TY - JOUR
AU1 - Kornyeyev, Dmytro
AU2 - Logan, Barry A.
AU3 - Tissue, David T.
AU4 - Allen, Randy D.
AU5 - Holaday, A. Scott
AB - Abstract The extent to which PSII photoinactivation affects electron transport (ΦPSII) and CO2 assimilation remains controversial, in part because it frequently occurs alongside inactivation of other components of photosynthesis, such as PSI. By manipulating conditions (darkness versus low light) after a high light/low temperature treatment, we examined the influence of different levels of PSII inactivation at the same level of PSI inactivation on ΦPSII and CO2 assimilation for Arabidopsis. Furthermore, we compared ΦPSII at high light and optimum temperature for wild-type Arabidopsis and a mutant (npq4-1) with impaired capacities for energy dissipation. Levels of PSII inactivation typical of natural conditions (<50%) were not associated with decreases in ΦPSII and CO2 assimilation at photon flux densities (PFDs) above 150 µmol m–2 s–1. At higher PFDs, the light energy being absorbed was in excess of the energy that could be utilized by downstream processes. Arabidopsis plants downregulate PSII activity to dissipate such excess in accordance with the level of PSII photoinactivation that also serves to dissipate absorbed energy. Therefore, the overall levels of non-photochemical dissipation and the efficiency of photochemistry were not affected by PSII inactivation at high PFD. Under low PFD conditions, such compensation is not necessary, because the amount of light energy absorbed is not in excess of that needed for photochemistry, and inactive PSII complexes are dissipating energy. We conclude that moderate photoinactivation of PSII complexes will only affect plant performance when periods of high PFD are followed by periods of low PFD. Introduction PSII is traditionally considered to be the primary target of environmental stresses that lead to photoinactivation (Barber and Andersson 1992, Aro et al. 1993), which we define as an irreversible or slowly reversible decline in the amount of functional PSII complexes. The inactivated PSII complexes cannot use absorbed light energy for photochemistry. As a result, the efficiency of light utilization in the combined pool of active and inactive PSII complexes decreases. In addition to photoinactivation, several regulatory mechanisms are responsible for reversible decreases (down-regulation) in the quantum efficiency of charge separation in PSII. Those mechanisms are associated with light-induced processes occurring in the antennae and reaction centers (see review by Horton et al. 1994), and are thought to reduce the probability of photoinactivation of PSII reaction centers (Horton et al. 1996). One of the approaches to distinguish between photoinactivation and down-regulation is to analyze the relaxation kinetics of non-photochemical quenching of chlorophyll fluorescence (Quick and Stitt 1989, Walters and Horton 1993). Several components of non-photochemical quenching have been identified (reviewed by Maxwell and Johnson 2000, Müller et al. 2001): qE, high energy state quenching (ΔpH dependent); qT, a component associated with state transitions [reduction in the absorption cross-section of PSII after phosphorylation of its light-harvesting complex (LHCII)]; and qI, a component dependent on the photoinactivation of PSII. However, the application of non-photochemical quenching coefficients has the following disadvantages. (i) The sample cannot be moved during experimentation. This requirement significantly complicates the measurements in the field and in the case when multiple samples must be measured. (ii) The coefficients cannot be directly compared with the efficiency of photochemistry. (iii) Non-photochemical chlorophyll fluorescence quenching coefficients provide an estimation of non-photochemical dissipation relative to the dark-acclimated state, and the comparison of two samples with different histories would be problematic (Maxwell and Johnson 2000). An alternative approach exists to study the different processes that regulate photosynthesis; it is known as energy partitioning analysis and is based on the estimation of the quantum yields of the different processes involved in excitation energy utilization in PSII complexes (Genty et al. 1989, Weis and Lechtenberg 1989, Demmig-Adams et al. 1996). We have introduced a modification of this methodology to allow the estimation of the contribution of regulatory and photoinactivation components of non-photochemical dissipation (NPDREG and NPDPI, respectively) to overall energy partitioning in PSII complexes (Kornyeyev et al. 2001, Kornyeyev et al. 2002). Subsequently, other researchers have developed similar approaches (Hikosaka et al. 2004, Hendrickson et al. 2005). In the present research, the analysis of energy partitioning in PSII complexes was used to study the changes in the regulation of PSII activity during and after a photoinactivation treatment. Since an effective PSII repair cycle (Melis 1991, Aro et al. 1993) prevents a substantial loss of functional PSII complexes under most field conditions during the growing season, the extent of PSII inactivation usually does not exceed 50% in experiments conducted under natural conditions (Demmig-Adams et al. 1996, Werner et al. 2001, Jiao et al. 2003, Kornyeyev et al. 2005). The influence of this level of PSII photoinactivation on the rate of electron transport and CO2 assimilation during exposure to stress and, especially, following stress is not well understood. Therefore, our research was focused on the impact of moderate levels of PSII photoinactivation on these processes. Under conditions that lead to photoinactivation, light energy absorbed by PSII antennae exceeds the amount required for electron transport. As a result, the activity (efficiency) of PSII is down-regulated through activation of thermal dissipation, a process that safely converts excess absorbed light energy to heat before it can lead to PSII photoinactivation (Horton et al. 1996). We hypothesize that down-regulation of PSII compensates for the extent of PSII photoinactivation to maintain the combined photoinduced decline in PSII efficiency in accordance with the demand downstream of PSII. This hypothesis can be used to explain the effects of PSII inactivation on the rate of electron transport and CO2 fixation under different levels of photon flux density (PFD). While rates of CO2 uptake and oxygen evolution at low PFD correlate with the light-induced decline in the amount of functional PSII complexes (Ögren and Sjöström 1990, Werner et al. 2001), the reported effects of PSII inactivation under moderate to saturating PFD are inconsistent. On the one hand, it has been shown that a moderate decline in PSII activity does not result in a decrease in the light-saturated rates of CO2 assimilation of willow leaves (Salix sp.) (Ögren and Sjöström 1990) and of natural phytoplankton assemblages (Behrenfeld et al. 1998) or the rate of oxygen evolution of Capsicum annuum leaves (Lee et al. 1999). Lee et al. (1999) concluded that PSII complexes occur in excess of the number needed to sustain light- and CO2-saturated electron transport by about 40%. On the other hand, a correlation between PSII photoinactivation and the rates of electron transport and CO2 assimilation has been observed for the leaves of Chenopodium album treated with an inhibitor of PSII repair (Hikosaka et al. 2004) and for leaves of rice subjected to different periods of low temperature and high PFD to achieve various levels of PSII inactivation (Hirotsu et al. 2005). The goal of this study was to evaluate the effect of PSII photoinactivation on the energy partitioning in PSII complexes and on CO2 assimilation at optimum temperature following a high PFD, chilling treatment. Since not only PSII, but PSI as well undergoes inactivation during illumination at suboptimum temperatures (Sonoike 1999), deconvoluting the effects of PSII photoinactivation from those of PSI presents challenges. Because PSII recovery at low PFD is more rapid than the recovery of PSI, we were able to establish different levels of PSII activity at the same level of PSI activity in Arabidopsis leaves by varying the conditions of recovery. Three hours of low PFD treatment following an exposure to chilling and high PFD conditions allowed for significant PSII recovery with no measurable PSI recovery, whereas 3 h of darkness prevented the recovery of both PSII and PSI. Thus, we were able to develop different PSII activities with a constant PSI activity and relate absorbed energy utilization to alterations in PSII photoinactivation. To test our hypothesis stated above further, we compared the response to a photoinactivation treatment of wild-type Arabidopsis with that of a mutant (npq4-1) impaired in its ability to conduct thermal dissipation. Results Effect of PSII inactivation on energy partitioning in leaf discs after chilling stress Exposure of leaf discs to 2,000 µmol m–2 s–1 and 5°C resulted in similar levels of photoinactivation of PSI and PSII (Fig. 1). The levels of PSI inactivation [the percentage of the amount of photooxidizable P700 left after the light treatment as determined by the differential absorbance changes (810 nm minus 860 nm) selective for absorbance changes caused by P700 (Klughammer and Schreiber 1998)] and PSII inactivation (the decline in values of Fv/Fm) were determined after 3 h of dark acclimation. The values of Fv/Fm that we obtained for the untreated, dark-acclimated, leaves (0.786 ± 0.016) were comparable with values of this parameter reported by other researchers for healthy, dark-acclimated Arabidopsis leaves (Havaux and Kloppstech 2001, Yoshida et al. 2002). When leaf discs were subsequently exposed to 24°C and a low PFD, conditions that favored recovery processes, the dark-acclimated Fv/Fm increased, indicating that PSII activity was restored up to about 84% of the pre-stress level in 3 h. No change in PSI activity was detected during the recovery period (Fig. 1). Our PSI observations are consistent with the data reported for Arabidopsis plants by other researchers (Zhang and Scheller 2004) who estimated PSI activity in isolated thylakoids by measuring the rate of NADP+ photoreduction. Therefore, one can conclude that the 3 h period of recovery under low PFD in our experiment had no substantial effect on the activity of PSI and that the leaf discs treated in this manner and those maintained in the dark differed in PSII, but not in PSI activity. When illuminated at 25°C, the levels of ΦPSII were significantly (P ≤ 0.05) higher for unstressed leaf discs in comparison with the leaf discs previously subjected to low temperature and high PFD (Fig. 2). For the previously stressed discs, ΦPSII determined at PFDs above 150 µmol m–2 s–1 was not significantly different for those discs allowed 3 h of low-light recovery and those maintained in darkness following the stress, supporting our suggestion that moderate inactivation of PSII complexes does not affect the rate of electron transport at high PFD. Only at PFDs below 150 µmol m–2 s–1 were values of ΦPSII higher for the leaf discs with the higher PSII activity. According to our hypothesis, similar levels of ΦPSII at high PFD in leaf samples that differ in PSII activity can be explained by the adjustments in NPDREG to compensate for non-photochemical dissipation associated with PSII inactivation. In order to examine this idea, we used the chlorophyll fluorescence data obtained from leaf discs exposed to 700 µmol m–2 s–1 to estimate the relative amount of the light energy that was dissipated through NPDREG and NPDPI. NPDPI is analogous to qI. Both parameters depend on the amount of non-functional PSII complexes. However, unlike qI, NPDPI represents the portion of the total light energy absorbed by all PSII complexes that is dissipated and reflects the light-induced changes in non-photochemical dissipation that are not readily reversible in the dark. NPDREG combines all non-photochemical processes that are light induced and easily reversible in the dark (qE and qT). Therefore, it can be used to assess overall down-regulation of PSII activity (or the NPDREG of excitation energy in the pool of PSII complexes). NPDREG and NPDPI are related to the pool of PSII complexes being measured in the leaf, not to the individual complexes. For a thorough presentation of the derivation of these parameters, refer to the Materials and Methods section and to Kornyeyev et al. (Kornyeyev et al. 2001, Kornyeyev et al. 2002). As expected, the higher level of PSII inactivation (lower Fv/Fm) led to a higher level of NPDPI in comparison with a lower level of PSII inactivation (higher Fv/Fm) (Fig. 3). However, the total level of light-induced, non-photochemical dissipation in PSII complexes (NPDREG + NPDPI) for the two populations of leaf discs was similar at 700 µmol m–2 s–1. Consequently, NPDREG was lower for leaf discs undergoing dark recovery (low Fv/Fm), compensating for their higher NPDPI. Energy partitioning in PSII of wild-type plants and mutants during a photoinactivation treatment at 25°C Similar changes in the levels of NPDREG and NPDPI were observed during a high PFD treatment (1,500 µµmol m–2 s–1) at a leaf temperature of 25°C for detached Arabidopsis leaves (Fig. 4). No significant changes in Fv′/Fm′ were detected between 20 and 150 min of illumination (Fig. 4A), indicating that the overall level of non-photochemical quenching was stable. At the same time, an increase in NPDPI coincided with a decrease in NPDREG (Fig. 4B). The quantum yield of electron transport in PSII complexes (ΦPSII) did not decline during the illumination period (Fig. 4A), even though Fv/Fm decreased to 69 ± 13% of the initial level after 150 min (not shown). While the leaves exhibited a substantial loss of PSII activity, the decrease in the amount of photooxidizable P700 was less dramatic. After 150 min of exposure to 1,500 µmol m–2 s–1 and 25°C, 84.8 ± 3.7% (means ± SD, n = 4) of the initial PSI activity was detected, providing a circumstance where the leaves had a relatively high PSI activity and a low PSII activity. We used an additional method to manipulate levels of PSII photoinactivation during a high PFD treatment at 25°C. We exposed lincomycin-treated, detached leaves of wild-type Arabidopsis and npq4-1 mutant plants lacking PsbS protein to 1,000 µmol m–2 s–1 at 25°C. After 40 min of illumination, NPDREG levels of 0.54 ± 0.09 and 0.27 ± 0.05 were observed for wild type and the mutants, respectively (means ± SD, n = 4–5). Thus, the development of the regulatory non-photochemical quenching in PSII complexes of npq4-1 plants was considerably inhibited, resulting in a higher sensitivity of PSII to light exposure than for wild-type Arabidopsis (Fig. 5A). During the treatment, no significant differences in PSI photoinactivation were observed between npq4-1 and wild type (88.7 ± 1.4 and 88.8 ± 6.0% of the initial activity for wild type and the mutants, respectively; means ± SD, n = 5, P = 0.974), but Fv/Fm for the mutants was lower than for the wild type (Fig. 5A). Despite the genotypic difference in PSII photoinactivation, ΦPSII was not significantly different for wild type and npq4-1 plants throughout the period of illumination at a PFD of 1,000 µmol m–2 s–1 (Fig. 5B). The effect of PSII inactivation on CO2 assimilation We studied the response of CO2 assimilation to the changes in the level of PSII activity for attached leaves subjected to a low temperature and high PFD treatment (2 h at 2,000 µmol m–2 s–1 and 5°C) followed by recovery under low light (30 µmol m–2 s–1) or in the dark at room temperature (∼24°C). After a post-stress recovery under these different conditions, the attached leaves did not differ significantly with respect to photoinactivation of PSI. Of the initial PSI activity, 76.7 ± 6.3 and 75.5 ± 3.2% remained in the leaves subjected to recovery after stress in darkness and under low light, respectively (means ± SD, n = 3, P = 0.795). However, values of Fv/Fm were different, averaging 0.665 ± 0.021 and 0.421 ± 0.072 (means ± SD, n = 3, P = 0.005) for the two types of leaf samples, respectively. This difference was due to the effective recovery of PSII activity under the low light treatment. The values of Fv/Fm did not change significantly after the gas exchange measurements were completed, being 0.652 ± 0.016 for leaves recovering under low light (P = 0.455) and 0.413 ± 0.041 for leaves recovering in darkness (P = 0.868) (means ± SD, n = 3). Therefore, significant differences in Fv/Fm between the two variants remained (P = 0.001). We found that the rates of CO2 assimilation for stressed Arabidiopsis plants (either after recovery in the dark or under low light) were significantly lower (P ≤ 0.05) than those rates obtained for control plants that were not subjected to the low temperature treatment (Fig. 6). CO2 assimilation measured above 150 µmol m–2 s–1 was not responsive to the differences in PSII activity. The high PFD at which CO2 assimilation saturated is consistent with the maximum PFD of 1,600 µmol m–2 s–1 that these plants experienced in the greenhouse. Discussion The various experiments that we conducted to test the effect of PSII inactivation on ΦPSII and CO2 assimilation during and after exposure to photoinactivating conditions support our hypothesis that a moderate decline in PSII activity exerts little control over electron transport and CO2 assimilation at moderate to high PFD for Arabidopsis. Our findings support the contention of some researchers that such a decline in PSII activity does not affect light-saturated photosynthesis measured as the rate of CO2 assimilation (Behrenfeld et al. 1998) or the rate of CO2-saturated oxygen evolution (Lee et al. 1999). In two studies showing a relationship between PSII inactivation (level of dark-acclimated Fv/Fm) and photosynthesis, either the rate of CO2 fixation was measured only at a low PFD (Werner et al. 2001) or the correlation between Fv/Fm and the maximum rate of CO2 assimilation at high PFDs was substantially weaker than the correlation between Fv/Fm and the initial slope of the light response curve of photosynthesis (at low PFDs) (Hikosaka et al. 2004). Recently, Hirotsu et al. (2005) reported a correlation between Fv/Fm and the post-stress rate of CO2 fixation and ΦPSII for rice leaves when they developed different PSII activities by varying the duration of exposure to high PFD and low temperature. This correlation could have been the result of parallel, independent changes in those parameters. It has been shown that chilling without photoinactivation can have an effect on Calvin–Benson cycle enzyme activities (Kingston-Smith et al. 1997). Also, a decrease in the efficiency of CO2 fixation could contribute to a decline in PSII activity. Nevertheless, the influence of PSII inactivation on electron transport and CO2 assimilation at high PFD cannot be excluded for a species, such as rice, whose PSII complexes may be more sensitive to these conditions than other photosynthetic components. In the study by Hirotsu et al. (2005), the amount of photooxidizable P700 remained at a high, constant level with increasing time of the stress treatment, and the ratio of the quantum yields of PSI and PSII increased, suggesting that electron transport was not restricted by PSI activity. When we imposed a chilling, high PFD stress on Arabidopsis leaves, PSI inactivation was considerable. However, the large difference (56%) in CO2 assimilation between unstressed and stressed leaves when PSI inactivation was only about 24% suggests that chilling in high light negatively affects other factors associated with CO2 assimilation by Arabidopsis. Since, at least in Arabidopsis and field-grown barley leaves (Fig. 1, Zhang and Scheller 2004, Teicher et al. 2000), inactivated PSI complexes do not undergo rapid repair, one might suggest that, depending on the initial ratio of PSII to PSI complexes, sustained PSI inactivation may potentially constrain electron transport during the recovery period. However, at the present time, there is no evidence to support such a suggestion. Our data indicate that the level of PSII inactivation in Arabidopsis leaves attained in our experiments, either because of chilling at high PFD or high PFD alone, affects the quantum yield for electron transport through PSII and the rate of CO2 assimilation only at low PFDs. Since low PFDs are favorable for PSII repair (Wünschman and Brand 1992), even in this environment, the effect may not be lasting or substantial. However, inactivation of PSII in the field might still influence the productivity of plants, since PSII recovery consumes metabolic resources of the plant cell. It appears that considerable PSII inactivation can occur without affecting ΦPSII when the absorption of light energy exceeds the capacity to utilize that energy, because PSII activity is regulated in response to downstream demands (Golding and Johnson 2003). This idea is in agreement with the observation that an inverse correlation between quantum efficiencies of linear electron transport and non-photochemical quenching of excitation energy in PSII complexes exists (Laisk et al. 1997, Melkonian et al. 2004). Under high PFD conditions, plants can down-regulate the quantum efficiency of photochemistry in PSII complexes through increased thermal energy dissipation (Horton et al. 1994, Müller et al. 2001). However, photoinactivation of PSII contributes to non-photochemical dissipation of excitation energy, as well (Walters and Horton 1993). Our data indicate that NPDREG compensates for NPDPI such that the overall level of dissipation matches the capacity of downstream electron sinks. As the amount of functional PSII complexes decreases (lower Fv/Fm), the need for regulated dissipation decreases and down-regulation (NPDREG) declines (Fig. 3, 4). In field experiments with cotton, the levels of NPDREG and NPDPI vary in opposite directions, while the total level of non-photochemical dissipation remains stable throughout the middle part of a solar day (Kornyeyev et al. 2005). Such compensation (NPDREG) is not necessary in low PFD circumstances where the amount of light energy absorbed is not in excess of that needed for photochemistry and inactive PSII complexes are dissipating energy. It has been suggested previously that moderate PSII photoinactivation might contribute to the protection of the remaining functional PSII complexes (Öquist et al. 1992, Flexas et al. 2001, Lee et al. 2001). Indeed, the share of the NPDPI increases during a photoinactivation treatment. However, if NPDPI does not increase, the total level of non-photochemical dissipation could still be realized through its regulatory component (NPDREG), as we have observed shortly after the beginning of illumination (Fig. 4). Therefore, the effect of PSII inactivation on PSII photoprotection may be apparent only in situations when the development of NPDREG is impaired, such as in the case of the mutants that lack PsbS protein or at low temperature and, perhaps, low concentrations of chloroplastic ascorbate that may lead to a reduction in the activity of violaxanthin de-epoxidase of the xanthophyll cycle. In those cases, NPDPI would be a major route towards attaining the total level of non-photochemical dissipation needed. Conclusions Moderate levels of PSII photoinactivation do not affect electron transport and CO2 assimilation in Arabidopsis leaves at moderate and high PFDs. At moderate to high PFDs, the level of PSII down-regulation decreases to compensate for an increase in the amount of photoinactivated, non-functional PSII complexes so that the overall PSII quantum yield remains unaffected. PSII inactivation reduces electron transport and CO2 assimilation only at low PFDs. Thus, PSII inactivation most probably influences the performance of plants only when they experience periods of intense light followed by periods of low light. Materials and Methods Plant material Wild-type Arabidopsis thaliana (var. Columbia) plants and Arabidopsisnpq4-1 plants lacking the chloroplast protein PsbS (Li et al. 2000) were grown in 0.5 l pots in a greenhouse at ∼28/24°C (day/night) with a natural photoperiod and a maximum daily PFD of 1,600 µmol m–2 s–1. Npq4-1 seed was generously supplied by Krishna K. Niyogi, University of California, Berkeley, CA, USA. Plants were fertilized with Hoagland’s solution once a week. The upper, fully expanded leaves of 4- to 6-week-old plants were used for fluorescence and gas exchange measurements. Experimental treatments To develop leaves with different levels of PSII activity and the same PSI activity, dark-acclimated leaf discs were exposed for 2 h to high PFD (2,000 µmol m–2 s–1) at 5°C in the temperature-controlled chamber of an oxygen electrode (Hansatech, King’s Lynn, Norfolk, UK). CO2 was supplied by a flow of humidified, ambient air. The leaves used in the experiments had been subjected to dark acclimation overnight. To assess the extent of PSII and PSI photoinactivation, some discs were removed from the chamber and placed on wetted paper in a Petri dish in the dark at room temperature (∼24°C) for 3 h before being analyzed for PSII and PSI activity, as described below. To develop PSII activities higher than the activities of these darkened leaf discs, some discs were allowed to recover for 3 h at 24°C and a PFD of 30 µmol m–2 s–1 to stimulate PSII repair processes. Following this recovery period, these discs were placed in the dark for 0.5–1 h before assessing PSI and PSII activity. Since the rate of PSII recovery was higher than that of PSI, two types of samples differing in PSII activity, but with the same low PSI activity, were obtained. To assess the effect of different levels of PSII inactivation on CO2 assimilation, gas exchange measurements (see below) were conducted on attached leaves previously subjected to low temperature and high PFD (2 h at 2,000 µmol m–2 s–1 and 5°C). The pots for these plants were covered with thermal insulating material and placed on a slightly warm surface during the photoinhibitory treatment to minimize the impact of low temperature on the root system. Only the targeted leaf and several nearby leaves were illuminated. The humidity did not drop below 60%. After the treatment, the pots were transferred to a bench in the laboratory with a temperature of approximately 24°C for recovery under a low PFD (30 µmol m–2 s–1) followed by darkness or recovery in darkness, only, for at least 3 h to obtain different levels of PSII activity (i.e. different levels of dark-acclimated Fv/Fm) at the same PSI activity. To determine the response to ΦPSII, leaf discs were exposed to a stepwise increase in the illumination from the lowest to the highest PFD. The leaf discs were acclimated to a desired PFD level for at least 10 min (20 min at PFDs <100 µmol m–2s–1), during which time Chl a fluorescence parameters stabilized. A similar procedure was used to obtain the response of CO2 assimilation to increasing PFD for attached leaves. Following a period of light acclimation (10–20 min), gas exchange parameters were monitored for the next several minutes until stable values were obtained before switching manually to the next PFD. The rate of CO2 assimilation for each sample was calculated as an average of 5–10 values recorded within 2 min after 10–20 min of acclimation to a desired PFD. No significant decline in PSII activity was detected as a result of illumination during the measurements (see Results). The impact of the light-induced decline in PSII activity on electron transport and the different components of non-photochemical dissipation of excitation energy was also investigated for detached Arabidopsis leaves subjected to a photoinactivating treatment of 1,500 µmol photons m–2 s–1 at 25°C for 2.5 h. Where noted, leaves were pre-treated with lincomycin to inhibit chloroplast protein synthesis and PSII repair processes. The leaves were harvested before sunrise by cutting their petioles under water. They were then immediately transferred to microfuge tubes containing 1 mg ml–1 lincomycin (863 U mg–1) and kept in the dark for 3–4 h at room temperature (∼24°C). At the end of this dark incubation period, the concentration of lincomycin in the bulk leaf tissue (CI) was 1.7–2.1 mM as estimated from the formula: CI = CS(WS/WL), where CS is the inhibitor concentration in the solution, WS is the weight of the solution taken up by a leaf, and WL is the fresh weight of the leaf (Bilger and Björkman 1994). Chlorophyll fluorescence measurements Chl a fluorescence emission from leaves and leaf discs was measured with a pulse amplitude-modulated fluorometer (PAM 101/103, Heinz Walz GmbH, Effeltrich, Germany). The experimental protocol described by Schreiber et al. (1986) and the nomenclature of Van Kooten and Snel (1990) for basic fluorescence parameters were used. Measurements of Fo and Fo′ were performed after the application of low-intensity (approximately 3 W m–2) far-red light. Saturating light pulses of 1 s duration were provided by a KL 1500 light source (Schott, Wiesbaden, Germany). For the measurements of Fv/Fm in dark-acclimated samples, the surface of the leaf or leaf disc was directly in contact with the fiber optic cable during the measurements of Fo in order to allow the application of the lowest measuring light possible from the PAM 101/103, minimizing QA reduction during illumination by the measuring light that can lead to an underestimation of Fv/Fm. For the measurements of Chl fluorescence emitted by light-acclimated leaves or leaf discs, the samples were kept on wetted paper attached to the temperature-controlled surface of the oxygen electrode chamber (Hansatech, King’s Lynn, Norfolk, UK). The leaf temperature was maintained at 25°C. The actual quantum yield of electron transport through PSII was determined as ΦPSII = (Fm′ – F)/Fm′ (Genty et al. 1989). The decrease in the ratio of variable (Fv = Fm – Fo) to maximal fluorescence (Fm) in dark-acclimated leaves was applied as a widely recognized measure of PSII photoinactivation (Tyystjrävi and Aro 1996). Fv/Fm is more suitable for the assessment of the decline in PSII functional activity than the determination of the D1 protein content for the following reasons: (i) the light treatment can cause damage to other PSII proteins (Andersson and Barber 1996, Henmi et al. 2004); (ii) chloroplasts contain D1 protein that is not associated with active PSII complexes (Melis 1991); (iii) the loss of D1 protein was shown to lag behind the loss of PSII activity during photoinactivation at low temperature (Aro et al. 1990, Schnettger et al. 1994); and (iv) it was shown that the extent of D1 loss does not correlate with the decline in PSII activity (Krieger et al. 1998). In addition, a close correlation between Fv/Fm and the amount of functional PSII reaction centers has been demonstrated under conditions that cause a decline in Fv/Fm as a result of a photoinactivation treatment (Park et al. 1995, Lee et al. 2001). In some cases, a decrease in Fv/Fm may be the result of sustained photoprotective changes in the quantum efficiency of photochemistry in PSII complexes. Since such changes are not readily reversible in the dark and diminish PSII activity even after dark acclimation, they can be considered as a form of inactivation of PSII complexes. Calculation of the regulatory and photoinhibitory components of non-photochemical dissipation in PSII complexes According to Genty et al. (1989), the ratio of variable to maximal fluorescence for light-acclimated leaves (Fv′/Fm′) gives an estimate of the quantum efficiency of excitation energy capture in PSII complexes with open reaction centers (see also Kitajima and Butler 1975, Harbinson et al. 1989, Rohácek 2002). A decrease in Fv′/Fm′ (Fv′ = Fm′ – Fo′) during illumination is associated with the development of non-photochemical quenching of excitation energy in PSII complexes. Some of the processes causing the decrease in the quantum efficiency of photochemistry in PSII complexes can be relaxed in the dark. Therefore, they form a regulatory component of non-photochemical dissipation. At the same time, the inactivation of PSII also contributes to the decrease in Fv′/Fm′. This photoinactivation (‘photoinhibitory’) component of non-photochemical dissipation is not readily reversible in the dark, because the reactivation/repair of PSII complexes requires light energy for protein synthesis (Wünschman and Brand 1992). The deconvolution of non-photochemical dissipation is usually performed by means of the kinetic analysis of the relaxation of the fluorescence quenching (Quick and Stitt 1989, Walters and Horton 1993). We avoided the use of the coefficients of non-photochemical fluorescence quenching for estimating the influence of different processes on light energy utilization in PSII complexes, because to determine these coefficients would have required the maintenance of the positions of the sample and fiber optic cable supplying the measuring beam throughout the experiment. One should also keep in mind that normalization of Fm using Fo levels in order to calculate non-photochemical quenching coefficients in a situation where the sample had been moved relative to the fluorometer was not possible in our case, because photoinactivation affected Fo levels as well (Shen et al. 1996). A simple determination of the remaining functional PSII complexes using changes in Fv/Fm or 1/Fo – 1/Fm (Lee et al. 2001) was not sufficient to estimate the contribution of the different types of non-photochemical dissipation to the utilization of the light energy absorbed by PSII antennae. Therefore, the application of the methodology known as energy partitioning in PSII was more appropriate for this study. This approach was introduced and further developed in the following publications: Genty et al. (1989), Weis and Lechtenberg (1989) and Demmig-Adams et al. (1996). The partitioning of total non-photochemical energy dissipation into its components represented the next logical step based on knowledge of the existence of different processes involved in the dissipation of excitation energy in PSII complexes. The following equations were used to estimate the contribution of NPDREG and NPDPI in the distribution of excitation energy in PSII complexes (further details can be found in Kornyeyev et al. 2001 and Kornyeyev et al. 2002): NPDREG = 1 – (Fv′/Fm′)/(Fv/Fm)PI (1) NPDPI = [1 – (Fv/Fm)PI/(Fv/Fm)](Fv′/Fm′)/(Fv/Fm)PI (2) Parameters Fv/Fm, Fv′/Fm′ and (Fv/Fm)PI reflect the quantum efficiency of photochemistry in PSII centers with open reaction centers before illumination, during illumination and after dark acclimation following illumination, respectively. The NPDREG relaxes in the dark, and the ratio of the quantum efficiencies of photochemistry in the presence and in the absence of down-regulation [(Fv′/Fm′)/(Fv/Fm)PI] was used to calculate the magnitude of this component (equation 1). The changes in Fv/Fm caused by the light treatment [expressed as the ratio (Fv/Fm)PI/(Fv/Fm)] were used to estimate the level of the photoinactivation component. Since the development of NPDREG during the illumination period affects the energy partitioning and lowers the contribution of NPDPI, the coefficient (Fv′/Fm′)/(Fv/Fm)PI was added to equation 2 to account for such changes. The quantum efficiency of photochemistry in PSII complexes under the different circumstances listed above can be expressed as a ratio of rate constants of different processes involved in the utilization of excitation energy (this approach was described in Kitajima and Butler 1975). Fv/Fm = kp/(kp+ kf + kcon) for a dark-acclimated non-stressed sample (3) (Fv/Fm)PI = kp/(kp + kf + kcon + kpi) for a dark-acclimated stressed sample (4) Fv′/Fm′ = kp/(kp + kf + kcon + kpi + kreg) for a light-acclimated sample (5) kp and kf are the rate constants of photochemistry and fluorescence, respectively. kcon, kreg and kpi are the rate constants of different components of thermal dissipation (constitutive, regulatory and photoinactivation, respectively). Constitutive thermal dissipation (kcon) is intrinsic to the structural characteristics of the PSII light-harvesting system. The existence of constitutive thermal dissipation in PSII complexes (thermal dissipation in the dark-acclimated state) is taken into account in a number of the most recent mathematical models dealing with the conversion of light energy in PSII complexes (Oxborough and Baker 2000, Rohácek 2002, Hendrickson et al. 2004, Hikosaka et al. 2004). In terms of these rate constants, the expression for NPDREG and NPDPI can be obtained by replacing the ratios Fv/Fm, Fv′/Fm′ and (Fv/Fm)PI in equations 1 and 2 with the corresponding ratios of the rate constants (equations 3–5). NPDREG = kreg/(kcon + kf + kp + kpi + kreg) (6) NPDPI = kpi/(kcon + kf + kp + kpi + kreg) (7) Thus, NPDREG and NPDPI reflect the quantum efficiencies of the corresponding processes, validating the use of equations 1 and 2 for estimation of their contribution to the utilization of the excitation energy in PSII complexes. P700 activity The relative amounts of photooxidizable P700 in leaves and leaf discs were measured by means of a PAM 101/103 modulated fluorometer (Heinz Walz GmbH, Effeltrich, Germany) equipped with an ED-P700DW emitter-detector unit. P700+ formation was induced by illumination with saturating far-red light of high intensity (15 W m–2) and monitored as differential absorbance changes (810 nm minus 860 nm) selective for absorbance changes caused by P700 (Klughammer and Schreiber 1998). The far-red light was produced by a 102-FR lamp (Heinz Walz GmbH, Effeltrich, Germany) with a far-red-emitting diode and a far-red long pass filter (RG 9, 0.5 mm, Schott, Wiesbaden, Germany). The lamp has an emission maximum at 735 nm. A flash of saturating light (KL 1500 light source, Schott, Wiesbaden, Germany) was used to ensure the oxidation of all active reaction centers of PSI (Klughammer and Schreiber 1994). Gas exchange measurements A Li-Cor model LI-6400 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA) was used to conduct the gas exchange measurements. The response of the rate of CO2 assimilation to PFD was determined at a leaf temperature of 25°C and a CO2 concentration of 370 µmol mol–1. The analyses were performed by means of a stepwise increase in the illumination from lower to higher PFD, as described above. After the attached leaves had acclimated to a desired PFD level, the values of the rate of CO2 assimilation were monitored until a stable rate was obtained. Statistical analyses Pairs of means at each PFD or time point were compared using a Student’s t-test (two-tailed). Means were considered significantly different for P ≤ 0.05. The data were processed using Microsoft Excel 2000 software. Acknowledgments This study was supported by a grant from the Southwest Consortium for Plant Genetics and Water Resources (United States Department of Agriculture funds). View largeDownload slide Fig. 1 Changes in PSI and PSII activity for Arabidopsis leaf discs during a recovery period following a 2 h exposure to 5°C and a PFD of 2,000 µmol m–2 s–1. PSI activity was estimated as the amount of photooxidizable P700, and PSII activity was estimated as the variable to maximum chlorophyll fluorescence (Fv/Fm) of dark-acclimated leaf discs. The average value of Fv/Fm measured before the photoinactivation treatment was 0.786 ± 0.016. Data are expressed as the mean ± SD (n = 5). View largeDownload slide Fig. 1 Changes in PSI and PSII activity for Arabidopsis leaf discs during a recovery period following a 2 h exposure to 5°C and a PFD of 2,000 µmol m–2 s–1. PSI activity was estimated as the amount of photooxidizable P700, and PSII activity was estimated as the variable to maximum chlorophyll fluorescence (Fv/Fm) of dark-acclimated leaf discs. The average value of Fv/Fm measured before the photoinactivation treatment was 0.786 ± 0.016. Data are expressed as the mean ± SD (n = 5). View largeDownload slide Fig. 2 The response of the quantum yield of electron transport in PSII complexes (ΦPSII) to increasing PFD for Arabidopsis leaf discs either exposed to a 2 h stress at 5°C and a PFD of 2,000 µmol m–2 s–1 or unstressed. Two levels of PSII inactivation were developed for the stressed leaf discs by subjecting them to either 3 h in darkness (stress + dark recovery) or under low PFD followed by dark acclimation (stress + low-light recovery) following the photoinactivation treatment. The levels of PSII activity in the samples are shown in Fig. 1. Data are expressed as the mean ± SD (n = 4–6). View largeDownload slide Fig. 2 The response of the quantum yield of electron transport in PSII complexes (ΦPSII) to increasing PFD for Arabidopsis leaf discs either exposed to a 2 h stress at 5°C and a PFD of 2,000 µmol m–2 s–1 or unstressed. Two levels of PSII inactivation were developed for the stressed leaf discs by subjecting them to either 3 h in darkness (stress + dark recovery) or under low PFD followed by dark acclimation (stress + low-light recovery) following the photoinactivation treatment. The levels of PSII activity in the samples are shown in Fig. 1. Data are expressed as the mean ± SD (n = 4–6). View largeDownload slide Fig. 3 The contribution of two routes of non-photochemical dissipation to the utilization of light energy absorbed by PSII antennae. NPDREG = regulated component (down-regulation of PSII that is reversible in the dark). NPDPI = photoinactivation component associated with PSII photoinactivation. Two levels of PSII inactivation were developed for stressed leaf discs, initially exposed for 2 h to 5°C and a PFD of 2,000 µmol m–2 s–1, by subjecting them to either a low PFD followed by dark acclimation (stress + low-light recovery) or 3 h of darkness only (stress + dark recovery), following the high PFD/low temperature treatment. Values of Fv/Fm and Fv′/Fm′ required to calculate NPDREG and NPDPI (see equations 1 and 2) were determined for leaf discs before the stress treatment and after acclimation to a PFD of 700 µmol m–2 s–1 at 25°C, respectively. To determine (Fv/Fm)PI, leaf discs were acclimated to darkness for approximately 1 h. Data are expressed as the mean ± SD (n = 4). View largeDownload slide Fig. 3 The contribution of two routes of non-photochemical dissipation to the utilization of light energy absorbed by PSII antennae. NPDREG = regulated component (down-regulation of PSII that is reversible in the dark). NPDPI = photoinactivation component associated with PSII photoinactivation. Two levels of PSII inactivation were developed for stressed leaf discs, initially exposed for 2 h to 5°C and a PFD of 2,000 µmol m–2 s–1, by subjecting them to either a low PFD followed by dark acclimation (stress + low-light recovery) or 3 h of darkness only (stress + dark recovery), following the high PFD/low temperature treatment. Values of Fv/Fm and Fv′/Fm′ required to calculate NPDREG and NPDPI (see equations 1 and 2) were determined for leaf discs before the stress treatment and after acclimation to a PFD of 700 µmol m–2 s–1 at 25°C, respectively. To determine (Fv/Fm)PI, leaf discs were acclimated to darkness for approximately 1 h. Data are expressed as the mean ± SD (n = 4). View largeDownload slide Fig. 4 Time courses of the quantum yield of electron transport in PSII complexes (ΦPSII) and the variable to maximum chlorophyll fluorescence of light-acclimated, detached Arabidopsis leaves (Fv′/Fm′) (A) and the light-induced changes in the contribution of two components of non-photochemical dissipation in the utilization of light energy absorbed by PSII antennae (B) during exposure to 1,500 µmol photons m–2 s–1 and 25°C. NPDREG = regulated component (down-regulation of PSII that is reversible in the dark). NPDPI = photoinactivation component associated with PSII photoinactivation. Data are expressed as the mean ± SD (n = 3–4). View largeDownload slide Fig. 4 Time courses of the quantum yield of electron transport in PSII complexes (ΦPSII) and the variable to maximum chlorophyll fluorescence of light-acclimated, detached Arabidopsis leaves (Fv′/Fm′) (A) and the light-induced changes in the contribution of two components of non-photochemical dissipation in the utilization of light energy absorbed by PSII antennae (B) during exposure to 1,500 µmol photons m–2 s–1 and 25°C. NPDREG = regulated component (down-regulation of PSII that is reversible in the dark). NPDPI = photoinactivation component associated with PSII photoinactivation. Data are expressed as the mean ± SD (n = 3–4). View largeDownload slide Fig. 5 The decline in PSII activity assessed as the variable to maximum chlorophyll fluorescence (Fv/Fm) (A) and changes in the quantum efficiency of electron transport in PSII complexes (ΦPSII) (B) during illumination at a PFD of 1,000 µmol photons m–2 s–1 and 25°C for dark-acclimated, detached Arabidopsis leaves (wild type and npq4-1 mutants lacking PsbS protein). Leaves were pre-treated with lincomycin to inhibit PSII repair. Data are expressed as the mean ± SD (n = 4–5). View largeDownload slide Fig. 5 The decline in PSII activity assessed as the variable to maximum chlorophyll fluorescence (Fv/Fm) (A) and changes in the quantum efficiency of electron transport in PSII complexes (ΦPSII) (B) during illumination at a PFD of 1,000 µmol photons m–2 s–1 and 25°C for dark-acclimated, detached Arabidopsis leaves (wild type and npq4-1 mutants lacking PsbS protein). Leaves were pre-treated with lincomycin to inhibit PSII repair. Data are expressed as the mean ± SD (n = 4–5). View largeDownload slide Fig. 6 The response of CO2 assimilation to increasing PFD (0–200 µmol m–2 s–1 in A and 400–2,000 in B) for attached Arabidopsis leaves either initially exposed to a 2 h stress at 5°C and a PFD of 2,000 µmol m–2 s–1 or unstressed. Two levels of PSII inactivation were developed for the stressed leaf discs by subjecting them to either a low PFD followed by dark acclimation (stress + low-light recovery) or 3 h in darkness (stress + dark recovery) following the photoinactivation treatment. Data are expressed as the mean ± SD (n = 3–6). View largeDownload slide Fig. 6 The response of CO2 assimilation to increasing PFD (0–200 µmol m–2 s–1 in A and 400–2,000 in B) for attached Arabidopsis leaves either initially exposed to a 2 h stress at 5°C and a PFD of 2,000 µmol m–2 s–1 or unstressed. 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TI - Compensation for PSII Photoinactivation by Regulated Non-photochemical Dissipation Influences the Impact of Photoinactivation on Electron Transport and CO2 Assimilation
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcj010
DA - 2006-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/compensation-for-psii-photoinactivation-by-regulated-non-photochemical-1ooKVKxzHn
SP - 437
EP - 446
VL - 47
IS - 4
DP - DeepDyve
ER -