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. 2018 Jun 15;59(10):1966–1975. doi: 10.1093/pcp/pcy118

Crassulacean Acid Metabolism Induction in Mesembryanthemum crystallinum Can Be Estimated by Non-Photochemical Quenching upon Actinic Illumination During the Dark Period

Tatsuya Matsuoka 1, Aya Onozawa 2, Kintake Sonoike 1,✉, Shin Kore-eda 3
PMCID: PMC6178971  PMID: 29917144

Abstract

Mesembryanthemum crystallinum, which switches the mode of photosynthesis from C3 to crassulacean acid metabolism (CAM) upon high salt stress, was shown here to exhibit diurnal changes in not only the CO2 fixation pathway but also Chl fluorescence parameters under CAM-induced conditions. We conducted comprehensive time course measurements of M. crystallinum leaf Chl fluorescence using the same leaf throughout the CAM induction period. By doing so, we were able to distinguish the effect of CAM induction from that of photoinhibition and avoid the possible effects of differences in foliar age. We found that the diurnal change in the status of electron transfer could be ascribed to the formation of a proton gradient across thylakoid membranes presumably resulting from diurnal changes in the ATP/ADP ratio reported earlier. The electron transport by actinic illumination thus became limited at the step of plastoquinol oxidation by the Cyt b6/f complex in the ‘night’ period upon CAM induction, resulting in high levels of non-photochemical quenching. The actinically induced non-photochemical quenching in the ‘night’ period correlated well with the degree of CAM induction. Chl fluorescence parameters, such as NPQ or qN, could be used as a simple indexing system for the CAM induction.

Keywords: Chl fluorescence measurements, Common ice plant (Mesembryanthemum crystallinum L.), Crassulacean acid metabolism (CAM), Non-photochemical quenching (NPQ), Photoinhibition, Salt stress

Introduction

Crassulacean acid metabolism (CAM) is a type of photosynthesis characterized by a diurnal change in carbon metabolism that enables CAM plants to carry out photosynthesis without opening stomata during light periods, resulting in avoidance of stress from water deficit. The diurnal change in CAM photosynthesis is usually divided into four phases. First, in Phase I in the dark (‘night’) period, CO2 is fixed by phosphoenolpyruvate carboxylase (PEPC) to form malate, which accumulates in the vacuole. Following a transition of the primary CO2-fixing enzyme from PEPC to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in Phase II, CO2 released by decarboxylation of malate is assimilated via the Calvin cycle in Phase III in the light (‘day’) period, during which time the stomata are closed (Black and Osmond 2003). When the malate supply is exhausted in the late afternoon, the stomata re-open and CO2 can be fixed directly from the atmosphere by the Calvin cycle (Phase IV). This diurnal change can be monitored by the concentration of malate in the leaves; the highest concentration will be at the end of Phase I, and the lowest in Phase IV. The cellular ATP/ADP ratio and the expression of PEPC also show distinct diurnal changes (Niewiadomska et�al. 2004, Freschi et�al. 2010).

Photosynthetic electron transport has also been reported to show diurnal changes in several CAM plants such as Kalancho� daigremontiana and Phalaenopsis hybrid ‘Atlantis’ (Winter and Demmig 1987, Mattos et�al. 1999, Griffiths et�al. 2008, Cela et�al. 2009, Pollet et�al. 2011), and electron transport in the CAM-inducible plant, Mesembryanthemum crystallinum, has been analyzed to examine the causal relationship between a diurnal change in carbon assimilation and electron transport (Keiller et�al. 1994, Sch�ttler et�al. 2002, Broetto et�al. 2007, Niewiadomska et�al. 2011). After a water deficiency-induced transition from C3 photosynthesis to CAM photosynthesis under conditions of high salt stress, M. crystallinum was shown to exhibit diurnal changes in the electron transport chain, as revealed by Chl fluorescence measurements. Specifically, higher non-photochemical quenching and lower photochemical quenching were observed in the Chl fluorescence of the CAM-induced plants during the dark period than during the light period, even when the photon flux density of actinic light was the same (Keiller et�al. 1994, Sch�ttler et�al. 2002, Broetto et�al. 2007, Niewiadomska et�al. 2011). Such differences were not observed in the C3 photosynthesizing M. crystallinum plants grown without salt stress. Thus, the non-photochemical quenching of Chl fluorescence can, in theory, be used to estimate the degree of CAM induction. Interestingly, this relationship has not been analyzed quantitatively in previous studies, probably due to the technical complexities of such measurements.

One challenge is the confounding effect of photoinhibition that occurs as a side effect of the salt stress used for CAM induction. The degree of photoinhibition could be assessed by measuring the maximum quantum yield of PSII, estimated as the Chl fluorescence parameter, Fv/Fm. However, the significance of the results of previous measurements of Fv/Fm has been debated, and the results seem to vary under different growth and experimental conditions (Keiller et�al. 1994, Broetto et�al. 2002, Sch�ttler et�al. 2002, Barker et�al. 2004, Broetto et�al. 2007). A plausible explanation for this discrepancy is the differing degrees of photoinhibition induced by the salt stress used for CAM photosynthesis induction. Another challenge is the effect of leaf position on the degree of CAM induction and/or Chl fluorescence. Indeed, the magnitude of CAM induction, inferred from accumulation of PEPC transcripts, has been reported to be greatly affected by leaf position (Piepenbrock and Schmitt 1991). Since previous Chl fluorescence studies of CAM photosynthesis were conducted using leaves at different positions, and of different ages, either in CAM-induced M. crystallinum (Keiller et�al. 1994, Niewiadomska et�al. 2011) or in obligate CAM plants (Winter and Demmig 1987, Mattos et�al. 1999, Griffiths et�al. 2008, Cela et�al. 2009, Pollet et�al. 2011), it has been difficult to determine the effect of foliar age on CAM induction.

To overcome these challenges, we conducted comprehensive time course measurements of M. crystallinum leaf Chl fluorescence using the same leaf throughout the CAM induction period. By doing so, we were able to distinguish the effect of CAM induction from that of photoinhibition and avoid the possible effects of differences in foliar age. Here we show that CAM induction accompanied the limitation of electron transfer from the plastoquinone pool to the cytochrome b6/f complex that induced non-photochemical quenching under actinic illumination. We also show that the Chl fluorescence parameters for non-photochemical quenching, such as NPQ and qN, can be used as an index of CAM induction.

Results

Changes in photosynthetic parameters over the course of CAM induction

Malate concentration in M. crystallinum leaves at the end of the dark period in diurnal light/dark cycles increased gradually for 2 weeks under salt stress conditions, induced by addition of 0.5 M NaCl to a hydroponic medium (Fig.�1A, filled circles). In contrast, when the concentration was measured in the middle of the light period (Fig.�1A, open circles), it was observed to be less affected by salt stress, showing the diurnal change that is typically seen in CAM plants (Fig.�1B, double circles). The malate concentration at the end of the dark period decreased again after 3 weeks (Fig.�1A, filled circles), presumably due to leaf senescence. These characteristic changes in the malate concentration were not observed in plants grown under normal conditions (Fig.�1, filled triangles in A and double triangles in B).

Fig. 1.

Fig. 1

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(A) Changes in malate concentration in M. crystallinum leaves over the course of crassulacean acid metabolism (CAM) induction. Filled circles, leaves from plants grown under CAM-inducing conditions where the malate concentration was determined at the end of the dark period; open circles, leaves under CAM-inducing conditions determined in the middle of the light period; filled triangles, control leaves where the malate concentration was determined at the end of the dark period; open triangles, control leaves determined at the middle of the light period. (B) Diurnal changes in malate concentration in M. crystallinum leaves. Measurements were carried out from day 15 to day 16 of the salt stress treatment. Double circles, leaves from plants grown under CAM-inducing conditions; double triangles, control leaves. Vertical bars represent � SD (n = 3).

To evaluate the status of photosynthetic electron transport under actinic light illumination during the course of CAM induction, we next monitored Chl fluorescence of the plants during the dark and light periods. Fv/Fm values, which represent an index of maximum quantum yield of PSII, of M. crystallinum leaves decreased during the first 3 d after the onset of the salt stress irrespective of the time of the day at which the measurements were conducted (Fig.�2A, circles). Such a decrease was not observed in the leaves grown without salt treatment (Fig.�2A, triangles). In order to elucidate the cause of the Fv/Fm decrease, we measured total Chl contents, as well as the Chl a/b ratio. Both factors showed little variation during the course of a 2 week CAM induction (Table�1), suggesting a constant antenna size. The reflectance spectra of the M. crystallinum leaf surfaces were not affected by the salt stress (Supplementary Fig. S1), supporting the results of the Chl content measurements. Meanwhile, Chl contents of CAM-induced leaves or control leaves showed a slight decrease after 3 weeks following the start of the salt stress conditions, possibly due to senescence.

Fig. 2.

Fig. 2

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Changes in Chl fluorescence parameters over the course of crassulacean acid metabolism (CAM) induction. (A) Fv/Fm, (B) NPQ, (C) qN, (D) qE, (E) qT + qI and (F) qP. Filled circles, leaves from plants grown under CAM-inducing conditions where measurements were performed at the end of the dark period; open circles, leaves from plants grown under CAM-inducing conditions where measurements were performed in the middle of the light period; filled triangles, control leaves where measurements were performed at the end of the dark period; open triangles, control leaves determined at the middle of the light period. Vertical bars represent � SD (n >4).

Table 1.

Chl content and Chl a/b ratios over the course of crassulacean acid metabolism (CAM) induction

Condition Days after the start of salt stress Chl
a + b (mg Chl g FW–1) a/b
Control 0 0.302 � 0.04 4.17 � 0.32
7 0.316 � 0.05 4.24 � 0.32
14 0.310 � 0.02 4.21 � 0.20
21 0.282 � 0.04 3.98 � 0.39
CAM-inducing 0 0.310 � 0.05 4.21 � 0.32
7 0.293 � 0.02 4.08 � 0.18
14 0.291 � 0.02 4.07 � 0.15
21 0.272 � 0.02 3.93 � 0.22
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Values are expressed as the mean � SD (n = 3).

Although the Fv/Fm values of CAM-induced leaves did not change diurnally, NPQ and qN (both of which are indices of non-photochemical quenching) showed a clear diurnal change. Under actinic illumination near the growth light level, NPQ (Fig.�2B, filled circles) and qN (Fig.�2C, filled circles) values of CAM-induced leaves in the dark period gradually increased during CAM induction. The increase of qN and NPQ persisted for 2 weeks, in parallel with the progression of CAM induction, unlike the change in Fv/Fm, which was observed only in the first 3 d. NPQ and qN values of the CAM-induced leaves in the light period also showed an increasing trend from day 6 to 10 after the onset of the salt stress, but the changes were not statistically significant (Fig.�2B, C, open circles). It must be noted that these parameters are determined under actinic illumination so that changes in the parameters do not necessary reflect the changes in electron transport in the dark. Instead, they are thought to reflect the metabolic interaction under actinic illumination.

To investigate the cause of qN development, we analyzed the relaxation kinetics of qN following cessation of exposure to actinic light. qE, the rapidly relaxing component of qN, showed a similar pattern to that of total qN over the course of CAM induction, in both the dark and light periods (Fig.�2D). qE comprised most of the qN, while qT and qI, which are slowly relaxing components of qN, were negligible, irrespective of the experimental conditions (Fig.�2E). In contrast, qP, an index of the oxidation state of the plastoquinone pool, decreased upon CAM induction only in the plants in the dark period (Fig.�2F, filled circles), mirroring the increase in NPQ and qN (Fig.�2B, C, filled circles).

The redox condition of the electron transport chain can also be analyzed by the absorbance kinetics of P700, the reaction center of PSI. Y(ND), an index of the donor side limitation of PSI, increased upon CAM induction in the dark period (Fig.�3B, filled circles), a pattern that is very similar to that of NPQ and qN (Fig.�2B, C). In contrast, Y(NA), an index of the acceptor side limitation of PSI, decreased upon CAM induction in the dark period (Fig.�3C, filled circles). Y(I), an index of the quantum yield of PSI, was not substantially affected by CAM induction (Fig.�3A).

Fig. 3.

Fig. 3

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Changes in the quantum efficiency of PSI in the course of crassulacean acid metabolism (CAM) induction; (A) Y(I), (B) Y(ND) and (C) Y(NA). Symbols are the same as in Fig.�1. Vertical bars represent � SD (n >4).

We also measured diurnal changes in Chl fluorescence (Fig.�4) or PSI parameters (Fig.�5) during a 24 h period after 15 d of salt stress. Fv/Fm showed no diurnal changes in the CAM-induced or control leaves (Fig.�4A, double circles and double triangles, respectively). The CAM-induced leaves showed consistently lower Fv/Fm values than the control leaves, suggesting the presence of PSII photoinhibition under salt stress. In contrast, clear diurnal changes were observed in the CAM-induced leaves (Figs.�4, 5, double circles) for NPQ (Fig.�4B), qN (Fig.�4C), qE (Fig.�4D), qP (Fig.�4F), Y(ND) (Fig.�5B) and Y(NA) (Fig.�5C). Such diurnal changes were not observed in the control leaves (Figs.�4, 5, double triangles). In all cases, the levels of the parameters determined in the light period in CAM-induced leaves were similar to those determined in the control leaves. The differences in the parameters between the CAM-induced leaves and the control leaves were only observed in the dark period, and were higher for qN, NPQ, qE and Y(ND), and lower for qP and Y(NA) in the CAM-induced leaves. We observed little difference in qT and qI (Fig.�4E) or Y(I) values between the CAM-induced and control leaves (Fig.�5A).

Fig. 4.

Fig. 4

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Diurnal changes in Chl fluorescence parameters from day 15 to 16 in leaves from plants grown under crassulacean acid metabolism (CAM)-inducing conditions. (A) Fv/Fm, (B) NPQ, (C) qN, (D) qE, (E) qT + qI and (F) qP. Double circles, leaves from plants grown under CAM-inducing conditions; double triangles, control leaves. Vertical bars represent � SD (n >4).

Fig. 5.

Fig. 5

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Diurnal changes of quantum efficiency of PSI from day15 to 16 under crassulacean acid metabolism (CAM)-inducing conditions. (A) Y(I), (B) Y(ND) and (C) Y(NA). Symbols are the same as in Fig.�1. Vertical bars represent � SD (n >4).

Relationship between CAM induction and NPQ, qN or qP development

The results obtained above indicated that several Chl fluorescence parameters in the dark period can be used for assessing CAM induction. To investigate this further, we examined the relationship between diurnal changes (i.e. the differences in quenching parameters between the values determined in the dark period and the values determined in the light period) of malate concentration and those of NPQ, qN and qP under different CAM induction conditions. In the experiments above, 0.5 M NaCl was used to induce salt stress and the fourth pair of true leaves was used for the measurements. Additionally, we measured the malate concentration and Chl fluorescence parameters for the second, third, fourth and fifth pairs of true leaves of plants grown in culture medium supplemented with 0.35 M NaCl (weak CAM-inducing conditions). A positive relationship between the diurnal changes of the malate concentration and both NPQ (Fig.�6A) and qN (Fig.�6B) values at the end of the dark period was observed both for the weak CAM-inducing conditions (Fig.�6, squares) and for the original CAM-inducing conditions (Fig.�6, circles). Additionally, the data points for both conditions could be fitted to the same approximate curve. We noted that the malate concentration increased by >20 μmol g FW–1, while the differences in NPQ and qN levels were saturated at approximately 0.7 (Fig.�6). A similar relationship between the changes in malate concentration and those in qP was also observed, albeit with a lower correlation (Fig.�6C). Although PSI parameters, such as Y(ND) and Y(NA), were examined only under one condition (0.5 M NaCl), the correlation between the diurnal changes in these parameters and the malate concentration was also high (Supplementary Fig. S2). The results showed that changes in Chl fluorescence parameters could be used to infer malate concentration, and thus for estimating the level of CAM induction, irrespective of the condition used for the CAM induction.

Fig. 6.

Fig. 6

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The relationship between quenching parameters and malate concentration. Diurnal changes in quenching parameters represent the differences between the values determined in the dark period and the values determined in the light period. Malate concentration was determined at the end of the dark period. (A) NPQ, (B) qN and (C) qP. Filled circles, leaves from plants grown under crassulacean acid metabolism (CAM)-inducing conditions (0.5 M NaCl); filled squares, leaves under weak CAM-inducing conditions (0.35 M NaCl).

Changes in the electron transport rate during the course of CAM induction

Although the observed differences in electron transport under actinic illumination could be ascribed to the passive changes brought about by redox conditions in the stroma, they could also reflect active change in the photosynthetic machinery in the thylakoid membranes. To distinguish between these two possibilities, we measured electron transfer through PSI, PSII and the whole chain in isolated thylakoid membranes prepared either in the dark period or in the light period. These three types of electron transport rate did not show any differences (Supplementary Fig. S3), irrespective of the light regime at the time of thylakoid membrane isolation (black bars for the dark period and white bars for the light period) or photosynthesis types of the leaves, i.e. determined either before the salt stress (Supplementary Fig. S3A), after 14 d of CAM-inducing conditions (Supplementary Fig. S3B) or after 14 d under control conditions (Supplementary Fig. S3C).

Discussion

Photoinhibition is induced by salt stress independent of CAM induction

Our analyses of photosynthetic parameters over a time course after the application of a salt stress that induces CAM photosynthesis revealed three categories of responses. Group 1: Fv/Fm, which decreases in the first 3 d of CAM-inducing conditions and remains at the same level for the remainder of the period. Group 2: qN, NPQ, qE, qP, Y(ND) and Y(NA), which gradually change over the entire 14 d of CAM-inducing conditions. Group 3: qT + qI and Y(I) do not show any significant change. Among these, the behavior of Fv/Fm is unique, not only in the time course of the change, but also since there is a lack of diurnal change. It appears that the decrease in this parameter, which represents the maximum quantum yield of the PSII reaction centers, was independent of CAM induction, and was presumably induced by the photoinhibition triggered by the salt stress.

Salt stress is known to induce photoinhibition of photosynthesis in many plant species (Murata et�al. 2007, Stepien and Johnson 2009). Although M. crystallinum is a halophyte, and thus more tolerant of high salt conditions than many other plant species, the 0.5 M NaCl treatment that was used to induce CAM photosynthesis also apparently induced photoinhibition. In previous studies, contradictory results have been reported regarding the changes in Fv/Fm during the course of CAM induction in M. crystallinum by salt stress: Fv/Fm decreased in some reports (Keiller et�al. 1994, Sch�ttler et�al. 2002, Broetto et�al. 2007) while no change was observed in others (Broetto et�al. 2002, Barker et�al. 2004) after 14 d of exposure to the salt stress. In any event, we concluded that the degree of photoinhibition itself is independent of the induction of CAM photosynthesis.

Non-photochemical quenching that persists during the dark period should be considered as a cause of the decrease in Fv/Fm. Our fluorescence decay kinetic measurements revealed that neither the slowly relaxing component of non-photochemical quenching, qT + qI, nor the rapidly relaxing component of non-photochemical quenching, qE, could be categorized as belonging to Group 1, suggesting that the decrease in Fv/Fm was not caused by the development of rapid non-photochemical energy dissipation. Since a decrease in Fv/Fm was observed in the dark period, as well as in the light period, it follows that Fv/Fm was not be restored to normal values during the 12 h dark period. It has been reported in some tree species, such as Pinus ponderosa and Picea pungens, that low Fv/Fm values resulting from photoinhibition persist until the next morning under cold stress (Adams and Demmig-Adams 1994, Adams et�al. 1994). Similarly, the influence of persisting non-photochemical quenching in salt-stressed M. crystallinum can be manifested as an apparent irreversible photoinhibition. A change in the light-harvesting systems may also contribute to the change in Fv/Fm. However, the Chl content of the M. crystallinum leaves was relatively constant over the course of up to 14 d of CAM induction in our study (Table�1). In addition, the reflectance spectrum of the leaf surfaces of M. crystallinum was not affected by CAM induction until 14 d following the onset of the salt stress (Supplementary Fig. S1). These results indicate that the efficiency of light harvesting was not decreased by CAM induction and was not the cause of the decrease in Fv/Fm.

Chl fluorescence measurement as a tool to monitor CAM induction

Following the initial phase of photoinhibition manifested as a decrease in Fv/Fm, subsequent gradual changes over a period of 14 d after the start of a salt treatment in the Group 2 parameters, qN, NPQ, qE, qP, Y(ND) and Y(NA), were mainly observed during the dark period. Thus, the changes in the later phase of the salt treatment can be ascribed to the influence of CAM induction. There were also indications of small changes in these parameters during the light period, especially at around 7 d after the start of the salt stress. However, these changes were not statistically significant and became less conspicuous at around 14 d after the start of the treatment, suggesting that the changes are not directly linked to the CAM induction. Thus, we used the diurnal changes in these parameters (i.e. the difference in the values of these parameters between the light period and dark period) as indices of CAM induction. We note that the effect of CAM induction on the redox condition of chloroplasts in the dark period could be monitored by Chl fluorescence measurements, although the photosynthetic parameters themselves were determined under actinic illumination (see the next section for details).

Among the Group 2 parameters that may reflect CAM induction, we selected Chl fluorescence parameters, NPQ, qN and qP, to examine the potential correlation with CAM induction. Diurnal changes in NPQ and qN increased in parallel with the accumulation of malate, as did changes in qP, albeit with a lower correlation (Fig.�6). Since this relationship was observed under different CAM-inducing conditions, as well as with leaves at different ages (i.e. leaf positions), we conclude that these parameters can be used for assessing the CAM induction in a wide range of experiments. Among these, NPQ is the simplest parameter to determine because it can be calculated using only Fm, Fm' and Fs values, thus making continuous recording possible. We also note that the level of NPQ was saturated in the leaves containing malate at >20 μmol g FW–1. Thus, NPQ may be useful for indexing the early CAM induction but not for very high accumulation of malate. NPQ can also be affected either by salt stress or by leaf senescence. In these cases, however, the NPQ level would not show diurnal changes. We employed diurnal changes of NPQ, not the absolute values of NPQ, for indexing CAM induction with the aim of focusing on the changes arising solely from CAM induction, since a gradual increase of NPQ by CAM induction was mainly observed in the dark period. We observed that NPQ values in the day period increased at around 7 d after the start of the salt stress, as stated above. The use of diurnal changes in NPQ values may also serve to eliminate other possible changes unrelated to CAM induction.

Effects of CAM induction on photosynthetic electron transport

Judging from the photochemical quenching of Chl fluorescence (qP) measured in the dark period, the plastoquinone pool was more reduced under actinic illumination in CAM-induced plants than in control plants grown without salt stress (Fig.�2F, filled circles). Under the same conditions, the downstream electron transport components, including the PSI reaction center P700 and the acceptor side of PSI, were oxidized (Fig.�3B, C). It therefore appears that oxidation of the plastoquinone pool at the QO site of the Cyt b6/f complexes is the rate-limiting step of electron transport. Since the parameters representing the development of non-photochemical quenching (i.e. NPQ and qN) increased under the same conditions (Fig.�2B, C), energy dissipation also seemed to be induced under actinic illumination. The main component of non-photochemical quenching is energy dependent qE (Fig.�2D), and we conclude that the proton gradient across the thylakoid membranes is developed by actinic illumination in the dark period after CAM induction. In completely CAM-induced M. crystallinum, an increase in NPQ (Keiller et�al. 1994, Niewiadomska et�al. 2011), a reduction of plastoquinone (Keiller et�al. 1994, Sch�ttler et�al. 2002) and an oxidation of P700 (Niewiadomska et�al. 2011) during the dark period have previously been reported. Since we observe very similar temporal changes during the CAM induction process for all these parameters (Figs.�2, 3), we assume a causal relationship between the changes in these parameters. The development of a proton gradient must be the first event, and this would lead to the development of non-photochemical quenching and the suppression of electron transport upon plastoquinol oxidation. Finally, the suppression of plastoquinone oxidation would lead to reduced PSII electron acceptors and oxidized electron transport components around PSI.

Photosynthetic parameters changed just after the beginning of the dark period and remained at similar levels during this period. These observations suggest that the regulation of the electron transport would be more energetic than metabolic. As a basic cause of the development of a proton gradient across the thylakoid membranes, several lines of evidence suggest the involvement of a high ATP/ADP ratio in chloroplasts. It has been reported that the ATP/ADP ratio was different among various CAM plant species (K�ster and Winter 1985, Li and Nose 2004). In CAM-induced M. crystallinum, the ATP/ADP ratio was higher in the dark period than in the light period in whole cells (Niewiadomska et�al. 2004) as well as in chloroplast stroma (Krieger et�al. 1998). Moreover, it has also been reported that cytosolic diurnal changes in the ATP/ADP ratio correlated with stromal diurnal changes in the ATP/ADP ratio (Gardestrom and Wigge 1988, Kromer et�al. 1993, Igamberdiev et�al. 2001). Thus, we speculate that the activity of a chloroplast proton ATPase would be suppressed in the presence of excess ATP, leading to the accumulation of protons in the thylakoid lumen. This, in turn, would suppress further oxidation of plastoquinol due to the high proton concentration in the thylakoid lumen.

We observed that the photosynthetic electron transport activity, determined using isolated thylakoid membranes, was similar in the dark and the light periods, irrespective of the induction of CAM (Supplementary Fig. S3). This result is consistent with earlier reports suggesting that salt stress did not have a major effect on the photosynthetic activity of isolated thylakoid membranes (Demmig and Winter 1983, Kore-Eda et�al. 1996). Together, the results indicate that the change in electron transport activity could not be ascribed to the intrinsic nature of the thylakoid membranes, supporting the hypothesis that the high ATP/ADP ratio in the chloroplast stroma in the dark is the first event that triggers the change in electron transport under actinic illumination during CAM induction. At present, there is no evidence for the involvement of the circadian clock in the changes in photosynthetic electron transport. Rather, the changes could be explained solely by metabolic interaction between stroma and thylakoid membranes. However, we cannot exclude the possibility of involvement of the circadian clock.

Materials and Methods

Plant materials and growth conditions

Mesembryanthemum crystallinum seeds were provided by Dr. J. Cushman, University of Nevada, Reno, NV, USA. Plants were grown in a growth chamber (LH-350-SP, NK system) at 25�C/17�C (12 h light/12 h dark), with a photon flux density of about 200 μmol m–2 s–1 during the light period and a relative air humidity of approximnately 50%. The plants were grown for 2 weeks after sowing on vermiculite, and then transferred to hydroponic culture (Terashima et�al., 1991). After 4 weeks, the first measurements were carried out (day 0). Plants were kept either in the same hydroponic culture medium (control conditions) or in culture medium supplemented with 0.5 M NaCl (CAM-inducing conditions) for the 3 week duration of the experiments. Measurements were performed on the fourth pair of true leaves.

To evaluate the effectiveness of the Chl fluorescence parameter as an index for CAM induction, we also employed different growth conditions and leaf positions for the measurements shown in Fig.�6. The plants were grown in a growth chamber (GB48, Conviron Ltd.) at 27�C/20�C (12 h light/12 h dark) and relative air humidly of about 20%/25% with a photon flux density of 150 μmol m–2 s–1 during the light period. Two weeks after sowing, the NaCl concentration of the culture medium was increased to 0.05, 0.10, 0.20 and 0.35 M every 12 h, and kept in the last condition for 3 weeks (weak CAM-inducing conditions). We used the second, third, fourth and fifth pairs of true leaves for the measurements.

Quantification of l-malate

Malate concentration was estimated from the absorption changes due to the reduction of NAD+ to NADH catalyzed by malate dehydrogenase (M�llering 1985). A 1 g aliquot of a M. crystallinum leaf was homogenized with 10 ml of 5% HClO4 using a homogenizer (PT10-35, Kinematica AG) and the homogenate was centrifuged for 10 min at 800�g. The precipitate was suspended in 5% HClO4 and centrifuged again for 10 min at 800�g. Supernatants from the two centrifugation steps were pooled, and the pH was adjusted to 9.0 by the addition of 5 M KOH. After being left to stand on ice for 30 min, the neutralized samples were centrifuged for 10 min at 800�g to remove precipitates. The malate content of the samples was determined in a 1 ml reaction mixture containing 70 mM 3-amino-1-propanol, 50 mM glutamate, 2 mM NAD+, 0.5 U of glutamate oxaloacetate transaminase (EC 2.6.1.1) and 10 U of malate dehydrogenase (EC 1.1.1.37) (M�llering 1985).

Chl fluorescence measurements

Chl fluorescence was measured at room temperature with a pulse-modulation fluorometer (PAM-2500, Heinz Walz GmbH). Leaves were dark acclimated for 20 min prior to the measurements to determine the minimal fluorescence level of the dark-acclimated samples (Fo). An 800 ms flash of saturating light (4,000 μmol m–2 s–1) from a red light-emitting diode (LED) integrated in the PAM-2500 fluorometer was used to determine the maximum fluorescence level (Fm). Continuous actinic light (209 μmol m–2 s–1) from the same LED source was used to determine the fluorescence levels under light-acclimated conditions. After actinic light illumination for 4 and 4.5 min, steady-state levels of the fluorescence were recorded and then pulses of the saturating light were applied. The average of the two steady-state levels of fluorescence was regarded as Fs while the average of the fluorescence levels upon saturating light was regarded as Fm'. Fo', the minimal fluorescence level of the light-adapted samples, was calculated as Fo′ = Fo/{[(Fm – Fo)/Fm] + (Fo/Fm′)} (Oxborough and Baker 1997). The Chl fluorescence parameters were calculated from Fo, Fo', Fs, Fm, Fm' and Fs as follows. Fv/Fm = (Fm – Fo)/Fm, Fv'/Fm' = (Fm' – Fo')/Fm', qP = (Fm'– Fs)/(Fm' –Fo'), Y(II) = (Fm'– Fs')/Fm', qN = 1 – (Fm' – Fo')/(Fm – Fo) and NPQ = (Fm – Fm')/Fm' (Baker 2008).

For the analysis of the relaxation of non-photochemical quenching, pulses of saturating light were applied 3.5 and 4 min after the actinic light was turned off. The average increases in the fluorescence values upon the saturating pulses at the two time points was regarded as FvRelax. The rapidly relaxing component of non-photochemical quenching, qE, and the slowly relaxing component of non-photochemical quenching, qT + qI, were calculated by qE = 1 – Fv'/(FvRelax) and qT + qI = 1 – F–Relax/Fv, respectively (Demmig and Winter 1988, Quick and Stitt 1989).

Redox kinetics of P700 and assessment of quantum yield of PSI

Redox kinetics of P700 and quantum yield of PSI were measured by the absorbance change at 820 nm with a spectrophotometer (PAM 101/102, Heinz Walz GmbH) equipped with an emitter/detector unit (ED-P700DW, Heinz Walz GmbH) as previously described (Schreiber et�al. 1988; Kudoh and Sonoike 2002). Relative absorbance levels of leaves under white actinic light (Ps), those of leaves in the dark (Po), those of leaves upon a saturating pulse under far red light (Pm) as well as those of leaves upon a saturating pulse under white actinic light (Pm') were used for calculation of the parameters; Y(I) = (Pm' – Ps)/(Pm – Po), Y(ND) = (Ps – Po)/(Pm – Po), Y(NA) = (Pm– Pm')/(Pm – Po) (Klughammer and Schreiber 1994). Leaves were dark acclimated for 20 min prior to the measurements. A 50 ms flash of saturating light (4,000 μmol m–2 s–1) from a multi-turnover light source (XMT-103, Heinz Walz GmbH) was used to determine Pm under far-red light from an LED (102-FR, Heinz Walz GmbH) or Pm' under white actinic light (200 μmol m–2 s–1) provided from a light source (KL-1500, Schott). Two flashes of the saturating light were applied after 2.5 and 3 min following the onset of white light, and the average of the relative absorbance levels was used as a measure of Pm'.

Isolation of thylakoid membranes

Mesembryanthemum crystallinum thylakoid membranes were isolated as previously described (Sonoike 1995). The fourth pair of true leaves were sampled at 2 h before the end of the dark period and 6 h after the beginning of the light period. After removing leaf stalks and major veins, about 10 g of leaf tissue was homogenized with a buffer (10 ml) containing 0.4 M sucrose, 50 mM Tris–HCl (pH 7.5), 10 mM NaCl and 5 mM MgCl2 using a homogenizer (PT10-35, Kinematica AG, Luzern, Switzerlan). The homogenates were filtered by filtration through a nylon mesh (pore size of 20 μm), the filtrate was centrifuged for 2 min at 12,000�g at 4�C and the pellet was suspended in 2 ml of buffer containing 0.4 M sucrose, 50 mM HEPES–HCl (pH 7.6), 10 mM NaCl and 5 mM MgCl2 using a soft brush. All procedures were performed on ice.

Measurements of the photosynthetic electron transport rate

Photosynthetic electron transport rates of isolated thylakoid membranes were determined at 25�C with liquid-phase Clark-type oxygen electrode (Oxygraph plus, Hansatech) as oxygen evolution (PSII electron transport) or oxygen consumption (PSI and whole chain electron transport) as previously described (Ogawa et�al. 2013). Saturating white light at 1,500 μmol m–2 s–1 provided from a light source (PICL-NRX, Nippon P.I.) was used as actinic light. The reaction mixture contained 10 μg Chl ml–1 thylakoid membranes, 0.4 M sucrose, 50 mM HEPES–HCl (pH 7.6), 10 mM NaCl, 5 mM MgCl2 and 10 mM methylamine with the following additional reagents: 0.4 mM 2,6-dimethylbenzoquinone (DMBQ) for PSII electron transport; 1 mM KCN, 1 mM ascorbic acid, 70 μM DCIP (2,6-dichloroindophenol), 10 μM DCMU and 100 μM methyl viologen for PSI electron transport; or 1 mM KCN and 100 μM methyl viologen for whole chain electron transport.

Measurements of reflectance

Square areas (1 cm�1 cm) were excised from the third or fourth pair of true leaves 10 min before the end of the dark period. The reflectance of the leaf surface was measured from 350 to 750 nm using an integrating sphere (ISV-722, JASCO) connected to a spectrophotometer (VP-650ST, JASCO).

Quantification of Chl content

After the measurement of reflectance, each 1 cm�1 cm leaf square was homogenized in 1.2 ml of distilled water using a pestle and mortar. A 200 μl aliquot of the suspension was mixed with acetone (800 μl), and the mixture was centrifuged for 5 min at 800�g, 24�C. Chl contents of the supernatant were determined according to Porra et�al. (1989).

Funding

This work was supported by The Ministry of Education, Culture, Sports, Science and Technology (MEXT) [Grant-in-Aid for Scientific Research on Innovative Areas Nos. JP16H06552 and JP16H06553 to K.S.] and the Japan Society for the promotion of Science (JSPS) [KAKENHI grant Nos. JP16H04809 and JP16K14759 to K.S.].

Disclosures

The authors have no conflicts of interest to declare.

Supplementary Material

Supplementary Data
Click here for additional data file. (1.5MB, pdf)

Glossary

Abbreviations

CAM

crassulacean acid metabolism

Fv/Fm

maximum quantum yield of PSII

Fv′/Fm′

effective quantum yield of open PSII

NPQ

a fluorescence parameter representing non-photochemical quenching based on the Sturn–Volmer equation

PAM

pulse amplitude modulation

PEPC

phosphoenolpyruvate carboxylase

qN

a fluorescence parameter representing non-photochemical quenching

qP

a fluorescence parameter representing photochemical quenching

qE

rapidly relaxing component of qN

qT and qI

slowly relaxing component of qN

Y(I)

quantum yield of electron transfer through PSI

Y(II)

effective quantum yield of electron transfer through PSII

Y(ND)

quantum yield of non-photochemical energy dissipation due to donor side limitation of PSI

Y(NA)

quantum yield of non-photochemical energy dissipation due to acceptor side limitation of PSI

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Associated Data

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Supplementary Materials

Supplementary Data
Click here for additional data file. (1.5MB, pdf)

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