady state indicate after 2 min and 60 min of light exposure, respectively. (A) 420 mmol m2 s1. (B) 1150 mmol m2 s1.
Role of PsbS under naturally fluctuating light
plots converged toward the same region of the relative value of 1, while there was a slight negative correlation, especially in 2012. As shown previously, II was not affected by the PsbS level during induction (Ishida et al. 2011). We conclude that energy allocated to electron transport throughout the day was not affected by the content of PsbS under naturally fluctuating light conditions.
Discussion Molecular nature of (NPQ
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In this study, we divided the quantum yield of dissipation into two components, light-dependent regulative dissipation (NPQ) and the basal intrinsic decay of excited chlorophyll (f,D), according to Hendrickson et al. (2004). We demonstrated that NPQ in the induction phase was dependent on the presence of PsbS, while it was independent of PsbS in the steady state. This observation was consistent with a previous report in Arabidopsis, which showed the same extent of qE in both the presence and absence of PsbS protein in the steady state in isolated chloroplasts due to a similarly high trans-membrane proton gradient (Johnson and Ruban 2010, Johnson and Ruban 2011). Thus, the main portion of NPQ in the steady state could be ascribed to slowly-forming qE independent of PsbS.
Molecular nature of (f,D
Fig. 3 Changes in chlorophyll fluorescence parameters under regularly fluctuating light. II, NPQ, and f,D according to Hendrickson et al. (2004), and qP in the WT and psbS plants are shown. An actinic light of 125 mmol m2 s1 was applied at an initial 30 min from induction to the steady state followed by successive 5-min light-fluctuation treatments. The light intensity was changed in the order 420 (light grey bars), 125 (dark grey), 1150 (white) and 125 mmol m2 s1 as one cycle. A saturation pulse was applied 30, 100 and 300 sec after the light intensity was changed. The time from the end of the initial 30 min low-light exposure is shown. Means ± SE (n = 5).
transport regardless of the amount of daily light absorption. To verify this, we plotted the daily efficiency of electron transport in psbS, relative to that in the WT plant, against the integrated PAR in each plant (Fig. 6). As a result, almost all of the
This parameter represents the quantum yield of the basal decay of excited chlorophyll molecules accompanied by the release of heat and chlorophyll fluorescence (Hendrickson et al. 2004). Our result suggests that f,D might be associated with energy dissipation that results from photoinhibition at least during the steady state period. In the WT, f,D was almost independent of the applied light intensity at 60 min L during the steady state, while f,D in psbS at 1150 mmol m2 s1 was approximately 15% higher than that at 420 mmol m2 s1. In the npq4 mutant of Arabidopsis, an increase in photoinhibition was observed (Li et al. 2002). Thus, the relatively high f,D in psbS under high light can be explained by a relatively high photoinhibition as estimated from the low Fv00 /Fm00 value after high-light treatment; the Fv00 /Fm00 values 10 min after high light in the WT and psbS were 0.66 and 0.61, respectively (Fig. 1B).
Components of (NPQ In the experiment shown in Fig. 5, NPQ was separated into fast-relaxing (fast) and slowly-relaxing (slow) components based on a proposal by Kasajima et al. (2009) with a minor modification. Our data suggest that energy dissipation associated with qE (including both fast forming and slowly forming types) is mainly reflected in the quantum yield NPQ, and that associated with photoinhibition (qI) is closely related to the increase in f,D as discussed above. It has been reported that energy dissipation associated with photoinhibition is reflected
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Fig. 5 The allocation of daily total absorbed light energy in PSII. The ratios of the integrated values of the flow of electron transport (II) and thermal dissipations (slow, fast and f,D) to the daily total absorbed light energy in PSII are shown. II and f,D were calculated from the respective quantum yields recorded every 5 min, while slow and fast were determined hourly, as described in the text. A typical high-light condition day (A: Oct 3, 2012) and low-light condition day (B: Sept 15, 2012) are shown. The integrated values of the daily absorbed light energy in PSII on high- and low-light days were 12.8 and 8.4 mol quanta m2, respectively.
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Fig. 4 Typical diurnal fluctuation in the quantum yields on a sunny day. II, NPQ, and f,D according to Hendrickson et al. (2004) and PAR (Sept 13, 2012) are shown in the upper and lower panels, respectively. Values for Fs, Fm’, and PAR were recorded every 5 min from dawn to sunset. (A) WT. (B) psbS.
Role of PsbS under naturally fluctuating light
Effect of the suppressed level of PsbS on photosynthetic electron transport
in the sum of NPQ and f,D (Hendrickson et al. 2004). According to this idea, it may not be valid to express the quantum yield of energy loss associated with photoinhibition as slow. Thus, we postulate that the quantum yield slow might be an indicator of photoinhibition only when f,D is kept at the same level.
Sampling theorem of the diurnal analysis of energy allocation in PSII To track changes in the energy allocation of absorbed light energy in PSII, it is important to consider the sampling theorem. In this study, we adopted a 5-min interval for the quantum yield analysis of naturally fluctuating light conditions. This is because the more frequent application of a saturating pulse tended to elevate f,D in the WT, probably due to accumulated photoinhibitory effects of the pulses (data not shown). Apparently, changes in fluorescence parameters within a short time scale can be lost by our measurements. Indeed, a large difference in fluorescence parameters between psbS and the WT was observed at 2 min after the onset of illumination (Figs. 1A, B), suggesting that some important information could have been lost. However, it should be noted that once large changes occurred in NPQ and f,D upon rapid changes in light intensity, the levels of NPQ and f,D were largely maintained for at least 5 min (Figs. 1, 3). Furthermore, the large changes in NPQ in the WT and the large changes in f,D in psbS under artificial illumination shown in Figs. 1 and 3 were also clearly seen under naturally fluctuating light (Fig. 4). This suggests that the present assay methods can provide general information on the changes in NPQ and f,D. However, we have to admit that we underestimated the changes in NPQ and f,D.
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Fig. 6 Relationship between the daily efficiency of electron transport in psbS plants and the total absorbed light energy in PSII over an entire day. The fraction of II to the daily integrated value of absorbed light energy in PSII (II/ (PAR 0.42)) in psbS relative to that in the WT plant set as 1, is shown. Each point represents a single measurement. The results obtained in successive years (2012 and 2013) are shown.
The fluctuation treatment in Fig. 3 shows higher II in psbS than in the WT upon fluctuation from high to low light, probably due to the slow relaxation of PsbS-dependent NPQ in the WT. This means that II can be controlled, at least partially, by PsbS. In contrast, upon fluctuation from low to high light, the WT showed higher II than psbS, possibly due to the relaxation of both NPQ and excitation pressure (visualized as an increase in qP) during high-light illumination, which was found only in the WT and not in psbS. In addition, a rapid increase in f,D was found upon fluctuation from low to high light only in psbS, which can be another reason of lower II in psbS. In the evaluation of the efficiency of daily energy utilization in PSII, the electron transport flux in psbS can be identical to that in the WT regardless of the light condition (Fig. 6 and Supplementary Fig. S1). The results of the light fluctuation experiments in Fig. 3 suggests that this comparable electron transport flux between psbS and WT can be attributed, in part, to compensation between a higher II in psbS than in the WT upon fluctuation from high to low light and a lower II upon fluctuation from low to high light. The energy loss associated with photoinhibition in psbS was higher than that in the WT on a high-light day, but no significant difference was found between the two genotypes on a low-light day (Fig. 5). In summary, we did not find any light conditions in which psbS showed higher II than the WT. If we assume that the higher f,D in psbS resulted in greater photoinhibition and thus the suppression of II, the suppression or fine tuning of the amount of PsbS does not seem to improve the overall photosynthetic yield.
Materials and Methods Plants Both of the two psbS genes in Japonica rice cv. Nipponbare (Oryza sativa L.) were silenced by RNAi (Ishida et al. 2011). The chlorophyll content was estimated by SPAD-502 Plus (Konica Minolta, Osaka, Japan). The seedlings of WT and the T4 generation of PsbS-suppressed lines (psbS) were grown in a growth chamber under fluorescent lights with an intensity of 300 mmol m2 s1 at the top of the canopy, using a 14-h photoperiod, a constant temperature of 30 C, and a relative humidity (RH) of 60–80%. The 1-month-old seedlings were transplanted to 1-L pots. Fertilizers (0.83 g L1 KCl, 2.38 g L1 (NH4)2SO4 and 2.86 g L1 Ca(H2PO4)2H2O + CaSO4) were applied before transplantation. The pots were continuously waterlogged throughout the experiment. For the experiment under naturally fluctuating light, plants that had been cultured in a growth chamber for 1 month as described above were cultivated in the semi-open greenhouse of the Graduate School of Agriculture of Kyoto University, Japan, from July to October. Eight- to 14week-old plants were used for the experiment.
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Measurement of chlorophyll fluorescence in a growth chamber
Measurements of chlorophyll fluorescence in a greenhouse Methods for the repeated monitoring of chlorophyll fluorescence under naturally fluctuating light in the field have been reported previously (Ishida et al. 2014). At predawn, Fo and Fm were determined by measuring light exposure and a saturating pulse, respectively. During daytime, Fs and Fm0 were recorded by measuring light and a saturating pulse, respectively. The maximum fluorescence level after dark treatment during daytime (Fm00 ) was measured by the application of a saturating pulse. These chlorophyll fluorescence parameters were measured with a MONITORING-PAM fluorometer (Walz, Effeltrich, Germany). A Walz MINI-PAM fluorometer was used for the measurement of Fm00 . For this measurement, a section of the leaf was treated by darkness for 20 min before the application of a saturating pulse. With the use of the allocation model mentioned above, diurnal changes in energy allocation in PSII and PAR were monitored in upright leaves of the WT and psbS on the same day. From predawn to after sunset, Fs and Fm0 values and PAR were measured every 5 min, and Fm00 values were measured every hour. The energy flux in PSII (mmol m2 s1) was calculated from the quantum yield and PAR. For instance, the flux of electron transport JPSII was calculated by multiplying the estimated energy absorption in PSII (= PAR 0.84 0.5) by the quantum efficiencies II. In this equation, 0.84 indicates the assumed absorbance in leaves, and 0.5 is the assumed proportion of
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Supplementary data Supplementary data are available at PCP online.
Funding This work was supported in part by the Network of Centers of Carbon Dioxide Resource Studies in Plants (NC-CARP) and the project “Functional analysis of genes relevant to agriculturally important traits in rice genome” of the Ministry of Agriculture and Fisheries of Japan.
References Brooks, M.D., Sylak-Glassman, E.J., Fleming, G.R. and Niyogi, K.K. (2013) A thioredoxin-like/b-propeller protein maintains the efficiency of light harvesting in Arabidopsis. Proc. Natl Acad. Sci. USA 110: 2733–2740. Demmig-Adams, B. (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim. Biophys. Acta 1020: 1–24. Genty, B., Briantais, J.-M. and Baker, N.R. (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990: 87–92. Goral, T.K., Johnson, M.P., Duffy, C.D.P., Brain, A.P.R., Ruban, A.V. and Mullineaux, C.W. (2002) Light-harvesting antenna composition controls the macromolecular organization, regulation and dynamics of thylakoid membranes in Arabidopsis thaliana. Plant J. 69: 289–301. Goral, T.K., Johnson, M.P., Duffy, C.D.P., Brain, A.P.R., Ruban, A.V. and Mullineaux, C.W. (2012) Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis. Plant J. 69: 289–301. Hendrickson, L., Furbank, R.T. and Chow, W.S. (2004) A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence. Photosynth. Res. 82: 73–81. Horton, P., Ruban, A.V. and Walters, R.G. (1996) Regulation of light harvesting in green plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 655–684. Horton, P., Ruban, A.V. and Wentworth, M. (2000) Allosteric regulation of the light harvesting system of photosystem II. Philos. Trans. R. Soc. Lond. B. 355: 1361–1370. Horton, P. and Ruban, A.V. (1992) Regulation of photosystem II. Photosynth. Res. 34: 375–385. Ishida, S., Morita, K., Kishine, M., Takabayashi, A., Murakami, R., Takeda, S. et al. (2011) Allocation of absorbed light energy in PSII
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Quenching analysis of chlorophyll fluorescence (Schreiber et al. 1986) was performed with a MONITORING-PAM fluorometer (Walz, Effeltrich, Germany). For the experiment under regulated actinic light, leaves were dark-adapted in the growth chamber for over 1 h prior to measurement. Plants were then moved to a laboratory adjacent to the growth chamber for measurement. The minimum fluorescence level in the absence of NPQ (Fo) was determined by measuring light illumination in the dark. The steady-state fluorescence level (Fs) was recorded under actinic light illumination. The maximum fluorescence level in the absence of NPQ (Fm) and the maximum fluorescence level in the presence of NPQ (Fm0 ) were measured by the application of a saturating pulse for 0.6 s. The minimum fluorescence level in the presence of NPQ (Fo0 ) was calculated from Fm, Fo and Fm0 , as 1/(1/Fo 1/Fm 1/Fm0 ) (Oxborough and Baker 1997). The quantum yield of photochemistry in PSII during light illumination (II) was calculated as (Fm0 Fs)/Fm0 . Total dissipation (1 II) was divided into two components, i.e. basal constitutive decay of excited chlorophyll (f,D), calculated as Fs/Fm, and light-dependent regulative dissipation (NPQ), calculated as Fs/Fm0 – Fs/Fm, as proposed by Hendrickson et al. (2004). Photochemical quenching (qP) was calculated as (Fm0 Fs)/(Fm0 Fo0 ).
absorbed light energy based on the assumption that absorbed light energy is distributed equally into PSII and PSI (Melis et al. 1987, Weis and Lechtenberg 1989). The daily integrated flux of each de-excitation pathway (II, NPQ or f,D) was calculated from the respective energy flux values as shown previously (Ishida et al. 2014). The energy flux of each process (JII, JNPQ or Jf,D) was integrated over an entire day, assuming that the absorbed light level did not change within 5 min intervals.
Role of PsbS under naturally fluctuating light
Noctor, G., Rees, D., Young, A. and Horton, P. (1991) The relationship between zeaxanthin, energy-dependent quenching of chlorophyll fluorescence and transthylakoid pH-gradient in isolated chloroplasts. Biochim. Biophys. Acta 1057: 320–330. Oxborough, K. and Baker, N.R. (1997) Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components—calculation of qP and Fv0 /Fm0 without measuring Fo0 . Photosynth. Res. 54: 135–142. Quick, W.P. and Stitt, M. (1989) An examination of factors contributing to non-photochemical quenching of chlorophyll fluorescence in barley leaves. Biochim. Biophys. Acta 977: 287–296. Rees, D., Young, A., Noctor, G., Britton, G. and Horton, P. (1989) Enhancement of the pH-dependent dissipation of excitation energy in spinach chloroplasts by light-activation; correlation with the synthesis of zeaxanthin. FEBS Lett. 256: 85–90. Rees, D., Noctor, G., Ruban, A.V., Crofts, J., Young, A. and Horton, P. (1992) pH dependent chlorophyll fluorescence quenching in spinach thylakoids from light-treated or dark-adapted leaves. Photosynth. Res. 31: 11–19. Ruban, A.V. and Horton, P. (1995) An investigation of the sustained component of nonphotochemical quenching of chlorophyll fluorescence in isolated chloroplasts and leaves of spinach. Plant Physiol. 108: 721–726. Ruban, A.V., Young, A.J. and Horton, P. (1993) Induction of nonphotochemical energy dissipation and absorbance changes in leaves. Evidence for changes in the state of the light harvesting system of photosystem II in vivo. Plant Physiol. 102: 741–750. Sarvikas, P., Hakala, M., Pa¨tsikka¨, E., Tyystja¨rvi, T. and Tyystja¨rvi, E. (2006) Action spectrum of photoinhibition in leaves of wild type and npq12 and npq41 mutants of Arabidopsis thaliana. Plant Cell Physiol. 47: 391–400. Schreiber, U., Schliwa, U. and Bilger, W. (1986) Continuous recording of photochemical and non-photochemical chlorophyll quenching with a new type of modulation fluorometer. Photosynth. Res. 10: 51–62. Walters, R.G. and Horton, P. (1991) Resolution of components of nonphotochemical chlorophyll fluorescence quenching in barley leaves. Photosynth. Res. 27: 121–133. Walters, R.G. and Horton, P. (1993) Theoretical assessment of alternative mechanisms for non-photochemical quenching of PSII fluorescence in barley leaves. Photosynth. Res. 36:2, 119–139. Weis, E. and Lechtenberg, D. (1989) Fluorescence analysis during steady-state photosynthesis. Philos. Trans. R. Soc. B: Biol. Sci. 323: 253–268. Zhu, X.G., Ort, D.R., Whitmarsh, J. and Long, S.P. (2004) The slow reversibility of photosystem II thermal energy dissipation on transfer from high to low light may cause large losses in carbon gain by crop canopies: a theoretical analysis. J. Exp. Bot. 55: 1167–1175.
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to thermal dissipation in the presence or absence of PsbS subunits in rice. Plant Cell Physiol. 52: 1822–1831. Ishida, S., Uebayashi, N., Tazoe, Y., Ikeuchi, M., Homma, K., Sato, F. et al. (2014) Diurnal and developmental changes in energy allocation of absorbed light at PSII in field-grown rice. Plant Cell Physiol. 55: 171–182. Johnson, M.P. and Ruban, A.V. (2010) Arabidopsis plants lacking PsbS protein possess photoprotective energy dissipation. Plant J. 61: 283–289. Johnson, M.P. and Ruban, A.V. (2011) Restoration of rapidly reversible photoprotective energy dissipation in the absence of PsbS protein by enhanced delta pH. J. Biol. Chem. 286: 19973–19981. Kasajima, I., Takahara, K., Kawai-Yamada, M. and Uchimiya, H. (2009) Estimation of the relative sizes of rate constants for chlorophyll deexcitation processes through comparison of inverse fluorescence intensities. Plant Cell Physiol. 50: 1600–1616. Kiss, A.Z., Ruban, A.V. and Horton, P. (2008) The PsbS protein controls the organization of the photosystem II antenna in higher plant thylakoid membranes. J. Biol. Chem. 283: 3972–3978. Krause, G.H. and Weis, E. (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 313–349. Ku¨lheim, C. and Jansson, S. (2005) What leads to reduced fitness in non-photochemical quenching mutant? Physiol. Plant 125: 202–211. Ku¨lheim, C., Agren, J. and Jansson, S. (2002) Rapid regulation of light harvesting and plant fitness in the field. Science 297: 91–93. Li, X.-P., Mu¨ller-Moule´, P., Gilmore, A.M. and Niyogi, K. (2002) PsbSdependent enhancement of feedback de-excitation protects photosystem II from photoinhibition. Proc. Natl. Acad. Sci. USA 99: 15222–15227. Maxwell, K. and Johnson, G.N. (2000) Chlorophyll fluorescence—a practical guide. J. Exp. Bot. 345: 659–668. Melis, M., Spangfort, M. and Andersson, B. (1987) Light-absorption and electron transport balance between Photosystem II and Photosystem I in spinach chloroplasts. Photochem. Photobiol. 45: 129–136. Mu¨ller, P., Li, X.-P. and Niyogi, K.K. (2001) Non-photochemical quenching. A response to excess light energy. Plant Physiol. 125: 1558–1566. Nilkens, M., Kress, E., Lambrev, P., Miloslavina, Y., Mu¨ller, M., Holzwarth, A.R. et al. (2010) Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis. Biochim. Biophys. Acta 1797: 466–475. Niyogi, K.K. (1999) Photoprotection revisited: Genetic and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 333–359.
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The PsbS protein plays an important role in dissipating excess light energy as heat in photosystem II (PSII). However, the physiological importance of PsbS under naturally fluctuating light has not been quantitatively estimated. Here we investigated energy allocation in PSII in PsbSsuppressed rice transformants (psbS) under both naturally fluctuating and constant light conditions. Under constant light, PsbS was essential for inducing the rapid formation of light-inducible thermal dissipation (NPQ), which consequently suppressed the rapid formation of basal intrinsic decay (f,D), while the quantum yield of electron transport (II) did not change. In the steady state phase, the difference between the wild type (WT) and psbS was minimized. Under regularly fluctuating light, the reduced PsbS resulted in higher II upon the transition from high light to low light and in lower II upon the transition from low light to high light, indicating that II was, to some extent, controlled by PsbS. Under naturally fluctuating light in a greenhouse, rapid changes in II were compensated by NPQ in the WT, but by f,D in psbS. As a consequence, a significantly lower NPQ integrated NPQ over a whole day) and higher f,D were found in psbS. Furthermore, thermal dissipation associated with photoinhibtion was enhanced in psbS. These results suggest that PsbS plays an important role in photoprotective process at the induction phase of photosynthesis as well as under field conditions. The physiological relevance of PsbS as a photoprotection mechanism and the identities of NPQ and f,D are discussed. Keywords: Energy allocation in PSII Naturally fluctuating light Non-photochemical quenching (NPQ) PsbS Thermal dissipation of absorbed light energy. Abbreviations: pH, transthylakoid pH gradient; psbS, PsbS-suppressed rice transformants; Fm (Fm0 , Fm00 ), the maximum fluorescence level achieved with a saturating pulse in the absence of NPQ (or in the presence of NPQ, or after a brief dark treatment during daytime); Fo (Fo0 ), the minimum fluorescence level achieved under a measuring light in the dark (or in the presence of NPQ); Fs, steady-state fluorescence level
in the light; fast (fast), the quantum yield (or daily integrated flux) of the fast-relaxing portion of light-inducible dissipation in the Kasajima model; f,D (f,D), the quantum yield (or daily integrated flux) of basal dissipation in the Hendrickson model; NPQ (NPQ), the quantum yield (or daily integrated flux) of light-inducible dissipation in the Hendrickson model; slow (slow), the quantum yield (or daily integrated flux) of the slowly-relaxing portion of light-inducible dissipation in the Kasajima model; II (II), the quantum rate (or daily integrated flux) of absorbed light energy in PSII allocated to electron transport; LHC, light-harvesting chlorophyll-protein complex; NPQ, non-photochemical quenching; PAM, pulse amplitude modulation; PAR, photosynthetically active radiation; PQ, plastoquinone; qE, energy quenching; qI, photoinhibitory quenching; qP, photochemical quenching; qT, state transition quenching; RH, relative humidity; SE, standard error; PAR, integrated PAR over an entire day; TD, thermal dissipation; VDE, violaxanthin deepoxidase; WT, wild type rice.
Introduction Under natural conditions, changes in light intensity follow diurnal and seasonal cycles and irradiance from the sun can be blocked by clouds, trees and buildings. Moreover, in a threedimensional canopy, leaves are exposed to fluctuating light (Zhu et al. 2004). Plants have evolved an efficient light-harvesting system in response to these fluctuating light conditions. The allocation of absorbed light energy in photosystem II (PSII) is highly controlled and some of the excess light energy is dissipated as heat, which can be monitored as non-photochemical quenching (NPQ) of chlorophyll fluorescence (Horton et al. 1996, Niyogi 1999, Maxwell and Johnson 2000). NPQ is the sum of at least three different mechanisms, i.e. qE (energy quenching), qT (state transition) and qI (photoinhibitory quenching), which are defined on the basis of their relaxation kinetics (Quick and Stitt 1989, Walters and Horton 1991). A dominant part of thermal dissipation that is associated with NPQ (NPQ-TD), qE-associated thermal dissipation (qE-TD),
Plant Cell Physiol. 55(7): 1286–1295 (2014) doi:10.1093/pcp/pcu069, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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Special Focus Issue – Regular Paper
Physiological Functions of PsbS-dependent and PsbS-independent NPQ under Naturally Fluctuating Light Conditions
Role of PsbS under naturally fluctuating light
these parameters were calculated from the chlorophyll fluorescence determined during the induction period. Thus, it is not yet clear how the mechanism of energy allocation changes under prolonged or fluctuating light illumination. The aim of this study was to estimate the physiological importance of PsbS under naturally fluctuating light conditions. We first obtained data on energy allocation in PSII, using the model proposed by Hendrickson et al. (2004), in PsbS-suppressed rice transformants under constant or regularly fluctuating light to understand the basic changes in energy allocation in the presence and absence of PsbS. Based on the data obtained above, we then analyzed the diurnal changes in energy allocation in plants cultivated in a semi-open greenhouse to reveal the mechanism of energy allocation under naturally fluctuating light conditions. As a result, drastic changes in energy allocation in PSII were induced depending on the amount of PsbS and the duration of illumination. Thus, we could confirm that PsbS protein plays an important role in the photoprotective process and its content is nearly optimized to adapt to natural conditions.
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protects reaction centers of PSII from photoinhibition. As shown previously, qE-type energy dissipation is fast-forming and rapidly reversible in the dark and is induced by a proton gradient across the thylakoid membrane (pH) caused by excess light (Krause and Weis 1991, Horton and Ruban 1992, Horton et al. 1996, Mu¨ller et al. 2001). PSII subunit S (PsbS) protein and violaxanthin de-epoxidase (VDE), which converts violaxanthin to zeaxanthin, are associated with this quenching process and are protonated at a low pH in the thylakoid lumen (Demmig-Adams 1990, Horton et al. 1996). The de-epoxidation state of xanthophyll pigments may regulate the affinity of LHCII for protons (Rees et al. 1992, Horton et al. 2000). The PsbS protein has been shown to enhance the dynamics of thylakoid membranes by reducing the ordering and increasing the fluidity of protein organization, which accelerates the reorganization and aggregation of LHCII (Goral et al. 2002, Kiss et al. 2008, Goral et al. 2012). However, a qE-type quenching process is an intrinsic property of LHCII, and thus can be induced independently of VDE or PsbS protein (Rees et al. 1989, Noctor et al. 1991, Johnson and Ruban 2010, Johnson and Ruban 2011). Therefore, PsbS facilitates reorganization of LHCII, which results in acceleration of qE induction. The most slowly relaxing component of NPQ, qI, supposedly reflects photoinhibitory quenching. However, there is no clear relationship between the extent of qI and photodamage to the PSII reaction center itself (Walters and Horton 1993). A recent study has shown that the thioredoxin-like/b-propeller protein SOQ1 could prevent the formation of a slowly reversible NPQ, and there might be other uncharacterized quenching pathways (Brooks et al. 2013). Additionally, some part of the NPQ induced by pH across thylakoid membranes, which is the same mechanism as in qE-type energy dissipation, had slow induction and relaxation kinetics, and thus was monitored as qI. This slow kinetics was associated with zeaxanthin, trapped protons and aggregated LHCII (Ruban et al. 1993, Ruban and Horton 1995, Nilkens et al. 2010). To maximize photosynthetic productivity under naturally fluctuating light, each component of NPQ-TD should be identified and the photoprotective process should be optimized. A modeling analysis has demonstrated that slowly-reversible qI-type energy dissipation prevented rapid recovery of the CO2 assimilation rate and reduced canopy photosynthesis (Zhu et al. 2004). Most studies on PsbS protein have been conducted under artificial and continuous light illumination, and no clear phenotype with respect to growth has been reported. However, a mutant of Arabidopsis thaliana npq4 that lacks PsbS protein showed a significant decrease in plant growth under field conditions and when grown under fluctuating light in a growth chamber (Ku¨lheim et al. 2002, Ku¨lheim and Jansson 2005). We previously showed, using the light energy allocation model in PSII proposed by Hendrickson et al. (2004), that the suppression of PsbS protein in rice resulted in a decrease in light-inducible dissipation (NPQ) and a consequent increase in basal intrinsic decay (f,D), whereas the allocation of energy to electron transport (II) did not change over a wide range of light intensities (Ishida et al. 2011). However,
Results Properties of "psbS We used a transgenic line of rice in which both of the two psbS genes had been silenced by RNAi (psbS; Ishida et al. 2011). At the T2 generation, quantitative reverse transcription-PCR (RTPCR) analysis revealed that the amounts of both gene transcripts were significantly reduced in this transgenic line, and no PsbS protein was detected by an immunoblot analysis (Ishida et al. 2011). The T4 generation of the transformant used in the present study also had no detectable PsbS protein by an immunoblot analysis (data not shown). The chlorophyll contents estimated by a SPAD chlorophyll meter in the 9-week-old wild type (WT) and psbS cultivated in a semi-open greenhouse were 42.6 ± 0.7 and 43.2 ± 0.7, respectively (means ± SE, n = 5). The aboveground dry weights of a plant grown in the greenhouse after the maturation of seeds in the WT and psbS were 48.9 ± 2.6 g and 41.1 ± 3.4 g, respectively, (means ± SE, n = 6) in the 2013 season.
Induction kinetics of (II, (NPQ and (f,D under constant light illumination We first investigated the time course of changes in II, NPQ and f,D under exposure to constant light (Figs. 1A, B). The parameter II represents the effective quantum yield of electron transport through PSII (Genty et al. 1989). The parameters NPQ and f,D represent the quantum yield of lightinducible energy dissipation and that of the intrinsic decay of excited chlorophylls, respectively (Hendrickson et al. 2004). There was no significant difference in Fv/Fm between the two genotypes in the dark-adapted state before light illumination (WT: 0.836 ± 0.001, psbS: 0.837 ± 0.001, means ± SE, n = 5).
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At 420 mmol m2 s1, NPQ in the WT plants was highest 2 min after the onset of actinic illumination (2 min L) and then gradually decreased to the steady state level at 60 min after the onset of light exposure (60 min L; Fig. 1A). In contrast, NPQ in psbS increased gradually and reached the steady state, at which the yield was almost identical to that in the WT. On the other hand, f,D was highest in both genotypes at 2 min L during the induction phase, and f,D in psbS was about
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two-fold greater than that in the WT. Subsequently, f,D gradually decreased after a rapid decrease at 4 min after the onset of illumination (4 min L) in both genotypes. The difference between them was minimized and the yield nearly returned to the darkadapted value in both genotypes at 60 min L. The quantum yield II showed an almost identical pattern regardless of the PsbS level; it was lowest at 2 min L, and then slowly increased to reach a steady state level at 60 min L after a
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Fig. 1 Changes in chlorophyll fluorescence parameters during induction from dark-adapted states. The quantum yields of light-inducible dissipation (NPQ), basal and constitutive dissipation (f,D) and electron transport (II) according to Hendrickson et al. (2004), and photochemical quenching (qP) were determined in both the wild type (WT) and the transgenic T4 plant in which the PsbS level was reduced (psbS). Leaves were dark-adapted for over 1 h before the measurement of Fo and Fm, and then actinic light of 420 (A) or 1150 mmol m2 s1 (B) was applied. After illumination for 60 min, the actinic light was turned off and leaves were kept in the dark for 12 min. Means ± SE (n = 5).
Role of PsbS under naturally fluctuating light
low light, II and f,D were at the same level in both genotypes at 60 min L (Fig. 2A). However, under high light, II in psbS was reduced at 60 min L, and this reduction was compensated by an increase in f,D (Fig. 2B). This reduced II and increased f,D may be associated with photoinhibition under high light, as discussed below.
Energy allocation in PSII under regularly fluctuating light To obtain insight into the energy allocation in PSII under fluctuating light, we conducted a series of experiments using low-, moderate- and high-light fluctuation after photosynthesis had reached a steady state (Fig. 3). Light-treated plants were subjected to light fluctuation where the light intensity was changed every 5 min, in the order 420, 125, 1150 and 125 mmol m2 s1 as one cycle. In this light fluctuation treatment, NPQ in the WT was much greater than that in psbS throughout light exposure. On the other hand, while f,D remained at the dark-treated level in the WT plant, a large increase was seen with an increase in light intensity in psbS. The changes in II under fluctuating light in psbS showed a characteristic pattern. Under moderate and high light (420 and 1150 mmol m2 s1), there was a 10–20% reduction in psbS, as was found in the induction phase upon the transition from dark to light shown in Figs. 1A, B. During light fluctuation treatment, the value of qP in psbS was consistently lower than that in the WT, and the difference between the two genotypes significantly increased under moderate and high light. This indicates that a higher reduction state of the PQ pool in psbS could reduce II under this condition. However, under exposure to low light (125 mmol m2 s1) just after moderate light illumination, there was no significant difference in II between the two genotypes. Furthermore, under low light just after high-light treatment (dark grey bar after the white bar in Fig. 3), II in psbS was higher 30 seconds after the light shift and gradually settled to a level comparable to that in the WT. This pattern in II could be observed even after the light fluctuation treatment was repeated several times.
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rapid increase between 2 to 4 min L. At 2 min L, II in psbS and the WT were nearly indistinguishable. Subsequently, II in the WT was slightly higher than that in the psbS, but the difference between the two genotypes was minimized at the steady state (60 min L). With regard to photochemical quenching (qP), the WT showed a significantly higher qP value than psbS at 2 min L, but the difference between the two genotypes became smaller at 60 min L (Fig. 1A). After the actinic illumination was turned off, there was no significant difference in these parameters between the two genotypes. To evaluate photoinhibition, we used the Fv00 /Fm00 value after a brief dark treatment instead of Fv/Fm, which is determined after an overnight dark treatment. The identical yield of II in the dark (i.e. Fv00 /Fm00 ) indicates that photoinhibition did not occur under this low-light illumination. At 1150 mmol m2 s1, the levels of NPQ in the WT and psbS at 2 min L were approximately 20% and 60% higher than the peak observed at 420 mmol m2 s1, respectively (Fig. 1B). However, under this high-light level, NPQ in the WT was not highest at 2 min L, and gradually increased to form a gentle peak around 10 min L. By contrast, in psbS, it gradually increased to a steady state level at 60 min L. The highest f,D in the first 2 min was about 20–30% higher than that at 420 mmol m2 s1 in both genotypes. After reaching the maximum at 2 min L, the yield in the WT decreased and returned to the dark-adapted value as in the pattern at 420 mmol m2 s1, while psbS showed an approximately 15% higher yield (P < 0.05) than the WT plant. The difference in f,D between the two genotypes was greater than that at 420 mmol m2 s1. The yield of II at 1150 mmol m2 s1 was approximately 40% lower than that at 420 mmol m2 s1 in both genotypes. At 2 min L during the induction phase, there was no significant difference between the two genotypes, after which psbS showed about 10% lower II than in the WT throughout the illumination period. Additionally, qP in psbS was significantly lower than that in the WT throughout light illumination, suggesting that the plastoquinone (PQ) pool remained more reduced than that in the WT. The lower II in the dark (i.e. Fv00 /Fm00 ) in both genotypes after high light than after low light indicates that photoinhibition occurred under high-light illumination, and the extent of the reduction in Fv00 /Fm00 in psbS after high light was much greater than that in the WT. It has also been reported that continuous illumination with high light causes a greater rate of photoinhibition in npq4, an Arabidopsis mutant in which the psbS gene is inactive, than in the WT (Li et al. 2002, Sarvikas et al. 2006). Rough estimates of the energy allocation in PSII, calculated from the same data set as for Figs. 1A, B, are shown in Fig. 2. Under both low and high light, at 2 min L during the induction phase, II was almost the same regardless of the amount of PsbS, and the lower level of NPQ in psbS than in the WT was compensated by an increase in f,D (left panels of Fig. 2). In contrast, at 60 min L during the steady state phase, the absence of PsbS had no effect on NPQ (right panels of Fig. 2). Under
Effect of the PsbS level on energy allocation in PSII under naturally fluctuating light Typical diurnal changes in the quantum yields for electron transport and dissipations (Sept 13, 2012) as well as PAR are shown in Fig. 4. In the WT plant, f,D remained constant regardless of the changes in light intensity. In contrast, NPQ rapidly tracked light fluctuation and showed a pattern identical to the light intensity. This rapid fluctuation pattern of NPQ was also observed in field-grown WT rice (Ishida et al. 2014). On the other hand, NPQ in psbS showed a smaller fluctuation while f,D responded more sensitively to changes in light intensity compared to the WT. This indicates that different dissipation mechanisms may respond to rapidly fluctuating light in the presence and absence of PsbS. These dissipation patterns
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were consistent with those in regularly fluctuating light treatment, as shown in Fig. 3.
Efficiency of the daily utilization of absorbed light energy in PSII We estimated the total daily allocation of absorbed light energy in PSII by calculating the integrated flux of de-excitation processes over an entire day (Fig. 5). In this analysis, we divided NPQ into slow and fast, according to Kasajima et al. (2009) with a minor modification: 00 slow ¼ 1=Fm 20 min dark 1=Fm x Fs fast ¼ NPQ slow In this model, it has been proposed that NPQ-TD associated with photoinhibition (qI-type NPQ) is mainly reflected in slow and NPQ-TD induced by pH (qE-type NPQ) is included in fast (Kasajima et al. 2009). On a high-light day (Oct 3, 2012, integrated PAR, 12.8 molm2), slow in psbS was twice as high as that in the WT plant (Fig. 5A). This is in agreement with a previous report that PsbS-suppressed Arabidopsis is more susceptible to photoinhibition than the wild type plant (Li et al. 2002). In contrast, fast in psbS was approximately 40% lower than
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that in the WT. Overall, NPQ (= slow + fast) in the WT was about 30% higher than that in psbS. The decrease in NPQ in psbS was nearly compensated by an increase in f,D. As a result, the lower level of PsbS had little impact on II under naturally fluctuating light. On a low-light day (Sept 15, 2012, integrated PAR, 8.4 mol m2), there was little difference in slow between psbS and the WT (Fig. 5B). This suggests that photoinhibition had little effect on the daily energy allocation under low-light conditions. fast in psbS was significantly lower than that in the WT. Additionally, the difference in f,D between the WT and psbS on a low-light day was smaller than that on a highlight day. As a result, II in psbS was at the same level as in the WT. Overall, the energy allocation under naturally fluctuating light was close to that in the induction phase as shown in Fig. 2, in that total NPQ (=slow + fast) and f,D in psbS were lower and higher, respectively, than those in the WT.
Relationship between DII in the PsbS-suppressed plant and integrated PAR Data in Fig. 5 indicate that the deficiency in PsbS did not result in any changes in integrated daily efficiency of electron
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Fig. 2 Rough estimates of energy allocation in PSII depending on the phase of photosynthesis and the light condition calculated from the data set as for Fig. 1 in the WT and psbS plants. Induction and the steady state indicate after 2 min and 60 min of light exposure, respectively. (A) 420 mmol m2 s1. (B) 1150 mmol m2 s1.
Role of PsbS under naturally fluctuating light
plots converged toward the same region of the relative value of 1, while there was a slight negative correlation, especially in 2012. As shown previously, II was not affected by the PsbS level during induction (Ishida et al. 2011). We conclude that energy allocated to electron transport throughout the day was not affected by the content of PsbS under naturally fluctuating light conditions.
Discussion Molecular nature of (NPQ
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In this study, we divided the quantum yield of dissipation into two components, light-dependent regulative dissipation (NPQ) and the basal intrinsic decay of excited chlorophyll (f,D), according to Hendrickson et al. (2004). We demonstrated that NPQ in the induction phase was dependent on the presence of PsbS, while it was independent of PsbS in the steady state. This observation was consistent with a previous report in Arabidopsis, which showed the same extent of qE in both the presence and absence of PsbS protein in the steady state in isolated chloroplasts due to a similarly high trans-membrane proton gradient (Johnson and Ruban 2010, Johnson and Ruban 2011). Thus, the main portion of NPQ in the steady state could be ascribed to slowly-forming qE independent of PsbS.
Molecular nature of (f,D
Fig. 3 Changes in chlorophyll fluorescence parameters under regularly fluctuating light. II, NPQ, and f,D according to Hendrickson et al. (2004), and qP in the WT and psbS plants are shown. An actinic light of 125 mmol m2 s1 was applied at an initial 30 min from induction to the steady state followed by successive 5-min light-fluctuation treatments. The light intensity was changed in the order 420 (light grey bars), 125 (dark grey), 1150 (white) and 125 mmol m2 s1 as one cycle. A saturation pulse was applied 30, 100 and 300 sec after the light intensity was changed. The time from the end of the initial 30 min low-light exposure is shown. Means ± SE (n = 5).
transport regardless of the amount of daily light absorption. To verify this, we plotted the daily efficiency of electron transport in psbS, relative to that in the WT plant, against the integrated PAR in each plant (Fig. 6). As a result, almost all of the
This parameter represents the quantum yield of the basal decay of excited chlorophyll molecules accompanied by the release of heat and chlorophyll fluorescence (Hendrickson et al. 2004). Our result suggests that f,D might be associated with energy dissipation that results from photoinhibition at least during the steady state period. In the WT, f,D was almost independent of the applied light intensity at 60 min L during the steady state, while f,D in psbS at 1150 mmol m2 s1 was approximately 15% higher than that at 420 mmol m2 s1. In the npq4 mutant of Arabidopsis, an increase in photoinhibition was observed (Li et al. 2002). Thus, the relatively high f,D in psbS under high light can be explained by a relatively high photoinhibition as estimated from the low Fv00 /Fm00 value after high-light treatment; the Fv00 /Fm00 values 10 min after high light in the WT and psbS were 0.66 and 0.61, respectively (Fig. 1B).
Components of (NPQ In the experiment shown in Fig. 5, NPQ was separated into fast-relaxing (fast) and slowly-relaxing (slow) components based on a proposal by Kasajima et al. (2009) with a minor modification. Our data suggest that energy dissipation associated with qE (including both fast forming and slowly forming types) is mainly reflected in the quantum yield NPQ, and that associated with photoinhibition (qI) is closely related to the increase in f,D as discussed above. It has been reported that energy dissipation associated with photoinhibition is reflected
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Fig. 5 The allocation of daily total absorbed light energy in PSII. The ratios of the integrated values of the flow of electron transport (II) and thermal dissipations (slow, fast and f,D) to the daily total absorbed light energy in PSII are shown. II and f,D were calculated from the respective quantum yields recorded every 5 min, while slow and fast were determined hourly, as described in the text. A typical high-light condition day (A: Oct 3, 2012) and low-light condition day (B: Sept 15, 2012) are shown. The integrated values of the daily absorbed light energy in PSII on high- and low-light days were 12.8 and 8.4 mol quanta m2, respectively.
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Fig. 4 Typical diurnal fluctuation in the quantum yields on a sunny day. II, NPQ, and f,D according to Hendrickson et al. (2004) and PAR (Sept 13, 2012) are shown in the upper and lower panels, respectively. Values for Fs, Fm’, and PAR were recorded every 5 min from dawn to sunset. (A) WT. (B) psbS.
Role of PsbS under naturally fluctuating light
Effect of the suppressed level of PsbS on photosynthetic electron transport
in the sum of NPQ and f,D (Hendrickson et al. 2004). According to this idea, it may not be valid to express the quantum yield of energy loss associated with photoinhibition as slow. Thus, we postulate that the quantum yield slow might be an indicator of photoinhibition only when f,D is kept at the same level.
Sampling theorem of the diurnal analysis of energy allocation in PSII To track changes in the energy allocation of absorbed light energy in PSII, it is important to consider the sampling theorem. In this study, we adopted a 5-min interval for the quantum yield analysis of naturally fluctuating light conditions. This is because the more frequent application of a saturating pulse tended to elevate f,D in the WT, probably due to accumulated photoinhibitory effects of the pulses (data not shown). Apparently, changes in fluorescence parameters within a short time scale can be lost by our measurements. Indeed, a large difference in fluorescence parameters between psbS and the WT was observed at 2 min after the onset of illumination (Figs. 1A, B), suggesting that some important information could have been lost. However, it should be noted that once large changes occurred in NPQ and f,D upon rapid changes in light intensity, the levels of NPQ and f,D were largely maintained for at least 5 min (Figs. 1, 3). Furthermore, the large changes in NPQ in the WT and the large changes in f,D in psbS under artificial illumination shown in Figs. 1 and 3 were also clearly seen under naturally fluctuating light (Fig. 4). This suggests that the present assay methods can provide general information on the changes in NPQ and f,D. However, we have to admit that we underestimated the changes in NPQ and f,D.
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Fig. 6 Relationship between the daily efficiency of electron transport in psbS plants and the total absorbed light energy in PSII over an entire day. The fraction of II to the daily integrated value of absorbed light energy in PSII (II/ (PAR 0.42)) in psbS relative to that in the WT plant set as 1, is shown. Each point represents a single measurement. The results obtained in successive years (2012 and 2013) are shown.
The fluctuation treatment in Fig. 3 shows higher II in psbS than in the WT upon fluctuation from high to low light, probably due to the slow relaxation of PsbS-dependent NPQ in the WT. This means that II can be controlled, at least partially, by PsbS. In contrast, upon fluctuation from low to high light, the WT showed higher II than psbS, possibly due to the relaxation of both NPQ and excitation pressure (visualized as an increase in qP) during high-light illumination, which was found only in the WT and not in psbS. In addition, a rapid increase in f,D was found upon fluctuation from low to high light only in psbS, which can be another reason of lower II in psbS. In the evaluation of the efficiency of daily energy utilization in PSII, the electron transport flux in psbS can be identical to that in the WT regardless of the light condition (Fig. 6 and Supplementary Fig. S1). The results of the light fluctuation experiments in Fig. 3 suggests that this comparable electron transport flux between psbS and WT can be attributed, in part, to compensation between a higher II in psbS than in the WT upon fluctuation from high to low light and a lower II upon fluctuation from low to high light. The energy loss associated with photoinhibition in psbS was higher than that in the WT on a high-light day, but no significant difference was found between the two genotypes on a low-light day (Fig. 5). In summary, we did not find any light conditions in which psbS showed higher II than the WT. If we assume that the higher f,D in psbS resulted in greater photoinhibition and thus the suppression of II, the suppression or fine tuning of the amount of PsbS does not seem to improve the overall photosynthetic yield.
Materials and Methods Plants Both of the two psbS genes in Japonica rice cv. Nipponbare (Oryza sativa L.) were silenced by RNAi (Ishida et al. 2011). The chlorophyll content was estimated by SPAD-502 Plus (Konica Minolta, Osaka, Japan). The seedlings of WT and the T4 generation of PsbS-suppressed lines (psbS) were grown in a growth chamber under fluorescent lights with an intensity of 300 mmol m2 s1 at the top of the canopy, using a 14-h photoperiod, a constant temperature of 30 C, and a relative humidity (RH) of 60–80%. The 1-month-old seedlings were transplanted to 1-L pots. Fertilizers (0.83 g L1 KCl, 2.38 g L1 (NH4)2SO4 and 2.86 g L1 Ca(H2PO4)2H2O + CaSO4) were applied before transplantation. The pots were continuously waterlogged throughout the experiment. For the experiment under naturally fluctuating light, plants that had been cultured in a growth chamber for 1 month as described above were cultivated in the semi-open greenhouse of the Graduate School of Agriculture of Kyoto University, Japan, from July to October. Eight- to 14week-old plants were used for the experiment.
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Measurement of chlorophyll fluorescence in a growth chamber
Measurements of chlorophyll fluorescence in a greenhouse Methods for the repeated monitoring of chlorophyll fluorescence under naturally fluctuating light in the field have been reported previously (Ishida et al. 2014). At predawn, Fo and Fm were determined by measuring light exposure and a saturating pulse, respectively. During daytime, Fs and Fm0 were recorded by measuring light and a saturating pulse, respectively. The maximum fluorescence level after dark treatment during daytime (Fm00 ) was measured by the application of a saturating pulse. These chlorophyll fluorescence parameters were measured with a MONITORING-PAM fluorometer (Walz, Effeltrich, Germany). A Walz MINI-PAM fluorometer was used for the measurement of Fm00 . For this measurement, a section of the leaf was treated by darkness for 20 min before the application of a saturating pulse. With the use of the allocation model mentioned above, diurnal changes in energy allocation in PSII and PAR were monitored in upright leaves of the WT and psbS on the same day. From predawn to after sunset, Fs and Fm0 values and PAR were measured every 5 min, and Fm00 values were measured every hour. The energy flux in PSII (mmol m2 s1) was calculated from the quantum yield and PAR. For instance, the flux of electron transport JPSII was calculated by multiplying the estimated energy absorption in PSII (= PAR 0.84 0.5) by the quantum efficiencies II. In this equation, 0.84 indicates the assumed absorbance in leaves, and 0.5 is the assumed proportion of
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Supplementary data Supplementary data are available at PCP online.
Funding This work was supported in part by the Network of Centers of Carbon Dioxide Resource Studies in Plants (NC-CARP) and the project “Functional analysis of genes relevant to agriculturally important traits in rice genome” of the Ministry of Agriculture and Fisheries of Japan.
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Quenching analysis of chlorophyll fluorescence (Schreiber et al. 1986) was performed with a MONITORING-PAM fluorometer (Walz, Effeltrich, Germany). For the experiment under regulated actinic light, leaves were dark-adapted in the growth chamber for over 1 h prior to measurement. Plants were then moved to a laboratory adjacent to the growth chamber for measurement. The minimum fluorescence level in the absence of NPQ (Fo) was determined by measuring light illumination in the dark. The steady-state fluorescence level (Fs) was recorded under actinic light illumination. The maximum fluorescence level in the absence of NPQ (Fm) and the maximum fluorescence level in the presence of NPQ (Fm0 ) were measured by the application of a saturating pulse for 0.6 s. The minimum fluorescence level in the presence of NPQ (Fo0 ) was calculated from Fm, Fo and Fm0 , as 1/(1/Fo 1/Fm 1/Fm0 ) (Oxborough and Baker 1997). The quantum yield of photochemistry in PSII during light illumination (II) was calculated as (Fm0 Fs)/Fm0 . Total dissipation (1 II) was divided into two components, i.e. basal constitutive decay of excited chlorophyll (f,D), calculated as Fs/Fm, and light-dependent regulative dissipation (NPQ), calculated as Fs/Fm0 – Fs/Fm, as proposed by Hendrickson et al. (2004). Photochemical quenching (qP) was calculated as (Fm0 Fs)/(Fm0 Fo0 ).
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