Optimum Dark Adaptation Period for Evaluating the Maximum Quantum Efficiency of Photosystem II in Ozone-Exposed Rice Leaves

Because the transient O3 injury of leaves is lost with time, the evaluation of O3 effect on the maximum quantum efficiency of PSII (Fv/Fm) is difficult. Thus, the authors examined Fv/Fm in rice leaves exposed to different O3 concentrations (0, 0.1, and 0.3 cm·m, expressed as O, O, and O) under different dark adaptation periods (0, 1, 5, 10, 20, and 30 min, expressed as D, D, D, D, D, and D) to ascertain its optimum time span. Fv/Fm was inhibited by O3; however in the O and O plants, it recovered during dark adaptation. In the O plants, Fv/Fm decreased gradually with time. F0 was found to be increased by O3, and it increased further in the O plants during dark adaptation. Under a high light intensity, Fm was decreased by O3, and the O3-induced damage to Fv/Fm was therefore more pronounced. However, the sensitivity of Fm was lower than that of F0. Consequently, the damage to PSII was mainly attributed to the inhibition of electron transport from QA to QB. The Fv/Fm ratio in the O plants was fully recovered at D, and in the O plants, Fv/Fm increased from D to D. The effects of O3 on the xanthophyll cycle-dependent quenching (fast relaxation phase) of qI disappeared when the dark adaptation period was greater than 20 min. However, it was difficult to distinguish the effects of O3 and other factors (e.g., light) before D. The current results demonstrate that the optimum dark adaptation period in rice leaves is 10 min because the effect of O3 remains maximal, while the effects of other factors on Fv/Fm disappear during this period. By accurate measurement of Fv/Fm, the physiology of O3 effect on PSII in rice leaves is precisely evaluated.


Introduction
In the Kanto region of Japan, where rice is cultivated as a staple summer crop, 10 -20 warnings regarding high photochemical oxidants (≧0.12 cm 3 •m −3 ) are received every growing season, and the hourly peak values of these substances are sometimes close to 0.2 cm 3 •m −3 [1].Overall, ozone (O 3 ) accounts for 90% or more of the total photochemical oxidants [2,3].When exposed to O 3 , rice plants suffer damage caused by the inhibition of net photosynthetic rate (P N ) and photosystem II (PSII) [4,5] as well as decreases in the contents of ribulose 1,5-bisphosphate carboxylase/oxygenase [6], the contents of chlorophyll and carotenoids [7], and nitrite reductase activity [8], in addition to visible leaf-related symptoms [4] and breakdown of the cellular ultrastructure [9].Moreover, O 3 suppresses growth [10], alters photoassimilate partitioning [11], and decreases grain yield [10,12,13]. Detrimental effects of O 3 have also been reported in many other crops and trees [14,15].
Because PSII of plants is deteriorated immediately after O 3 exposure [5], the monitoring of PSII is useful as a tool to evaluate O 3 damage and recovery from it.Recently, the examination of chlorophyll fluorescence measurements has become established practice for the diagnosis of changes in PSII caused by environmental stresses such as excessive light and water stress.Many studies assessing the effects of environmental stress on PSII have been conducted [16,17].By analyzing chlorophyll fluorescence measurements, Kobayakawa and Imai [5] showed that the maximum (F v /F m ) and operating (F q '/F m ') quan-tum efficiencies of PSII photochemistry were decreased by acute (5-h) O 3 exposure.In addition, PSII in rice leaves is adversely affected by chronic O 3 exposure [7].Among the indicators obtained through performing chlorophyll fluorescence measurements, F v /F m is used most frequently.F v /F m is determined from the easiest and simplest type of chlorophyll fluorescence measurement.However, before such measurements can be obtained, knowledge regarding the dark adaptation period is required.When a leaf is kept in the dark, the primary quinone acceptor of PSII (Q A ) becomes maximally oxidized.The PSII reaction centers can then perform photochemical reduction of Q A , and the minimal fluorescence in the darkadapted state (F 0 ) can be determined.Thereafter, when a leaf is exposed to a short actinic pulse of high PPFD (typically of less than 1 s at several thousand µmol m −2 •s −1 ), Q A reaches a maximally reduced state, and the maximal fluorescence in the dark-adapted state (F m ) can be determined.Baker [18] described variable fluorescence (F v ) as the difference between F 0 and F m values.Generally, the dark adaptation period for the leaves subjected to this treatment is 30 min.However, Sonoike [19] observed that if the dark adaptation period is prolonged, the effects of short-term stress on PSII will disappear.In fact, many researchers [20][21][22][23][24][25][26][27][28][29] have measured the effect of O 3 on F v /F m based on qualification of chlorophyll fluorescence under various dark adaptation periods, the shortest of which was 5 min [27], and the longest was 60 min [23,28].
This study was conducted to establish the optimum dark adaptation period for measurement of the PSII F v /F m in rice leaves under O 3 stress or stress free conditions to clarify the inhibition and recovery of photosynthesis by O 3 .

Chlorophyll Fluorescence Measurements
A fluorometer (LI-6400-40; Li-Cor Inc., Lincoln, NE, USA) attached to a portable photosynthesis and transpiration measurement system (LI-6400XT; Li-Cor Inc., Lincoln, NE, USA) and a portable fluorometer (MINI-PAM; Heinz Walz GmbH, Effeltrich, Germany) were used to measure the chlorophyll fluorescence of the eighth leaves from 0.1 -1.1 h after O 3 exposure for each of five replicate plants.Chlorophyll fluorescence parameters were determined in these plants by applying 0.2 and 7000 µmol·m −2 ·s −1 of measuring light and a saturating pulse (0.8 s).Prior to the fluorescence measurements, the leaves were kept in the dark for 0, 1, 5, 10, 20, or 30 min (expressed as D 0 , D 1 , D 5 , D 10 , D 20 , and D 30 , respectively).Subsequently, F 0 and F m were determined by irradiating the measuring light and saturating pulses, respectively.In addition to F v /F m , (1/F 0 ) − (1/F m ) was calculated as an indicator of PSII photoinactivation [18,19,31].The dark adaptation treatments and fluorescence measurements were conducted at 28˚C.In Exp. 2, chlorophyll fluorescence was measured using only a portable fluorometer (MINI-PAM; Heinz Walz GmbH, Effeltrich, Germany).

Statistical Analysis
All chlorophyll fluorescence-related data were subjected to a two-way analysis of variance (ANOVA).The data were further subjected to a multiple comparison by Tukey's test to clarify the effects of O 3 concentrations with elapsing dark adaptation period.Statistical analyses were performed using Excel Statistics 2010 for Windows software package (Social Survey Research Information Co. Ltd., Tokyo, Japan).Differences among treated samples were considered statistically significant at P ≤ 0.05 or P ≤ 0.01 compared with non-treated plant group at each time interval.Appropriate standard errors of the means (SE) were calculated, and the results are presented as line graphs.
sponses of the two applied fluorometers.The F 0 , F m and F v /F m values measured using one fluorometer (MINI-PAM) were slightly lower than those measured using the other fluorometer (LI-6400-40); however, the trends were similar (Figure 1).Thus, we employed MINI-PAM fluorometer in the replicate experiment (Exp.2) because it was easier to carry than the LI-6400-40 fluorometer.The F v /F m ratio was lowest at D 0 in all treatments, and F v /F m was found to be decreased in an O 3 -concentration-dependent manner.In the O 0 plants, F v /F m increased from D 0 to D 10 and was then nearly constant from D 10 to D 30 .The F v /F m ratio in the O 0.1 plants increased from D 0 to D 10 , as in the O 0 plants, but then increased further after D 10 .In the O 0.3 plants, F v /F m increased from D 0 to D 5    [18,19].In the present study, the F 0 values recorded in the O 0.1 and O 0.3 plants were generally higher than in the O 0 plants at all measurement times, and the values increased gradually with the progression of dark adaptation (Figures 1(c), (d) and 2(b)).Previous studies in snap bean [20,28] and Betula pendula [24] support the current results that F 0 was increased by O 3 exposure.The increase in F 0 and decrease in F m are induced by the inhibition of electron transport from Q A to Q B and the oxygen-evolving system from the manganese cluster to the tyrosine residue of the D1 protein, respectively [19].Therefore, it appeared that the damage that occurred to PSII in the O 0.  1 and 2) examining the O 3 inhibition of F v /F m , F 0 , F m , and (1/F 0 ) − (1/F m ) support the description provided above regarding the fluorometers (LI-6400-40 and MINI-PAM), with special reference to the dark adaptation period.In Exp. 2, F v /F m was found to be decreased by O 3 treatment, while F 0 was increased (Figures 2(a) and (b)), as observed in Exp. 1.However, the trend found for F m differed from that observed in Exp. 1.The value of F m recovered dramatically with the period of darkness; however, because the decrease that occurred at D 0 was more pronounced than in Exp. 1, this recovery was insufficient (Figure 2 Statistical analyses (Tables 1 and 2) of the results obtained using a single fluorometer (MINI-PAM) supported the existence of differential responses to early dark adaptation periods in the two experiments.In Exp. 2, F 0 , F m , and (1/F 0 ) − (1/F m ) showed lower significant levels than those of Exp. 1 when compared to each of the control plants (O 0 ) at early dark adaptation periods.

Discussion
Consistent with the results of a previous study by our group [5], F v /F m was found to be adversely affected by O 3 in the present study.The F v /F m ratios in all plants   [32] reported that the intercepted radiation is a major determining factor in O 3 inhibition.While the decrease in F v /F m was induced by an increase in F 0 in the present study, in previous studies, a decrease in F v /F m was found to be induced by a decrease in F m in O 3 -exposed lettuce [25] and tobacco [26].Guidi et al. [22] measured the effect of O 3 on chlorophyll fluorescence in 14 bean cultivars and reported that the cause of the inhibition of F v /F m (e.g., increased F 0 and/or decreased Fm)depended on the cultivar.Interestingly, the F v /F m was found to be decreased by both an increase in F 0 and a decrease in F m in chronically O 3 -exposed rice leaves [7].Therefore, the cause of the decrease in F v /F m depends on the species, cultivar, and environmental conditions involved.F v /F m is determined without irradiating actinic light.Therefore, the obtained values can only be decreased by the non-photochemical quenching coefficient (q N ).Three major components of q N have been identified in plant leaves in vivo: the energy (ΔpH)-dependent quenching coefficient (q E ), photoinhibitory quenching coefficient (q I ), and state-transition quenching coefficient (q T ).The relaxation kinetics of these components differ: the half time (t 1/2 ) is 1 min for q E , 5 -10 min for q T , and >30 min for q I [16,17].Because q T is suppressed under strong light, it should not be regarded as a photoprotective mechanism.In this study, the leaves of the plants were exposed to O 3 during sunny daytime hours.Therefore, F v /F m was decreased mainly by q E and q I .q I includes the xanthophyll cycle-dependent component and inactivation of the D1 protein component.The relaxation time of these two components differs: the t 1/2 of the former is shorter than 30 min, whereas that of the latter is longer than 1 h [16,17].In all plants, F v /F m recovered dramatically from D 0 to D 1 (Figures 1(a), (b) and 2(a)).This recovery was likely induced by the relaxation of q E .Consequently, in addition to the O 0.1 and O 0.3 plants, a decrease in F v /F m caused by q E also occurred in the O 0 (control) plants.Furthermore, as the degree of recovery observed in the O 0.3 plants from D 0 to D 1 was higher than in the O 0 and O 0.1 plants, q N was increased by a higher O 3 concentration (O 0.3 ).Additionally, in the O 0 plants, F v /F m did not change from D 10 onward (Figures 1(a), (b) and 2(a)).Consequently, it appeared that q I disappeared in the O 0 plants during a 10 min period of dark adaptation.Thus, because F v /F m decreased in the O 0 plants prior to D 5 , it is difficult to distinguish the effects of O 3 and other factors (e.g., light) prior to this time point.As F v /F m recovered in the O 0.1 plants (increased) from D 10 to D 20 , the xanthophyll cycle-dependent quenching (fast relaxation phase) of q I would also have been increased during that time.Consequently, if the dark adaptation period is greater than 20 min, the effects of O 3 on the fast relaxation phase of q I will disappear.However, when only the effects on D1 protein inactivation are to be evaluated, leaves must be maintained for more than 20 min in the dark.In the O 0.3 plants, F v /F m decreased as the dark adaptation period progressed from D 5 .Therefore, if the dark adaptation period is too long, F v /F m might differ from the value obtained immediately after O 3 exposure.
The results of these experiments imply that the optimum dark adaptation period for evaluating F v /F m of PSII in O 3 -exposed rice leaves is 10 min because the effect of O 3 is maximal at this time, and the effects of other factors on F v /F m disappear.
3 plants was induced mainly by the inhibition of electron transport from Q A to Q B .In addition, as (1/F 0 ) − (1/F m ) was found to be decreased in the O 0.3 plants with the period of darkness (Figures1(g), (h) and 2(d)), PSII was gradually inactivated even during dark adaptation.However, F m was observed to be decreased by O 3 treatment at any measurement time in Exp. 2. The F v /F m obtained at D 0 in Exp. 2 was lower than in Exp. 1.As the O 3 -induced decrease in F m observed in Exp. 2 (Figure 2(c)) was more pronounced than that in Exp. 1 (Figures 1(e) and (f)), it is possible that the detrimental ness (Figures 1(g) and (h)).Statistical analyses (Tables (c)).(1/F 0 ) − (1/F m ) was decreased by O 3 treatment, as in Exp. 1, and this value decreased gradually with the period of darkness in the O 0.3 plants (Figure 2(d)).

Table 2 . Statistical analyses of the chlorophyll fluoresence parameters in rice leaves between control plants (O 0 ) and O 3 -treated plants (O 0.1 or O 0.3 ) under different dark adaptation periods
In the O 0 and O 0.1 plants, F v /F m recovered with the period of darkness, however, because F v /F m decreased gradually with the period of darkness in the O 0.3 plants, we inferred that the inhibition of PSII by . *p < 0.05, **p < 0.01, ***p < 0.001.n.s., not significant by Turkey's test.F 0 , minimal fluorescence in the dark-adapted state; F m , maximal fluorescence in the dark adapted state; F v /F m , maximal quantum efficiency of PSII.

•O 3 . a
Optimum Dark Adaptation Period for Evaluating the Maximum Quantum Efficiency of Photosystem II in Ozone-Exposed Rice Leaves 1755 effect of O 3 was more pronounced in Exp. 2 due to the higher light intensities involved.In fact, the PPFD values recorded at 12:00 h in the natural light growth chamber were approximately 900 and 1100 µmol·m −2 ·s −1 in Exp. 1 and Exp. 2, respectively.Guidi et al. [23] also observed that the inhibition of P N and PSII activity by O 3 was greater under high light intensities (30 -1000 µmol•m −2 · s −1 PPFD).Similarly, Kobayakawa and Imai