Photoprotection of PSI by Far-Red Light Against the Fluctuating Light-Induced Photoinhibition in Arabidopsis thaliana and Field-Grown Plants

It has been reported that PSI photoinhibition is induced even in wild-type plants of Arabidopsis thaliana, rice and other species by exposure of leaves to fluctuating light (FL) for a few hours. Because plants are exposed to FL in nature, they must possess protective mechanisms against the FL-induced photodamage. Here, using A. thaliana grown at various irradiances, we examined PSI photoprotection by far-red (FR) light at intensities comparable with those observed in nature. Dark-treated leaves were illuminated by red FL alternating high/low light at 1,200/30 µmol m⁻² s⁻¹ for 800 ms/10 s. By this FL treatment without FR light for 120 min, the level of photo-oxidizable P700 was decreased by 30% even in the plants grown at high irradiances. The addition of continuous FR light during the FL suppressed this damage almost completely. With FR light, P700 was kept in a more oxidized state in both low- and high-light phases. The protective effect of FR light was diminished more in mutants of the NADH dehydrogenase-like complex (NDH)-mediated cyclic electron flow around PSI (CEF-PSI) than in the PGR5 (proton gradient regulation 5)-mediated CEF-PSI, indicating that the NDH-mediated CEF-PSI would be a major contributor to PSI photoprotection in the presence of FR light. We also confirmed that PSI photoinhibition decreased with the increase in growth irradiance in A. thaliana and field-grown plants, and that this PSI photodamage was largely suppressed by addition of FR light. These results clearly indicate that the most effective PSI protection is realized in the presence of FR light.

Introduction

In the field, daytime irradiance changes drastically with time. Even at an open site, irradiance changes depending on elevation of the sun, and cloud cover and movements. In canopy shade, irradiance fluctuation is further enhanced by the presence of vegetation.

In 2010, it was reported that seedlings of the pgr5 (proton gradient regulation 5) mutant of Arabidopsis thaliana did not grow and eventually died in fluctuating light (FL) consisting of alternating high and low light (Tikkanen et al. 2010). This was attributed to PSI photoinhibition (Suorsa et al. 2012). Surprisingly, PSI photoinhibition was observed not only in the pgr5 mutant but also in the wild-type plants of A. thaliana (Kono et al. 2014) and Oryza sativa (Yamori et al. 2016) after moderate FL treatments of the leaves for only a few hours, while PSI is fairly resistant to continuous light at very high irradiances (Munekage et al. 2002, Sonoike 2011). It was also found that repetitive hypersaturating or saturating flashes induce marked photoinhibition of PSI in A. thaliana (Tsuyama and Kobayashi 2009), Helianthus annuus (Sejima et al. 2014) and Triticum aestivum (Zivcal et al. 2015). FL-induced photoinhibition of PSI also causes a decrease in the CO₂ assimilation rate (Sejima et al. 2014, Zivcak et al. 2015, Yamori et al. 2016).

PSI photoinhibition by FL appears to occur when the electron flow on the acceptor side of PSI is limited and the rate of reactive oxygen species (ROS) formation exceeds the rate of ROS scavenging. The main cause of photoinhibition of PSI would be the destruction of the iron–sulfur centers by the ROS (Inoue et al. 1986, Sonoike and Terashima 1994, Tjus et al. 1999, Sonoike 2011). In particular, involvement of the hydroxyl radical (·OH), produced from hydrogen peroxide (H₂O₂) by the Fenton reaction, has been claimed (Sonoike 1996, Tjus et al. 2001). At much greater irradiance, singlet oxygen (¹O₂) was produced in PSI and would also be involved in the photodamge to PSI (Kok et al. 1965, Takagi et al. 2016).

Because light fluctuation is a potent stress factor for PSI (Kono et al. 2014, Sejima et al. 2014, Yamori et al. 2016), plants should have mechanisms to cope with the light fluctuation. The cyclic electron flow around PSI (CEF-PSI; Strand et al. 2016), the NADH dehydrogenase-like complex-dependent pathway (NDH-mediated CEF; Shikanai et al. 1998, Shikanai 2016) and the PGR5–PGRL1-mediated pathway (PGR5-mediated CEF; Munekage et al. 2002, Munekage et al. 2004, Hertle et al. 2013, Labs et al. 2016) have been proposed to protect PSI from the fluctuating growth light in A. thaliana (Suorsa et al. 2012) and in rice (Yamori et al. 2016). The results of the experiments employing short-term FL-treatments indicated that the PGR5-mediated CEF-PSI and the water–water cycle (WWC), also known as the Mehler–ascorbate peroxidase pathway (Asada 1999), were essential for protection of PSI (Kono et al. 2014, Allahverdiyeva et al. 2015, Kono and Terashima 2014).

These alternative electron flows induce non-photochemical quenching (NPQ) in the PSII antenna system and suppress the electron flow at the Cyt b₆/f complex, through generating a large ΔpH (Miyake 2010, Shikanai 2014, Tikhonov 2014). With respect to protection of PSI against FL-induced photoinhibition, CEF-PSI, especially the PGR5-mediated CEF-PSI, alleviates the acceptor-side limitation of PSI, and subsequently suppresses the electron flow at the Cyt b₆/f complex by generating a ΔpH, which eventually enhances the donor-side limitation of PSI (Kono and Terashima 2016). This photoprotective regulation is effective in the high-light (HL) phase of FL. Sejima et al. (2014) found that the PSI photoinhibition in H. annuus by repetitive hypersaturating or saturating pulses (SPs) was suppressed by rather strong background light between the pulses, >600 µmol m⁻² s⁻¹. This protection of PSI by the release of the acceptor-side limitation of PSI would be due to activation of the reactions on the PSI acceptor side, including those of the Calvin–Benson cycle enzymes, by the relatively high irradiance of the background light or in the low-light (LL) phase (Kono and Terashima 2014). In fact, the background light at 200 µmol m⁻² s⁻¹ showed no photoprotective effect (Sejima et al. 2014).

In the field, the irradiance per unit wavelength of the visible light (400–700 nm) measured in an open site is comparable with that of far-red (FR) light 700–800 nm; Gates 1980), although spectra in Gates (1980) are all expressed in the unit of energy rather than irradiance per unit wavelength. In canopy shade, visible light is largely absorbed by the leaves, while FR light is hardly absorbed and thereby is transmitted considerably through the leaves (Tasker and Smith 1977, Smith 1982). Thus, irradiance per unit wavelength of FR light in the canopy shade becomes greater than that of visible light. For land plants, therefore, irradiance per unit wavelength of FR light received by their leaves is always greater than or comparable with that of visible light or so-called photosynthetically active radiation (PAR; 400–700 nm). Although the abundance of FR light is substantial, many researchers of photoinhibition tend to use PAR only. In particular, monochromatic red or blue light-emitting diodes (LEDs) have been used widely as light sources.

Although the alternative electron flows contribute to suppression of the PSI photoinhibition, these activities are insufficient (Kono and Terashima 2016, Yamori et al. 2016). However, the PSI photoinhibition needs to be almost fully suppressed, because, once PSI is damaged, repair and/or de novo PSI synthesis are very slow (Kudoh and Sonoike 2002). In this study, we put forward a hypothesis that FR light could contribute to the protection of PSI in the FL. We tested whether addition of FR light to short-term FL suppresses PSI photoinhibition. We used A. thaliana grown in constant light in a growth cabinet. Erigeron annuus and Commelina communis collected in the field were also used.

Results

Protection of PSI and PSII by an additional far-red light from the FL-induced photoinhibitions

In the present study, the fluctuating actinic red light (FL), consisting of an alternating HL phase for 800 ms and a LL phase for 10 s, was used. HL at 1,200 µmol m⁻² s⁻¹ was consistently used, while irradiance of LL varied from 0 to 135 µmol m⁻² s⁻¹. Fig. 1 shows the effects of the irradiance level, 0, 30 or 135 µmol m⁻² s⁻¹, in the LL phase of the FL treatments on photoinhibitions of PSI and PSII. These FLs are referred to as FL-1200/0, FL-1200/30 and FL-1200/135. The measurements were made after the FL treatment for 30, 60, 90 and 120 min using the same leaf that had been dark-treated for 30 min. Photoinhibition of PSI and PSII was assessed as the reduction of P_m and that of F_v/F_m after dark treatment for 30 min. After the measurements of P_m and F_v/F_m, the leaf was treated with the FL for another 30 min, until the cumulated time of the FL treatment reached 120 min. In complete darkness, no recovery of P_m and F_v/F_m occurred (Supplementary Fig. S1).

Fig. 1

Effects of partial existence of FR light, added to either the HL or LL phase of the fluctuating red light, on photoinhibition of PSI (A) and PSII (B) in dark-treated leaves. FL alternating HL from red LEDs at 1,200 µmol m⁻² s⁻¹ for 800 ms, and LL from red LEDs at 0 (FL-1200/0, squares), 30 (FL-1200/30, circles) or 135 µmol m⁻² s⁻¹ (FL-1200/135, triangles) for 10 s was used. Following the FL treatment for 30, 60, 90 and 120 min, the functional PSI and PSII reaction centers, P_m and F_v/F_m, respectively, were determined after the dark treatment for 30 min. Data were normalized to the initial values measured in the dark before the light treatments. A series of measurements at 30, 60, 90 and 120 min was made with the same leaf. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C). The values represent the means ± SD (n = 3–5 leaves). Differences among the treatments at 120 min were analyzed by the Tukey–Kramer multiple comparison test (P < 0.05). Different letters indicate significant differences among the FL treatments.

All these FL treatments caused PSI photoinhibition, the extent of which increased with the treatment time (Fig. 1A). The extent also varied with the irradiance level in the LL phase. In the FL-1200/0 treatment, PSI showed the most striking photoinhibition, in which the P_m level at 120 min was decreased to 60% of that before the treatment. With the increase in the irradiance in the LL phase, PSI photoinhibition was more alleviated. Conversely, the extent of the PSII photoinhibition, though not marked, increased with the decrease in the irradiance level in the LL phase (Fig. 1B).

To examine the effect of FR light on PSI photoinhibition by the FL, we added FR light to FL-1200/30 (Fig. 2). FR light from LEDs (peaking at 740 nm, Supplementary Fig. S2) at 25.6 (high-light), 12.1 (moderate-FR) or 3.93 (low-FR) W m⁻², called hFR, mFR or lFR, respectively, was added continuously to FL-1200/30 and denoted as FL-(1200 + hFR)/(30 + hFR), FL-(1200 + mFR)/(30 + mFR) or FL-(1200 + lFR)/(30 + lFR). Fig. 2 shows the effects of these FL + FR treatments. The PSI photoinhibition was markedly alleviated by lFR light compared with the absence of FR (Fig. 1A). The additions of mFR or hFR light suppressed the PSI photoinhibition almost completely (Fig. 2A). For PSII, photoinhibition was also suppressed by the addition of FR light, especially hFR light.

Fig. 2

Suppressing effects of additional far-red (FR) light on photoinhibition of PSI (A) and PSII (B) by fluctuating light. The fluctuating red light used was FL-1200/30. FR light was added to the FL and the intensity of FR light was constant throughout the FL treatment. Three FR light intensities, high-FR light at 25.6 W m⁻² (hFR, circles), moderate-FR light at 12.1 W m⁻² (mFR, diamonds) and low-FR light at 3.93 W m⁻² (lFR, asterisks), were used. Following the treatments for 30, 60, 90 and 120 min, P_m and F_v/F_m, were determined after the dark treatment for 30 min. Data were normalized to the initial values measured in the dark before the light treatments. A series of measurements at 30, 60, 90 and 120 min was made with the same leaf. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C). The values represent the means ± SD (n = 3–5 leaves). Differences among the treatments at 120 min were analyzed by the Tukey–Kramer multiple comparison test (P < 0.05). Different letters indicate significant differences among the FL treatments.

Next, we added FR light in either the HL or the LL phase (Fig. 3); hFR light was used. FL treatments were FL-(1200 + hFR)/hFR, FL-(1200 + hFR)/30, FL-(1200 + hFR)/0 and FL-1200/hFR. P_m levels after the FL treatment for 120 min decreased to about 72% in FL-(1200 + hFR)/30 and FL-(1200 + hFR)/0, and 85% in FL-(1200 + hFR)/hFR (Fig. 3A). The decrease in P_m at 120 min in FL-(1200 + hFR)/hFR was similar to that in FL-(1200 + lFR)/(30 + lFR) in Fig. 2A and that in FL-1200/135 in Fig. 1A. By the FL-1200/hFR treatment, P_m at 30 min was decreased to 85% but the decrease was decelerated thereafter. For PSII, photoinhibition was slightly greater in FL-(1200 + hFR)/0 than those in the others. These results indicate that the presence of FR light in HL and LL phases suppresses PSI photoinhibtion. When FR light was present only in the HL phase the protective effects were not perfect. The presence of FR light in the LL phase without a red LED light also did not suppress PSI photoinhibition perfectly. In contrast to PSI, the presence of FR light in the HL phase did not contribute to the suppression of PSII photoinhibition. The presence of only FR light in the LL phase, FL-(1200 + hFR)/hFR, significantly alleviated the damage to PSII, compared with FL-(1200 + hFR)/0, and the extent of alleviation was the same as that in FL-(1200 + hFR)/30.

Fig. 3

Effects of partial existence of FR light in the HL or LL phase of the fluctuating red light on suppression of photoinhibitions of PSI (A) and PSII (B) by the FL treatments in dark-treated leaves. The four FL treatments were FL-(1200 + hFR)/hFR (circle), FL-(1200 + hFR)/30 (square), FL-(1200 + hFR)/0 (triangle) and FL-1200/hFR (diamond). The cycle of fluctuation is 800 ms in the HL phase and 10 s in the LL phase. FR light intensity was 25.6 W m⁻² (hFR). Following treatments for 30, 60, 90 and 120 min, P_m and F_v/F_m were determined after the dark treatment for 30 min. Data were normalized to the initial values measured in the dark before the light treatments. A series of the measurements at 30, 60, 90 and 120 min was made with the same leaf. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C). The values represent the means ± SD (n = 3–5 leaves). Differences among the treatments at 120 min were analyzed by the Tukey–Kramer multiple comparison test (P < 0.05). Different letters indicate significant differences among the FL treatments.

Fast kinetics of P700 signal and Chl fluorescence induced by a high-light pulse

To evaluate the effects of FR light on the electron flows in PSI and PSII in the FL treatment, we compared the rapid changes in 830 nm absorption and Chl fluorescence during the HL phase for 800 ms between the FL-1200/30 and FL-(1200 + hFR)/(30 + hFR) treatments (Fig. 4). Fig. 4 shows typical traces of the PSI and PSII responses to the 100th HL pulse which was given at about 1,080 s after the onset of the FL treatment. In the absence of FR light, the P700 signal showed a spike of P700⁺formation upon HL illumination (Fig. 4A, blue line), followed by P700⁺ reduction by the electrons derived from PSII to a plateau, and P700 was gradually oxidized till the cessation of the HL. Upon the transition from HL to LL, the P700 signal rapidly decreased and became stable at the same, almost fully reduced level as in the LL phase before the HL phase. In the presence of FR light, the level of P700⁺ in the LL phase was greater than that in the LL phase in the absence of FR light (Fig. 4B, blue line). Analyses of PSI quantum yields (Table 1) showed that preferential excitation of PSI by FR light alleviated the acceptor-side limitation of PSI, Y(NA), and increased the donor-side limitation of P700, Y(ND).

Table 1

PSI quantum yields measured at 10 s from the onset of LL phases after the 100th and 200th high-light pulses of the FL treatments

	Y(I)	Y(ND)	Y(NA)
After the 100th HL
FL-1200/30	0.562 ± 0.1058	0.006 ± 0.0680	0.432 ± 0.1082
FL-(1200 + FR)/(30 + FR)	0.387 ± 0.0341*	0.389 ± 0.0270**	0.224 ± 0.0204*
After the 200th HL
FL-1200/30	0.534 ± 0.132	0.009 ± 0.0085	0.456 ± 0.130
FL-(1200 + FR)/(30 + FR)	0.390 ± 0.0200*	0.361 ± 0.0229**	0.248 ± 0.0162*

At 10 s from the onset of the LL phase after either the 100th or the 200th high-light pulse of the FL-1200/30 or FL-(1200 + hFR)/(30 + hFR) treatment, a saturating pulse (SP) was applied. During the SP, FR light was illuminated for both of the FL treatments. After the SP, there was a dark interval for 1,100 ms, during which the full reduction level of the P700 signal was obtained. These full reduction levels were used to calibrate the P700 signals of Fig. 4. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C).

The values represent the means ± SD (n = 3–5).

Differences between the presence and absence of FR, detected by Student’s t-test, are indicated by *(P < 0.05) or **(P < 0.01).

Fig. 4

Changes in Chl fluorescence (red line) and 830 nm absorption (blue line) around the 100th HL pulse at 1,200 µmol m⁻² s⁻¹ for 800 ms in the absence (FL-1200/30, A) and presence [FL-(1200 + hFR)/(30 + hFR), B] of FR light. FR light intensity was 25.6 W m⁻² (hFR). Typical traces of the responses to the 100th HL pulse at about 1,080 s in the FL treatments are shown. The P700 signal was normalized to the P_m level before the FL treatment. To calibrate the P700 signal, the saturating pulse (SP) was applied at 10 s from the onset of the LL phase after the 100th HL pulse. During the SP, FR light was illuminated for both of the FL treatments. After the SP, a dark interval of 1,100 ms was given, during which full reduction in the level of the P700 signal was obtained. The zero value of the P700 signal on the vertical axis means the full reduction level of P700. PSI parameters determined by these SPs are shown in Table 1. Arrows indicate the start and end of the HL phase.

The difference between the maximum and minimum levels of the P700 signal during the HL in the presence of FR light was much greater than that in the absence of FR light. A separate assessment of PSI quantum yields revealed that Y(NA) during the HL treatment was much smaller and Y(ND) was greater in the presence of FR light (Table 2). Thus, a marked decrease in the P700 signal after the initial spike by the HL did not reflect gradual development of the acceptor-side limitation, Y(NA), but the reduction of P700⁺ by the electrons from PSII. After cessation of the HL, the P700 signal rapidly decreased, but the re-oxidation was accelerated by FR light.

Table 2

PSI quantum yields measured during the 100th and 200th high-light pulses of the FL treatments

	Y(I)	Y(ND)	Y(NA)
100th HL
FL-1200/30	0.318 ± 0.0170	0.253 ± 0.0380	0.428 ± 0.0227
FL-(1200 + FR)/(30 + FR)	0.246 ± 0.0296*	0.528 ± 0.0261**	0.232 ± 0.0146**
200th HL
FL-1200/30	0.270 ± 0.0210	0.220 ± 0.0486	0.488 ± 0.0690
FL-(1200 + FR)/(30 + FR)	0.236 ± 0.0227	0.479 ± 0.0686**	0.285 ± 0.0560**

At 10 s from the onset of the 99th or 199th high-light pulse of the FL-1200/30 or FL-(1200 + hFR)/(30 + hFR) treatment, HL at 1,200 µmol m⁻² s⁻¹ was illuminated for 2 s in the absence or presence of FR light, and a saturating pulse (SP) was applied at 1 s after the onset of the 100th or 200th HL. In this experiment, the 100th or 200th HL period was extended to 1 s instead of 800 ms. During the SP, FR light was illuminated for either of the FL treatments. After the cessation of the SP, there was dark interval for 1,100 ms, during which the full reduction level of P700 signal was obtained. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C).

The values represent the means ± SD (n = 3).

Differences between the presence and absence of FR, detected by Student’s t-test, are indicated by *(P < 0.05) or **(P < 0.01).

Chl fluorescence followed the changes in irradiance. Upon the transition from LL to HL, the rise in fluorescence was slower in the presence of FR light than its absence. In the presence of FR light, once the maximum levels were attained, the fluorescence level was slightly increased (Fig. 4B, red line) or constant (Supplementary Fig. S3) during the HL phases depending on the duration of the FL treatment, while a slight decrease was observed in the absence of FR light. After the cessation of the HL, the decrease in the fluorescence to the level in the LL phase before the HL was faster in the presence of FR light than in its absence because of the acceleration of the P700 oxidation by FR light (compare the traces in red in Fig. 4A and B).

Effects of the addition of FR light on photosynthetic parameters

To examine the effect of addition of the FR light to the red continuous actinic light on the PSI and PSII parameters, the light response curves in the steady state were obtained in the absence and presence of hFR light at 25.6 W m⁻² (Fig. 5). For the energy captured by PSI pigments, the quantum yields of the PSI photochemistry, Y(I), Y(ND) and Y(NA), were measured. The fluorescence parameters measured were the quantum yields of the PSII photochemistry, Y(II), non-regulated energy dissipation, Y(NO), and regulated energy dissipation, Y(NPQ). The same leaf was used in the series of measurements in the absence and presence of FR light, and 830 nm absorption and Chl fluorescence were simultaneously measured with the DUAL-PAM-100 (Walz).

Fig. 5

Responses of photosynthetic quantum yields of PSI (A) and PSII (B) in the leaves to continuous light at various irradiances of the red actinic light in the absence (filled and open symbols) and presence (colored symbols) of FR light. FR light intensity was 25.6 W m⁻² (hFR) throughout the measurement. For PSI parameters, Y(I) (, ), Y(ND) (, ) and Y(NA) ( , ) are shown. For the fluorescence parameters, Y(II) (, ), Y(NO) (, ) and Y(NPQ) ( , ) are shown. These parameters were obtained in the steady state attained at 3–10 min after the changes in the irradiance. A series of measurements in the absence and presence of FR light was conducted in the same leaf. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPaO₂, at 25°C). The values represent the means ± SD (n = 4 leaves).

In the absence of FR light, all parameters in PSI and PSII showed typical trends as observed in previous studies (Kono et al. 2014, Zivcak et al. 2015). In the presence of FR light, at low to moderate irradiances up to 500 µmol m⁻² s⁻¹, all PSI parameters, Y(I), Y(ND) and Y(NA), differed from those in the absence of FR light. Y(ND) values at the low irradiances were high and decreased with the irradiance. Y(NA) increased in a complementary manner. The alleviation of Y(NA) increased Y(I). At irradiances >500 µmol m⁻² s⁻¹, all PSI parameters in the presence of FR light showed almost the same values as those in its absence (Fig. 5A). The presence of FR light did not change the PSII parameters (Fig. 5B).

Roles of CEF-PSI in protection of PSI by FR light against FL-induced photoinhibition

To assess the effects of deficiency of the NDH-mediated CEF-PSI or the PGR5-mediated CEF-PSI on photoprotection by FR light, we compared P_m and F_v/F_m before and after the FL-1200/30 treatments with and without hFR light at 25.6 W m⁻² for 120 min in A. thaliana crr2-2 and crr4-3 mutants defective in the NDH complex and the pgr5 mutant defective in PGR5 (Fig. 6). In the absence of FR light, P_m values decreased to about 70% of those before the FL treatment in all these mutants, which was similar to the level observed in the wild type Columbia-0 (Col-0) after the FL-1200/30 treatment for 120 min. In pgr5, addition of hFR light to the FL markedly suppressed the PSI photoinhibition, and the P_m level was about 92% of that before the FL treatment. This suppression by FR light in pgr5 was slightly smaller than that in Col-0 with FR light, although the difference was not statistically significant. In crr2-2 and crr4-3, however, suppression effects of the PSI photoinhibition by hFR light were smaller and the P_m levels remained about 80%.

Fig. 6

Effects of a deficiency of the NDH-mediated or PGR5-mediated CEF-PSI on the FL1200/30-induced photoinhibition of PSI (A) and PSII (B) for 120 min in the absence and presence of FR light. The mutants of NDH-mediated CEF-PSI, crr2-2 () and crr4-3 (), the mutant deficient in PGR5, pgr5 (), and the wild-type plants, Col-0 (), were analyzed. P_m and F_v/F_m relative to the initial values before the light treatments are shown. FR light intensity was 25.6 W m⁻² (hFR). The data of Col-0 are taken from Fig. 2. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C). The values represent the mean ± SD (n = 3–5 leaves). Different lower case letters indicate statistically significant differences detected by the Tukey–Kramer multiple comparison test (P < 0.05).

Relationships between growth irradiance and tolerance of the FL-induced photoinhibition

Arabidopsis thaliana plants showed marked PSI photoinhibition by FL-1200/30 for 120 min (Fig. 1). To examine the effect of growth irradiance, we grew the Col-0 plants with white light at either 30, 60, 135, 240 or 500 µmol m⁻² s⁻¹. Fig. 7 shows the effects of the FL-1200/30 treatment for 120 min on these plants. All plants showed PSI photoinhibition by FL-1200/30 (Fig. 7A). The plants grown at 30 µmol m⁻² s⁻¹ showed the greatest PSI photoinhibition, and the plants grown at 60 µmol m⁻² s⁻¹ were the next most affected. The extents of PSI photoinhibition were similar among the plants grown at 135, 240 and 500 µmol m⁻² s⁻¹. The plants grown at 30 µmol m⁻² s⁻¹ showed marked photoinhibition after the FL-1200/30 for 30 min, but the extent did not increase with prolonged FL-1200/30 treatment. The extents of PSII photoinhibition were small and similar in all these plants (Fig. 7B).

Fig. 7

Effects of growth irradiance on the tolerance of the FL-induced photoinhibition. The plants were grown in constant light at 30 (crosses), 60 (diamonds), 135 (circles), 240 (triangles) and 500 (squares) µmol m⁻² s⁻¹ for 8 h per day. Following the FL-1200/30 treatment for 30, 60, 90 and 120 min, the levels of P_m and F_v/F_m were determined after dark treatment for 30 min. Data for 135 µmol m⁻² s⁻¹ are reproduced from Fig. 1. The leaves from 6-month-old plants in 30 µmol m⁻² s⁻¹, 10- to 12 week-old plants in 60 µmol m⁻² s⁻¹ and 5- to 7-week-old plants in 240 and 500 µmol m⁻² s⁻¹ were used. FR light was not included in the FL. Data were normalized to the initial values measured in the dark before the light treatments. A series of measurements at 30, 60, 90 and 120 min was made with the same leaf. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C). The values represent the means ± SD (n = 3–5 leaves). Differences among the treatments at 120 min were analyzed by the Tukey–Kramer multiple comparison test (P < 0.05). Different letters indicate significant differences among the growth irradiances.

FL-induced photoinhibition and protection by FR light in field-grown plants

To examine whether the FL-induced PSI photoinhibition occurs in field-grown plants and whether FR light is effective in protecting PSI, we used two field-grown plants. Erigeron annuus (Asteraceae) was sampled from an open site and a shaded site, and Commelina communis (Commelinaceae) was sampled from the same open site (for light environments of these sites, see hemispherical photographs in Supplementary Fig. S4). We collected leaves in the early morning and these detached leaves were kept in the dark for 60 min with their petioles in distilled water. Then, we illuminated these plants with FL-1200/30 for 120 min. Fig. 8 compares photoinhibition of these plants in the absence and presence of FR light. In the absence of FR light, the extents of PSI photoinhibition were different among the plants. Comparing the plants from the same open site, PSI photoinhibition was greater in C. communis than in E. annuus. PSI was more photoinhibited in E. annuus from the shaded site than in that from the open site (Fig. 8A). In the presence of mFR light at 12.1 W m⁻², however, PSI photoinhibition was hardly observed. For PSII, all plants showed slight photoinhibition. Some protection effects of the mFR light were observed (Fig. 8B).

Fig. 8

Photoprotection of PSI by FR light against the FL-1200/30-induced photoinhibition in field-grown plants. Gray bars, Erigeron annuus from an open site; black bars, E. annuus collected from a shaded site; and white bars, Commelina communis from the open site. P_m (A) and F_v/F_m (B) after the treatments for 120 min relative to the initial values before the treatments are shown. FR light intensity was 12.1 W m⁻² (mFR). The leaves were collected in the morning, and kept in the dark with their petioles in distilled water for 60 min. Measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 2°C). The values represent the mean ± SD (n = 3–5 leaves). Different lower case letters indicate statistically significant differences detected by the Tukey–Kramer multiple comparison test (P < 0.05).

Discussion

In this study, we focused on the photoprotection of PSI by FR light against FL-induced photoinhibition. For HL, the irradiance at 1,200 µmol m^–2 s^–1 was chosen to simulate the sunflecks in the field (Pearcy 1990). Also, background light at low irradiances, not complete darkness, was adopted. With this sunfleck-like FL, we demonstrated that wild-type A. thaliana plants and field-grown E. annuus and C. communis plants suffered the PSI photoinhibition (Figs. 1, 8) and that the addition of FR light, the intensity of which was adjusted to the realistic level observed in the field on sunny days, suppressed PSI photoinhibition almost completely (Figs. 2, 8).

In the absence of FR light, the donor-side limitation of PSI in the LL phase was small due to low irradiances of LL (Figs. 4A, 5B ). Because the duration of HL for 800 ms was short enough, the activation state of the acceptor-side reactions of PSI, including those of the Calvin–Benson cycle enzymes, was probably low at least in the initial phases of the FL treatment. In the presence of FR light, P700 tended to be oxidized in the LL phase, and this large donor-side limitation of P700 contributed to suppression of photoinhibition (Figs. 4B, 5B). On the other hand, FR light in the HL phase was effective especially when the acceptor-side reactions of PSI were little activated (Fig. 3; Table 2). The PSI photoprotection by FR light was not marked in CEF-PSI mutants, especially those of NDH-mediated CEF-PSI (Fig. 6). These results strongly indicate that the most effective PSI photoprotection requires FR light and the activity of CEF-PSI, especially NDH-mediated CEF-PSI.

Roles of FR light in LL and HL phases in protecting PSI

In the experiments using continuous red light (Fig. 5), additional FR light did not activate the Y(II). Thus, the rate of photosynthetic CO₂ fixation was not enhanced by FR light either. With respect to the photoprotection by FR light in the present experiments, the contribution of activation of the Calvin–Benson cycle by FR light would be small, because the irradiance level in the LL phase was low and thereby the acceptor-side reactions of PSI would not be very activated. The large Y(ND) in FR light during LL phases also cannot be attributed to the ΔpH-dependent down-regulations.

A more important photoprotective mechanism against FL-induced photoinhibtion would be the non-photochemical energy quenching within PSI by P700⁺ (Trissl 1997). Electrons can only flow to the acceptor side of PSI by excitation of P700. This implies that only the increment of CEF-PSI activity is insufficient for the protection of PSI. Because excess excitation energy is safely quenched by P700⁺ in moderately strong light, it effectively suppresses the electron flow to the acceptor side. The efficient quenching by P700⁺, namely high Y(ND), is therefore very important.

The absence of FR light in HL phases caused the marked PSI photoinhibition after the FL treatment for 30 min (Figs. 2, 3). In our previous paper, we reported that the FL-induced PSI photoinhibition occurred when Y(NA) was large (Kono et al. 2014). In the leaves kept in the dark or in low light, Y(NA) in the absence of FR light is large, because the PSI acceptor-side reactions are largely inactive (Fig. 5; Table 1). In such a situation, the CEF-PSI driven by FR light would alleviate the risk of the electron flow to oxygen. This is also valid in the HL phase of the FL treatment (Table 2). Therefore, in addition to the energy quenching by P700⁺, acceleration of CEF-PSI by the FR light will keep electrons in physiologically normal paths, namely the linear electron flow and CEF-PSI. Then electrons preferentially reduce ferredoxin and NADP⁺, and not O₂ (Chow and Hope 1998).

Our present results can be explained by involvement of these mechanisms, i.e. the energy quenching by P700⁺ and acceleration of CEF-PSI by FR light. The fraction of P700⁺ in the LL phase was greater in the presence of FR light than in its absence, as indicated by high Y(ND) (Figs. 4, 5; Table 1). The pool size of P700, plastocyanin, the Cyt b₆/f complex and plastoquinone may be a few dozen electrons per P700. These components tend to be oxidized when P700 is in the oxidized state (P700⁺). Though small, this oxidized pool in the LL phase due to the presence of FR light at least contributed to suppression of the complete reduction of P700 upon onset of the HL phase (Fig. 4). This effect may explain the difference in PSI photoinhibition between FL-(1200 + hFR)/hFR and FL-(1200 + hFR)/0 (Fig. 3). This is also supported by the finding that the extent of PSI photoinhibition decreased with the intensity of FR light (Fig. 2). Acceleration of CEF-PSI induced by FR light in the HL phase contributed to keeping a large fraction of P700⁺ during the HL phase [Table 2, high Y(ND)]. This large fraction of P700⁺ could efficiently quench the excitation energy.

Moreover, the results shown in Fig. 6 indicate that NDH-mediated CEF-PSI is important in the photoprotection of PSI by FR light. Previous studies have suggested that the NDH-mediated CEF-PSI functions, in particular, at low irradiances in rice (Yamori et al. 2011) and Marchantia polymorpha (Ueda et al. 2012). For A. thaliana, however, it was reported that even the complete disruption of plastid ndh genes did not affect photosynthesis (Hashimoto et al. 2003). It was also shown that the phenotype of an NDH-deficient mutant, pgr5∙crr4-3, of A. thaliana was apparent only when PGR5 was absent (Munekage et al. 2004). These findings imply a minor role for the NDH-mediated CEF-PSI. It has actually been suggested that, for C₃ plants in general, the PGR5-mediated CEF-PSI, rather than the NDH-mediated CEF-PSI, is the main player (Munekage et al. 2004, Okegawa et al. 2008, Wang et al. 2015). We also suggested the major role of the PGR5-mediated CEF-PSI in protection against FL-induced PSI photoinhibition (Kono et al. 2014, Kono and Terashima 2016). On the other hand, it has been reported that chloroplast NDH in A. thaliana formed a supercomplex with PSI, and that this supercomplex formation was required for the stability of the NDH complex (Ifuku et al. 2011, Peng et al. 2011), which would imply important function(s) of the chloroplast NDH in relation to PSI (Peng et al. 2009, Peng and Shikanai 2011). The present data (Figs. 4, 6) indicate that, in the presence of FR light, the NDH-mediated CEF-PSI would mainly contribute to keeping the large fraction of P700⁺ in the HL phases. PSI photoinhibition in the pgr5 mutant, which has the NDH-mediated CEF-PSI, was not completely suppressed, although the difference between the pgr5 and the wild type was not statistically significant. Also, in the presence of FR light, PSI photoinhibition in the mutants in the NDH-mediated CEF-PSI was suppressed to some extent, which could be due to the contribution of the PGR5-mediated CEF-PSI. Thus, some contribution of PGR5-mediated CEF-PSI in PSI photoprotection may be likely. The difference between the present study and the previous studies was attributed to the presence and absence of FR light: FR light was used in the present study. The effects of FR light could be different between the NDH- and PGR5-mediated CEF-PSI. Further investigations are needed to elucidate the detailed mechanisms of PSI photoprotection via the energy quenching by P700⁺ and the acceleration of CEF-PSI by FR light.

The photoprotective effects of FR light would be dependent on the capacity of the energy quenching by P700⁺ and of CEF-PSI. Thus, these protection mechanisms should have limitations. In fact, the A. thaliana Col-0 plants grown at 135 µmol m⁻² s⁻¹, which showed no PSI photoinhibition by the FL treatment with HL at 2,300 µmol m⁻² s⁻¹ in the presence of FR light, suffered marked photoinhibition when HL at 3,600 µmol m⁻² s⁻¹ was used (Supplementary Fig. S5).

State transitions have been known to contribute to preserving PSI in the FL regime. The stn7 mutant of A. thaliana lacks the state transition7 (STN7) kinase, which mediates the state transitions via the phosphorylation of the light-harvesting complex II (LHCII) proteins. Grieco et al. (2012) showed that, in fluctuating white light, growth of the mutant plants was significantly slower than that of the wild-type plants, while the mutant plants showed growth comparable with that of the wild type plants in continuous light. In the present study, FR light in LL phases might induce state 1, as in the stn7 mutant. We checked the effects of the state transitions using the stn7 mutant (Supplementary Fig. S6). P_m levels in stn7 after the treatments in both the presence and absence of hFR light were very similar to those in the Col-0 plants. These results indicate that the effect of the state transitions on photoinhibition by the FL used in the present study was small.

Roles of FR light in protecting PSII

In contrast to PSI, there was little net photoinhibition of PSII by the FL treatments (Fig. 1), possibly because the repair system of PSII was able to keep up with the photoinactivation of PSII as there was light in the LL phases. In complete darkness, there were no recoveries of F_v/F_m and P_m (Fig. 1; Supplementary Fig. S1). With respect to the photoinactivation of PSII, more damage might be observed if lincomycin, an inhibitor of synthesis of the 70 S type protein, had been present. However, even in the FL-1200/0 and FL-(1200 + hFR)/0 treatments for 120 min in which recovery would not occur, the decrease in F_v/F_m was about 5% of the initial level. Thus, the effect of the FL used in the present study on PSII photoinhibition would be small.

Roles of species differences and growth conditions in the FL-induced PSI photoinhibition

The data with A. thaliana and field-grown plants support that the HL-acclimated plants show more tolerance to FL-induced PSI photoinhibition (Figs. 7,8). We also used sunflower (H. annuus, Asteraceae, Supplementary Fig. S6). Two varieties of sunflower plants, which were grown under full sunlight in the field, showed no PSI photoinhibition after the FL-1200/30 treatment for 120 min. A marked PSI photoinhibition was observed only when HL was increased to 6,000 µmol m⁻² s⁻¹. It is worth studying the efficient protection mechanisms in these sunflower plants.

Impact of this study on the photosynthetic studies

It is important to realize the fact that plants in the field are always exposed to FR light in addition to visible light or so-called photosynthetically active radiation (PAR). Wild-type plants of A. thaliana, rice and some field-grown plants showed PSI photoinhibition by short-term FL treatment in the laboratory. In nature, light fluctuates, but plants do not appear to suffer from PSI photoinhibition. This suggests that there must be some mechanisms that protect PSI against the potential damaging effects of FL. In the present study, we clearly showed that the FR light is the key factor in the PSI protection, possibly through quenching excitation energy within PSI and driving CEF-PSI. Monochromatic FR light does not support high rates of photosynthetic O₂ evolution or CO₂ fixation, as was clearly shown by the ‘red drop’ (Emerson and Lewis 1943, Emerson et al. 1957, Brody and Emerson 1959). The roles of FR light have been largely neglected for this reason. In previous studies, photosynthetic activities were measured mostly in the absence of FR light. Because many commercially available systems for analyses of the photosynthetic activities use LEDs, such as red and blue LEDs, and red, green and blue (RGB) LEDs, to supply PAR, they do not adequately characterize photosynthetic functions, unless attention is paid to the roles of FR light.

We showed that the quantum yield of PSI increased in the presence of FR light when the irradiance level was <500 µmol m^–2 s^–1, suggesting that the true activities of PSI and PSII should be assessed in the presence of FR light as in the natural environment. The basic photosynthetic response to light, such as the light response curves and induction curves of photosynthetic parameters, should also be measured in light consisting of not only PAR but also background FR light. Furthermore, mechanisms of various photosynthetic responses, such as photoinhibitions and light acclimation, have been mainly examined using monochromatic LEDs or polychromatic PAR. Our results clearly show that FR light should be taken into account in future photosynthetic studies, especially ecophysiological studies seeking a mechanistic understanding of plant performance in nature.

Materials and Methods

Plant materials

Arabidopsis thaliana wild-type (Col-0) plants were pot-grown in a growth cabinet at room temperature of 23°C and relative humidity of 60%. In the 8 h photoperiod, light was provided by a bank of white fluorescent tubes and the irradiance at the plant level was 135 µmol m⁻² s⁻¹. Plants were irrigated 2–3 times weekly with deionized water for 2 weeks after germination, and afterwards with 1 : 500 strength Hyponex 6-10-5 solution (Hyponex Japan). Hyponex 6-10-5 contained 6.00% total nitrogen (2.90% ammonia-nitrogen and 1.05% nitrate-nitrogen), 10.0% water-soluble phosphate, 5.0% water-soluble potassium, 0.05% water-soluble magnesium, 0.001% water-soluble manganese and 0.005% water-soluble boron. Mature rosette leaves from 7- to 10-week-old plants were detached, and these detached leaves with their petioles kept in distilled water were used in all experiments. We detected no differences between the results obtained with the detached leaves and those with the attached leaves. All measurements were made in ventilated room air (40 Pa CO₂, 21 kPa O₂, at 25°C).

We also grew Col-0 plants at either 30, 60, 240 or 50 µmol m⁻² s⁻¹ with a day-length of 8 h. The plants were grown for 6 months, 10–12 weeks, and 5–7 weeks at 30, 60, and 240 and 500 µmol m⁻² s⁻¹, respectively. Fully expanded mature leaves were used. Growth periods were varied to obtain leaves with sufficient area for reliable measurements.

Arabidopsis thaliana mutants (crr2-2, Hashimoto et al. 2003; crr4-3, Kotera et al. 2005; pgr5, Munekage et al. 2002; and stn7, Bonardi et al. 2005) were also grown at 135 µmol m⁻² s^−1, and other environmental conditions were accomplished in the same manner as for the Col-0 plants. The stn7 mutant was used in Supplementary Fig. S6.

In July 2016, we also used E. annuus and C. communis grown on the campus of the University of Tokyo. Erigeron annuus and C. communis plants were sampled in an open site. Erigeron annuus plants were also sampled in a shaded site (for the hemispherical photographs of these sites, see Supplementary Fig. S4). The leaves were collected in the early morning, and these leaves were kept in the dark for 60 min with their petioles in distilled water.

Fluctuating light and far-red light treatments

In this study, the FL alternating between the HL phases at 1,200 µmol m⁻² s⁻¹ for 800 ms and LL phases at 0, 30 or 135 µmol m⁻² s⁻¹ for 10 s were used. The FL treatment is abbreviated such as FL-1200/30 in the text. For these light treatments, red LEDs (Excelitas/ELCOS GmbH) with the wavelength peak at 635 nm were used. Irradiance levels were software controlled (DualPAM V. 1.19). This enabled very quick changes in the irradiances. FR light was provided by LEDs with the wavelength peak at 740 nm at the intensity of 3.93 (low-FR), 12.1 (moderate-FR) and 25.6 (high-FR) W m⁻² (Supplementary Fig. S2). These intensities are appropriate levels corresponding to the levels observed in the field. FR light was supplied constantly throughout the FL treatments. When FR light was used in combination with red actinic light, the FL treatments are abbreviated as FL-(1200 + FR)/(30 + FR), FL-1200/FR, etc.

Chl fluorescence and 830 nm absorbance change measurements

Chl fluorescence and absorption changes at 830 nm were measured simultaneously using a DUAL-PAM-100 (equipped with a fluorometer for Chl fluorescence, and a near-infrared detector for P700 absorption analysis with emitters at 830 and 875 nm; Walz) in ventilated room air with the detached leaf with its petiole in the water. SPs from red LEDs (7,000 µmol m⁻² s⁻¹, 300 ms duration) were applied to determine the maximum Chl fluorescence with closed PSII centers after dark treatment (F_m) and during illumination (F_m′). The maximum photochemical quantum yield of PSII (F_v/F_m) and the effective quantum yield of PSII [Y(II)] in actinic light were calculated as (F_m − F₀)/F_m and (F_m′ − F_s′)/F_m′ (Genty et al. 1989), respectively, where F_s′ is the steady-state Chl fluorescence level in the actinic light from red LEDs.

Two other PSII quantum yields, Y(NPQ) and Y(NO), which represent the regulated and non-regulated energy dissipation in PSII, respectively, were calculated as F_s′/F_m′ − F_s′/F_m and F_s′/F_m, respectively (Hendrickson et al. 2004). These add up to unity with the photochemical quantum yield [i.e. Y(II) + Y(NPQ) + Y(NO) = 1; for details, see Klughammer and Schreiber (2008)]. F₀′ is the minimal fluorescence yield in actinic light and was estimated as F₀/(F_v/F_m + F₀/F_m′) according to Oxborough and Baker (1997).

With the Dual-PAM-100, P700⁺ was monitored as the absorption difference between 830 and 875 nm in a transmission mode. In analogy to the quantum yields of PSII, the quantum yields of PSI were determined using the SP method (Klughammer and Schreiber 1994). The maximum oxidizable P700, P_m, was determined by application of the SP in the presence of FR light at 740 nm. For A. thaliana and field-grown plants, P_m levels determined in the presence of high FR light at 12.1 and 25.6 W m⁻² were smaller than those in low FR light at 3.93 W m⁻². Therefore, we used low FR light at 3.92 W m⁻² to determine the P_m level. The decrease in P_m is an indicator of PSI photoinhibition. Y(I), Y(ND) and Y(NA) are determined in the light. These add up to unity with the photochemical quantum yield [i.e. Y(I) + Y(ND) + Y(NA) = 1]. To oxidize the intersystem electron carriers, FR light was applied from 200 ms before the start of the saturation pulse to its cessation.

Statistical analyses

The Tukey–Kramer multiple comparison test or Student’s t-test was used.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was partly supported by Japan Society for the Promotion of Science (JSPS) [KAKENHI Grant Number 26291055 (to I.T.) and 16H06552 (to W.Y.)].

Abbreviations

Abbreviations
 
• CEF-PSI

  cyclic electron flow around PSI

 
• FL

  fluctuating light

 
• FR

  far-red

 
• HL

  high light

 
• LED

  light-emitting diode

 
• LL

  low light

 
• NDH

  NADH dehydrogenase-like complex

 
• NPQ

  non-photochemical quenching

 
• PAR

  photosynthetically active radiation

 
• PGR5

  proton gradient regulation 5

 
• ROS

  reactive oxygen species

 
• SP

  saturating pulse

 
• WWC

  water–water cycle

 
• Y(I)

  effective quantum yield of PSI

 
• Y(II) effective quantum yield of PSII; Y(NA)

  acceptor-side limitation of PSI

 
• Y(ND)

  donor-side limitation of P700

 
• Y(NO)

  non-regulated energy dissipation

 
• Y(NPQ)

  regulated energy dissipation

Acknowledgments

We thank Professors Chikahiro Miyake, Kentaro Ifuku, Ko Noguchi and W.S. Chow for helpful discussions. We also thank anonymous reviewers for their critical comments on the early versions of the manuscript.

Disclosures

The authors have no conflicts of interest to declare.