PSII photochemistry and carboxylation efficiency in Liriodendron tulipifera under ozone exposure

Environmental and Experimental Botany 70 (2011) 217–226

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PSII photochemistry and carboxylation efficiency in Liriodendron tulipifera under ozone exposure Elisa Pellegrini, Alessandra Francini, Giacomo Lorenzini, Cristina Nali ∗ Department of Tree Science, Entomology and Plant Pathology “Giovanni Scaramuzzi”, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy

a r t i c l e

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Article history: Received 23 July 2010 Received in revised form 21 September 2010 Accepted 24 September 2010 Keywords: Air pollution Chlorophyll a fluorescence Oxidative stress Photosynthesis Xanthophyll cycle Urban forest

a b s t r a c t Liriodendron tulipifera is an important forest plant which is commonly used in urban environments as a shade tree. Young plants have been exposed (under controlled conditions) to 120 ppb of O3 for 45 consecutive days (5 h d−1 ). The aim of this investigation was to clarify if O3 limits the physiological performance of L. tulipifera. In treated plants, dynamics related to membrane injury, gas exchange and chlorophyll a fluorescence leads to: (i) increase in lipid peroxidation (maximum value of +78% 15 days after the fumigation, compared to controls); (ii) reduction of photosynthetic activity (up to 66% 28 days after the exposure), twinned with a partial stomatal closure and a store of CO2 in substomatal chambers; (iii) reduction in carboxylation efficiency (−11% at the end of exposure); (iv) damage to PSII, as demonstrated by the increase in the PSII excitation pressure (−57% 28 days after the treatment). On this basis, O3 should be considered very harmful to L. tulipifera, although the reduction of total chlorophylls content and the activation of xanthophyll cycle take place in order to attempt to regulate light absorbed energy limiting oxidative damage. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Tropospheric ozone (O3 ) is both an air pollutant and a greenhouse gas. Between the late 19th century and 1980, concentrations of background O3 in the mid-latitudes of the northern hemisphere doubled to approximately 60–75 ␮g m−3 and have since increased to 80 ␮g m−3 (EEA, 2010). Ozone can no longer be considered a mere local air quality issue, but it is a global problem, requiring a global solution (Royal Society, 2008). A recent analysis strongly indicates that O3 levels in the troposphere increased over western North America during April–May in the period 1995–2008 (Cooper et al., 2010). Even if in Europe O3 levels during summer 2009 were among the lowest in the past decades and observed exceedances were less spatially extensive than in previous years, the overall picture is still worrying. No exceedances of the information threshold value occurred in northern Europe. The highest 1 h ozone concentration of 284 ␮g m−3 was observed in France. The Directive’s long-term objective to protect human health (maximum O3 concentration of 120 ␮g m−3 over 8 h) was exceeded in all EU Member States and other European countries. Indeed, analyses clearly show that: (i) the exceedances occur most of all in the Mediterranean area, (ii) the year-to-year differences in the occurrence are induced substantially by meteorological variations, and (iii) the concentrations measured at rural background level remained unchanged

∗ Corresponding author. Tel.: +39 050 2210552; fax: +39 050 2210559. E-mail address: [email protected] (C. Nali). 0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.09.012

from 1997 to 2009 (EEA, 2010). Future scenarios are alarming, in terms of economic damage derived to the yield losses, especially in countries with economy based on agricultural production (Van Dingenen et al., 2009). Ozone is the most phytotoxic of the common air pollutants and its widespread distribution presents a risk for greater plant damage (Booker et al., 2009). Effects on trees have received greater attention and, for this reason, a high proportion of species have been assessed for sensitivity in terms of several response indicators, such as (i) crown condition, (ii) basal area and (iii) tree-ring increments, (iv) leaf morphology, (v) ramification structure and, recently, (vi) visible leaf injuries (Bussotti and Ferretti, 2009). Effects of O3 on forests are of particular interest, constituting approximately 30% of land area in the world and being important in terms of conservation of biodiversity and mitigation of climate change. Within CONECOFOR (Italian Integrated Programme for Forest Ecosystems Monitoring), a programme to implement the study on the effects of atmospheric pollution and climate change on forest ecosystems, Bussotti and Ferretti (2009) report that, over the period 2003–2007, a total of 45 plant taxa were found to be symptomatic; of these, 23 were not listed as O3 -sensitive either in the current literature or in the lists of sensitive species. Even so, trees have a marked ability to buffer the effects of O3 , thanks to their reserve organs which enable them to enact greater detoxification and defence mechanisms (Nunn et al., 2005). European forests are showing a long-term trend of increased productivity, because of a combination of factors including increased CO2 , N depositions and climate changes (Nabuurs et al., 2003). These factors ameliorate the resilience of

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trees against O3 , but O3 itself is considered a factor potentially capable of reducing the benefits of CO2 and N fertilization (Magnani et al., 2007). However, when functional changes of urban trees are investigated in response to O3 , it is necessary to stress that (i) conclusive proof of these effects is lacking in the literature, (ii) they are confronted by a range of stresses, (iii) the Directive 2008/50/EC states that urban monitoring stations have to be used only to evaluate the protection of human health, although (iv) biomonitoring suggests that urban O3 levels are high enough to damage plants (Nali et al., 2006), in particular in the Southern European countries around the Mediterranean (Paoletti, 2009). According to that, O3 toxic potential to urban forests should be better evaluated, in order to choose species for urban planting especially in cities of Mediterranean area. Ferretti et al. (2007) have reviewed O3 levels, uptake and plant response to O3 in Italy (considered as a hotspot for O3 and representative of the impacts of this pollutant on Mediterranean vegetation, because of its central position in the Mediterranean basin; Paoletti, 2009) and have concluded that, in comparison to central and northern European countries, O3 exposure at remote sites exceeds concentration-based critical levels of United Nation Economic Commission for Europe (UN/ECE), if expressed in terms of AOT40. Among possible reasons, there are establishments of current critical levels in terms of cut-off value and accumulation level, environmental limitations to O3 uptake and inherent characteristics of Mediterranean vegetation. Originally described by Linnaeus, Liriodendron tulipifera (yellow poplar or tulip tree) is one of two species in the Genus Liriodendron in the Magnolia family. A hardwood native to eastern North America, it is has been introduced to many temperate parts of the world, from Europe to South America, from Australia to South Africa and China. It is fast-growing and can grow to more than 50 m, making it a valuable timber tree and is also recommended as a shade tree in urban forestry. A tulip tree (named “Alley Pond Giant” or “Queens Giant”) is “probably the oldest living thing” in New York City, with an estimated age of 450 or more, according to a sign posted in Alley Pond Park (Queens, NYC) (Kilgannon, 2004). Long considered an O3 -sensitive species, yellow poplar is used as a bioindicator of O3 in US forests (Davis and Skelly, 1992), even if some authors considered this species a false positive in bioindicators of air pollution (Hacker and Neufeld, 1992). The long-term effect of O3 on the photosynthesis of trees in urban area has received little attention. Response to O3 of yellow poplar has been studied in potted seedlings under environmentally controlled conditions showing both inhibition and stimulation in growth and physiological processes (Rebbeck and Scherzer, 2002), while most other studies report no significant O3 effects on photosynthesis of yellow poplar seedlings in short-term controlled environment exposures (Rebbeck et al., 2004). At the physiological level, O3 can induce: (i) modification in stomatal conductance to water vapour and in the rate of CO2 photoassimilation (Andersen, 2003); (ii) a decrease in photosynthetic efficiency, as an effect on the light reactions of photosynthesis, which would be reflected by increase in chlorophyll fluorescence and heat dissipation; (iii) a reduction in carboxylation efficiency by an alteration of the biochemical activity of the Calvin cycle (Calatayud et al., 2003). At the biochemical level, O3 causes damage to: (i) photosystems (Cascio et al., 2010); (ii) CO2 fixation sites (Nali et al., 2004); (iii) chlorophyll pigment system (Ranieri et al., 2001); (iv) electron transport and (v) membranes, causing leakage and subsequent ionic imbalance (Francini et al., 2007). Similar conclusions were drawn mostly for crops, but the degree to which crop responses could describe O3 effects in long-lived trees is unclear. The problem is not easy to solve since most of our knowledge comes from studies of young trees growing in controlled or semi-controlled conditions, whose physiology and responses may be different from those of adult trees

(Samuelson and Kelly, 2001). In addition, the toxicology of O3 is very complex, many factors such as species, cultivars, clones, provenances and leaf age playing an important function in determining the overall plant response (Nali et al., 1998). In order to better understand how the main species of urban forest respond to O3 , 1-year-old saplings of L. tulipifera has been exposed to a chronic fumigation with 120 ppb of O3 for 45 consecutive days (5 h d−1 ) in standardized conditions of growth and treatment (to minimize effects of factors altering O3 uptake by the leaves and their response to pollutant). Ozone dose was selected on the basis of a preliminary screening in order to know the toxicity threshold in terms of visible injury. The aim of this investigation was to clarify if O3 limits the physiological performance of L. tulipifera. It is useful to underline that Rebbeck and Scherzer (2002) have reported that the response of field-grown saplings exposed to O3 over 5 years appears to be very similar to responses observed in shorter-term exposures under more controlled conditions. Demonstrating these similarities in O3 responses should greatly enhance the predictive modeling effort to scale the impacts of O3 from juvenile to mature trees (Kolb and Matissek, 2001). 2. Materials and methods 2.1. Plant material, cultural practices and ozone exposure One-year-old saplings of L. tulipifera, grown in plastic pots containing a mix of steam sterilized soil and peat (1:1), were placed for 1 month in a controlled environment facility at a temperature of 20 ± 1 ◦ C, a RH of 85 ± 5% and a photon flux density at plant height of 500 ␮mol photon m−2 s−1 provided by incandescent lamps, during a 12 h photoperiod. Uniform plants, selected when they were 35 cm tall (fourth leaf fully expanded), were placed in a controlled environment fumigation facility under the same climatic conditions as the growth chamber. The entire methodology has been performed according to Francini et al. (2008). Plants were exposed to 120 ± 13 ppb of O3 (for O3 , 1 ppb = 1.96 ␮g m−3 , at 20 ◦ C and 101.325 kPa) for 45 consecutive days (5 h d−1 , in form of a square wave between 09:00 and 14:00). Analyses were performed at 8, 15, 28, 39 and 45 days from the beginning of exposure (FBE). 2.2. TBARS determination and symptom evaluation TBARS (thiobarbituric acid reactive substances) assay, determined according to Hodges et al. (1999), quantifies oxidative stress by measuring the peroxidative damage to membrane lipids that occurs with free radical generation and that results in the production of MDA (malondialdehyde), which reacts with thiobarbituric acid. Leaf discs (0.3 g) of tissue samples were homogenised in 2.5 ml of trichloroacetic acid 0.1% and centrifuged at 10,000 × g for 10 min. The supernatant was collected and 1 ml was mixed with 4 ml of 20% trichloroacetic acid and 0.5% thiobarbituric acid. The mixture was heated at 95 ◦ C (30 min), quickly cooled and centrifuged at 10,000 × g for 10 min. The supernatant was used to determine MDA concentration at 532 nm using a UV–vis spectrophotometer (Biochrom 4060). To correct the measure for possible interference by MDA-sugar complexes, which also absorb around 532 nm, an aliquot of the sample extract was incubated without thiobarbituric acid (TBA) and the absorbance of the solution at 532 nm was subtracted from that containing TBA reagent. Moreover, the absorbance of the sample was also read at 440 nm in addition to 532 and 600 nm. Calculations were performed utilizing the equation TBARS (nmol ml−1 ) = (A–B/157,000) × 106 , where A = [(A532+TBA ) − (A600+TBA ) − [(A532−TBA ) − (A600−TBA )] and B = [(A440+TBA ) − (A600+TBA ) × 0.0571].

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Leaf injury mature symptoms were evaluated manually at the end of the fumigation, on the basis of the percentage of necrotic area on the adaxial surface by overlaying a transparent plastic grid (4 mm) and counting the percentage of intersections covering injured areas with respect to healthy ones. 2.3. Gas exchange and chlorophyll a fluorescence parameters Foliar CO2 and water vapour exchanges were measured with an open infra-red gas exchange system (CIRAS-1, PP-Systems) equipped with a Parkinson leaf chamber, able to clamp single leaves. Details are reported in Francini et al. (2007). Measurements were performed at ambient CO2 concentrations (340–360 ppm) at 80% RH. The chamber was illuminated by a quartz halogen lamp and the leaf temperature was maintained at 26 ± 0.4 ◦ C. Photosynthetic activity at saturating light level (Amax ) was measured at 1200 ␮mol photons−2 s−1 (determined as saturating by preliminary light response curve). The calculation of intercellular CO2 concentration (Ci ) was based on the equations described in von Caemmerer and Farquhar (1981). Stomatal limit (Ls ) values were calculated by the formula of Ls = 1 − Ci /Ca , where Ca is the external CO2 concentration and intrinsic water use efficiency (WUEi ) was determined as the ratio between Amax and stomatal conductance to water vapour (Gw ) (sensu Volkova et al., 2011). Leaf photosynthetic CO2 assimilation responses to irradiance were calculated using the Smith equation (Tenhunen et al., 1976) and determined at a CO2 concentration of 350 ppm. Apparent quantum yield (˚a ) was calculated from the initial slope dA /dPPFD (PPFD, photosynthetic photon flux density) of the curve by linear regression using values got with PPFD below 300 ␮mol m−1 s−1 . Photosynthetic CO2 assimilation rate was recorded after stabilization at each light intensity (0–1200 ␮mol photon m−2 s−1 ). The response of leaf net CO2 assimilation rate (A) to Ci (where Ci < 200 ␮mol mol−1 ) was analyzed according to the mechanistic model of CO2 assimilation proposed by Sharkey (1985). The slope dA /dCi of the regression equation was taken as quantum efficiency (˚CO2 ) of the leaf. The assimilation chamber conditions were maintained at a relative humidity of 63 ± 7% and a temperature of 25 ± 1.1 ◦ C. The maximum carboxylation rate of Rubisco (Vcmax ), the light-saturated rate of electron transport (Jmax ) and the daytime respiration (Rd ) was calculated in according to Dubois et al. (2007). Modulated chl a fluorescence measurements and the status of the electron transport of photosytem II (PSII) were carried out with a PAM-2000 fluorometer (Walz) on the same leaves used for gas exchange dark-adapted for 40 min using a dark leaf-clip. Minimal fluorescence, F0 , when all PSII reaction centers were open, was determined using the measuring modulated light which was sufficiently low (<1 ␮mol m−2 s−1 ) without inducing any significant variable fluorescence. The maximal fluorescence level, Fm , when all PSII reaction centers were closed, was determined by applying a saturating light pulse (0.8 s) at 8000 ␮mol m−2 s−1 in dark adapted leaves. Fluorescence induction was started with actinic light (about 400 ␮mol m−2 s−1 ) and superimposed with 800 ms saturating pulses [10,000 mol m−2 s−1 photon flux density (PFD)] at 20 s intervals to determine maximal fluorescence in the light ). Minimal fluorescence in the light-adapted state adapted state (Fm  (F0 ) was determined immediately after turning off the actinic source in the presence of a far-red (>710 nm) background for 10 s to ensure maximal oxidation of PSII electron acceptors. The intensity of actinic light was maintained at about 400 ␮mol m−2 s−1 and saturating flashes of white light 15,000 ␮mol m−2 s−1 and 800 ms duration were given every 20 s. The saturation pulse method was used for analysis of quenching (qP ) and no-photochemical quenching (qNP ) components as described by Schreiber et al. (1986).  ), that is an estimation of the efficiency The value of ˚exc (Fv : Fm of excitation energy transfer to open PSII traps, was computed

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 and F  in the light-adapted (where Fv is the difference between Fm 0 state). The actual quantum yield of PSII (˚PSII ) was computed as  − F )/F  , where F achieved (F − F  ), is the steady-state fluo(Fm s s t m 0 rescence yield in the light-adapted state, as in Rohacek (2002). The apparent electron transport rate through PSII (ETR) was computed as qP × ˚PSII × PFD × 0.5 × 0.84 (Schreiber et al., 1986). Details are reported in Francini et al. (2007). The coefficient of photochemical quenching (qL ) is a measurement of the fraction of open PSII reaction centres based on the lake model of PSII antenna pigment organisation. This was defined by Kramer et al. (2004) as qP × F0 /Fs . The fraction of absorbed light that was thermally dissipated in PSII antennae (%D) and utilised in PSII photochemistry  ) × 100 and (F  /F  ) × q × 100; (%P) was estimated from 1 − (Fv /Fm v m P the fraction of light absorbed by PSII that is not used in photochemistry nor dissipated in the PSII antenna (%X) was estimated  ) × (1 − q ) × 100, according to Demmig-Adams et al. from (Fv /Fm P (1996).

2.4. Pigment analysis Pigment analysis was performed by HPLC according to Ciompi et al. (1997). 30 mg of leaves previously utilised for gas exchange analysis and fluorescence measurements were homogenised in 3 ml of 100% HPLC-grade methanol overnight. The supernatant was filtered through 0.2 ␮m Minisart SRT 15 filters and immediately analyzed. The extraction was carried out as quickly as possible, in dimmed green light. HPLC separation was performed at room temperature with a Dionex column (Acclaim 120, C18, 5 ␮m particle size, 4.6 mm internal diameter × 150 mm length). The pigments were eluted using 100% solvent A (acetonitrile/methanol, 75/25, v/v) for the first 12 min to elute all xanthophylls, including the resolution of lutein from zeaxanthin, followed by a 3 min linear gradient to 100% solvent B (methanol/ethylacetate, 68/32 v/v), 15 min with 100% solvent B, which was pumped for 15 min to elute chlorophyll b and chlorophyll a and ␤-carotene, followed by 2 min linear gradient to 100% solvent A. The flow-rate was 1 ml min−1 . The column was allowed to re-equilibrate in 100% solvent A for 10 min before the next injection. The pigments were detected by their absorbance at 445 nm. To quantify the pigment content, known amounts of pure standard were injected into the HPLC system and an equation, correlating peak area to pigment concentration, was formulated. 2.5. Statistical analysis A minimum of three plants per treatment were used in each of the three repeated experiments. Following performance of the Shapiro–Wilk W test, data were analyzed using two- or oneway analysis of variance (ANOVA) and comparison among means was determined by least significant difference (LSD) post-test (P ≤ 0.05). The mean differences related to ˚a , ˚CO2 , maximum rate of Rubisco-limited carboxylation (Vcmax ), maximum CO2 saturated photosynthetic rate-limited by electron transport (Jmax ) and daytime respiration (Rd ) were compared by paired-sample ttest (P ≤ 0.05). Linear correlations were applied to: ETR vs Amax , Jmax vs Vcmax and fraction of light thermally dissipated in the antenna (%D) versus de-epoxidation index value (DEPS). Analyses were performed by NCSS 2000 Statistical Analysis System Software. 3. Results 3.1. Visible injury and membrane damage Fourty five days FBE, leaves showed severe minute (Ø 1–2 mm) roundish dark-blackish necrosis located in the interveinal area of the adaxial surface. The injured area was about 40% of the total

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3.2. Dynamics of gas exchange and chlorophyll a fluorescence

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Fig. 1. Time course of TBARS (thiobarbituric acid reactive substances) in Liriodendron tulipifera leaves exposed to ozone (120 ppb for 45 consecutive days, 5 h d−1 ). Data are shown as mean ± standard deviation. The measurements are carried out on plants maintained in filtered air (open circle) and 8, 15, 28, 39 and 45 days from the beginning of exposure (closed circle). Different letters indicate significant differences (P ≤ 0.05). In the box, results of two-way ANOVA are reported, asterisks showing the significance of factors/interaction for: ***P ≤ 0.001, **P ≤ 0.01.

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surface (range 37–42%). No damage was observed in unfumigated (control) plants. Membrane integrity was significantly affected by O3 (Fig. 1). According to the two-way ANOVA test, the interaction between O3 and time was significant, as well as separate factors. In treated plants, an evident increase of peroxidation was observed starting to 8 days FBE (+54% of TBARS levels in comparison with air filtered material). This increase reached a maximum of +78% 15 days FBE.

Gas exchange parameters at light saturation level are reported in Fig. 2. According to the two-way ANOVA test, the interaction between O3 and time was significant for all parameters, as well as the effects of both factors. Starting from 8 days FBE, Amax significantly decreased (−32% compared to controls) and this reduction was maintained during the entire period of fumigation with values in the range between −46 and −66% (Fig. 2A). This decrease was twinned with lower values of Gw (−8, −16, −25, −22 and −13%, 8, 15, 28, 39 and 45 days FBE, respectively) (Fig. 2B). A strong increase in Ci (Fig. 2C) and, consequently, a decrease in Ls (Fig. 2D) were also prolonged during the exposure period (+8, +15, +13, +27 and +32%, for Ci , and −21, −41, −42, −43 and −64%, for Ls , respectively, 8, 15, 28, 39 and 45 days FBE). WUEi ranged between 0.016 and 0.027 in treated plants and between 0.033 and 0.037 in controls (data not shown). Irradiance response curves of CO2 assimilation rate were performed in leaves exposed to filtered air and to O3 15 and 45 days FBE. Control leaves showed typical light response curves, while treated plants strongly decreased their photosynthetic rate. The reduction was already evident 15 days FBE, the light saturation reaching for irradiance values above 300 ␮mol m−2 s−1 and no recovery was observed by increasing fumigation time up to 45 days. At this irradiance, CO2 assimilation approached light saturation with values of maximal photosynthetic CO2 fixation of about 2 ␮mol m−2 s−1 (data not shown). In treated leaves, ˚a was significantly lower than controls in both times of measurement (−80 and −50%, respectively, 15 and 45 days FBE) (Table 1). Referring to parameters derived from CO2 response curve of CO2 assimilation rate (Table 1), ˚CO2 was not affected in the first 15 days of fumigation. After prolonged exposure, this value became lower than that observed in control plants (−33%). Both Vcmax and Jmax decreased regardless the time of exposure (−10 and −11%, for the first, and −7 and −22%, for the last parameter, 15 and 45 days FBE). Relationships between Vcmax and Jmax in both treated and untreated materi-

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Fig. 2. (A–D) Time course of gas exchange parameters in Liriodendron tulipifera leaves exposed to ozone (120 ppb for 45 consecutive days, 5 h d−1 ). Data are shown as mean ± standard deviation. The measurements are carried out on plants maintained in filtered air (open circle) and 8, 15, 28, 39 and 45 days from the beginning of exposure (closed circle). Different letters indicate significant differences (P ≤ 0.05). In the boxes, results of two-way ANOVA are reported, asterisks showing the significance of factors/interaction for: ***P ≤ 0.001, **P ≤ 0.01. Abbreviations: Amax = photosynthetic activity at saturating light level; Gw = stomatal conductance to water vapour; Ci = intercellular CO2 concentration; Ls = stomatal limit.

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Table 1 Foliar gas exchange parameters estimated from irradiance (˚a , apparent quantum yield) and from CO2 [˚CO2 , quantum efficiency; Vcmax , maximum rate of carboxylation (␮mol CO2 m−2 s−1 ); Jmax , light-saturated rate of electron transport (␮mol electrons m−2 s−1 ); Rd , daytime respiration (␮mol CO2 m−2 s−1 )] response curves of CO2 assimilation rate in Liriodendron tulipifera plants exposed to ozone (120 ppb for 45 consecutive days, 5 h d−1 ). Controls were kept in charcoal-filtered air. Measurements were made 15 and 45 days from the beginning of exposure on fully expanded leaves. Data are shown as mean ± standard deviation. For each parameter, asterisks show that the difference between the control and ozonated plants is significant. Days of treatment Control Ozone P Control Ozone P

15

45

˚a

˚CO2

0.05 ± 0.001 0.01 ± 0.001 0.04 ± 0.001 0.02 ± 0.002

0.03 0.02 ns 0.03 0.02

***

***

***

± 0.001 ± 0.002 ± 0.001 ± 0.001

Vcmax

Jmax

Rd

38.5 ± 1.74 34.7 ± 1.40

73.7 ± 2.23 68.9 ± 1.30

*

*

31.1 ± 1.82 27.6 ± 1.12

68.9 ± 3.03 53.9 ± 2.78

−3.1 −3.3 ns −2.6 −4.4

*

**

**

± 0.29 ± 0.19 ± 0.44 ± 0.36

ns = P > 0.05. *** P ≤ 0.001. ** P ≤ 0.01. * P ≤ 0.05.

als were close and linear and did not differ significantly between them (control: y = 0.75x + 45.1, R2 = 0.86; ozonated: y = 2.09x − 3.49, R2 = 0.98). Rd was not consistently affected by O3 15 days FBE, but at the end of fumigation the values were lower as compared to filtered air material (−69%). Coming to chlorophyll fluorescence parameters (Fig. 3), interactions between O3 and time were always significant, as well as the effects of both single factors, with the exception of time

A

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for (1 − qP ). In treated leaves, sensitivity to oxidative stress was determined as changes in Fv /Fm , that provides an estimate of the maximum quantum efficiency of PSII photochemistry (Butler, 1978). In dark-adapted leaves of controls this ratio was in the mean value of 0.818 ± 0.0119 (Fig. 3A), that lies in the range (0.800 ≤ Fv /Fm ≤ 0.860) reported by Björkman and Demming (1987) for healthy plants. Treated plants slowly reduced this ratio, being −8% (compared to control) 45 days FBE. This decrease was due

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Fig. 3. (A–F) Time course of chlorophyll a fluorescence parameters (arbitrary units) in Liriodendron tulipifera leaves exposed to ozone (120 ppb for 45 consecutive days, 5 h d−1 ). The measurements are carried out on plants maintained in filtered air (open circle) and 8, 15, 28, 39 and 45 days from the beginning of exposure (closed circle). Different letters indicate significant differences (P ≤ 0.05). In the boxes, results of two-way ANOVA are reported, asterisks showing the significance of factors/interaction for: ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, ns = P > 0.05. Abbreviations: Fv /Fm = variable and maximal fluorescence ratio; (1/F0 ) − (1/Fm ) = indicator of PSII functionality; ФPSII = actual quantum yield of PSII; ˚exc = efficiency of excitation energy transfer to open PSII traps; (1 − qP ) = reduction state of QA ; qL = coefficient of photochemical quenching.

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Table 2 Fluorescence quenching parameters in Liriodendron tulipifera plants exposed to ozone (120 ppb for 45 consecutive days, 5 h d−1 ). Controls were kept in charcoal-filtered air. Measurements were made 8, 15, 28, 39 and 45 days from the beginning of exposure on fully expanded leaves. Data are shown as mean ± standard deviation. For each parameter, different letters indicate significant differences (P ≤ 0.05). Asterisks show the significance of factors/interaction in the two-way ANOVA. Days of treatment %P

%D

%X

0 8 15 28 39 45 0 8 15 28 39 45 0 8 15 28 39 45

Control 37 37 38 39 41 38 46 45 46 44 41 46 19 19 18 19 18 16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.0 1.5 2.9 3.3 1.8 4.6 1.4 2.4 1.0 2.0 1.1 1.0 3.1 2.0 1.9 1.4 0.9 1.5

Ozone c c c c c c b b b ab ab b ab ab ab ab ab a

37 25 28 29 26 24 46 50 51 50 50 53 19 25 23 23 25 21

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.0 3.1 2.7 2.4 2.9 4.2 1.4 1.7 2.3 3.3 1.4 1.6 3.1 2.6 1.1 2.0 1.6 0.9

P c ab ab b ab a b cd cd c c c ab d cd cd d bc

Ozone

***

Time

*

Ozone × time

***

Ozone

***

Time

**

Ozone × time

*

Ozone

***

Time

*

Ozone × time

*

Abbreviations: %P, fractions of light absorbed by PSII antenna that are used in photochemistry; %D, fractions of light absorbed by PSII antenna that are thermally-dissipated; %X, fractions of light absorbed by PSII antenna not used in photochemistry nor dissipated in the antenna. *** P ≤ 0.001. ** P ≤ 0.01. * P ≤ 0.05.

to a strong and significant increase in F0 prolonged during all the time of exposure associated to Fm similar to controls or lower (data not shown). PSII functionality can be measured conveniently as (1/F0 ) − (1/Fm ), because the decline of this parameter is used as an indicator of photoinactivation of PSII complexes (Lee et al., 1999). Already 8 days FBE (1/F0 ) − (1/Fm ) values significantly decreased in comparison to controls (−21%), reaching progressively the lowest value (−34.0%) 39 days FBE (Fig. 3B). Referring to quenching analysis, ˚PSII values were significantly reduced in treated plants already 8 days FBE (−37% in comparison to controls) to decrease to −44% 39 days FBE (Fig. 3C). Similar patterns were recorded for ETR (data not shown) and ˚exc (Fig. 3D), which reflects the intrinsic efficiency of open PSII reaction centers in the light-adapted state (Calatayud et al., 2006). The relationship between ETR and Amax gives an indication of the capacity of plants to protect PSII from oxidative damage (Lovelock and Ball, 2002), being dependent on all factors that influence stomatal opening like leaf temperature, light level and oxidative stress (Berry and Björkman, 1980): 45 days FBE, the significant correlation in both treated and untreated materials suggests that the O3 -induced effects were well established, as demonstrated by the development of leaf necrosis (y = 23.1x − 44.3, R2 = 0.86, untreated; y = 50.7x − 47.1, R2 = 0.66, treated). Starting from 8 days, FBE (1 − qP ) increased (+52% in comparison to controls) up to end of fumigation, reaching the maximum value of +65% (Fig. 3E). Two-way ANOVA related to qNP showed that O3 factor was the only significant one (P ≤ 0.05). So data were then re-analyzed by one-way ANOVA to highlight the differences due to O3 : the mean value of control plants was 0.725 ± 0.0971 vs 0.788 ± 0.0588 of ozonated ones (Fratio = 7.04, P = 0.011). In untreated leaves, qL reached a mean value of 1.70 ± 0.046, while it strongly decreased in fumigated ones (−34, −44, −57, −50 and −33%, respectively, 8, 15, 28, 39 and 45 days FBE) (Fig. 3F). From the fluorescence quenching parameters, it is possible to estimate the fraction of light absorbed by the PSII antenna used in photochemistry (%P, equivalent to ˚PSII ), a second fraction thermally dissipated in the antenna (%D) and a third fraction not used in photochemistry nor dissipated in the antenna (%X): Table 2 shows results following O3 exposure. Control leaves dissipated and used approximately 45 and 38% of the light absorbed by PSII, respectively. In treated ones, already 8 days FBE only 25% of the absorbed

light of PSII was used in primary photochemistry and 50% was dissipated. A similar trend was observed until the end of fumigation. The fraction %X reached a mean value of 18% in control plants and of 23% in treated ones. 3.3. Leaf pigments Fig. 4 shows the results of leaf pigments content. Interactions, as well as both separate factors, were significant regardless of examined parameter. O3 induced a marked decrease in the total content of chlorophylls starting from 8 days FBE (−24% in comparison to controls), reaching the maximum value of −27% 15 days FBE (Fig. 4A). Lutein (Fig. 4B) and ␤-carotene (Fig. 4C) followed the same trend of chlorophylls (Fig. 4B and C): the levels of these pigments showed significant difference in treated plants in comparison to controls 8 days FBE (−25 and −35%, respectively), with a minimum value at the end of fumigation (−57 and −44%, respectively). The levels of the xanthophylls cycle pigments (VAZ) (Fig. 4D) and the total content of xanthophylls (lutein + neoxanthin + VAZ) (Fig. 4E) also showed a marked decrease starting from 8 days FBE (−24 and −22%, respectively), with values of −28 and −54% at the end of fumigation. DEPS significantly increased, reaching the maximum value at the end of the treatment (+26%, in comparison to controls) with, consequently, a marked activation of the cycle (Fig. 4F). The indication of the role played by xanthophylls cycle in dissipating excess light energy could be explained by the significant correlation in both treated and untreated materials between the efficiency of thermal energy dissipation (%D) and DEPS (y = 0.99x + 24.49, R2 = 0.74, untreated; y = 0.91x + 29.99, R2 = 0.91, treated). 4. Discussion Trees have several positive effects on the urban environment: they reduce runoff and noise and mitigate gaseous air contaminants (like CO2 emitted from combustion, Janssens et al., 2003, and O3 , Paoletti, 2009) and particulate matter (Lorenzini et al., 2006). This ecological function can be modified by adverse factors such as low rainfall and dry soil, extreme temperature, parasite infestations and high sunlight radiation. In particular, O3 is considered a major environmental problem for vegetation in relation to its wide

C

c c

c

c

12 c

ab

6

ab

b

9

ab

a

P Ozone *** Time ** Ozone x Time *

12 10

2

10

b

b

b

b

b

b

8 b 6 4 2

a

a a

a

a

P Ozone *** Time * Ozone x Time *

1.0 0.8

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P Ozone *** Time *** Ozone x Time ***

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d cd 25

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DEPS

Total xanthophylls (μ g mg-1 FW)

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0

E

8

223

0 12

β -carotene (μg mg-1 FW)

B

d

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Lutein (μ g mg-1 FW)

15

VAZ ( μg mg-1 FW)

A

Chlorophyll content (μg mg-1 FW)

E. Pellegrini et al. / Environmental and Experimental Botany 70 (2011) 217–226

bc

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ab 20 ab

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49

0

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49

Time (day)

Fig. 4. (A–F) Time course of leaf pigments content in Liriodendron tulipifera leaves exposed to ozone (120 ppb for 45 consecutive days, 5 h d−1 ). The measurements are carried out on plants maintained in filtered air (open circle) and 8, 15, 28, 39 and 45 days from the beginning of exposure (closed circle). Different letters indicate significant differences (P ≤ 0.05). In the boxes, results of two-way ANOVA are reported, asterisks showing the significance of factors/interaction for: ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05. Abbreviations: DEPS = de-epoxidation index; VAZ = violaxanthin + antheraxanthin + zeaxanthin.

geographical distribution and its chemical activity and, considering that in urban atmosphere during the warm season the concentrations of this pollutant may be well above 100 ppb for many hours (Wang et al., 2009), it is important to understand functional changes of urban trees in response to this pollutant. Very interesting is the case of L. tulipifera, an ornamental species characteristic of urban environment that at the moment is well studied in terms of sensitivity to oxidative stress. This plant has been previously classified as sensitive to O3 in terms of visible symptoms (Mahoney et al., 1984). Studies at different spatial and temporal scales showed conflicting results: some authors reported a significant increase of photosynthetic rate and an enhanced growth of this tree at the stage of flowering and producing seed (Chappelka et al., 2003) and also after O3 exposure with a seasonal 24 h mean ambient O3 concentrations in the range of 32–46 ppb over five seasons (Rebbeck et al., 2004). On the contrary, no alterations of the physiological processes due to chronic treatment (in open-top chambers) with twice ambient O3 for 10 weeks were observed by Loats and Rebbeck (1999). Discrepancies in the literature are probably due to the different conditions of experiments: most studies reporting no significant effects on photosynthesis were carried out on seedlings exposed to short term O3 fumigations in controlled environment, while an evident impact of the pollutant on growth and physiological process was observed when treatments were performed during flowering in open-top chambers.

In this study, the behaviour of L. tulipifera seedlings to chronic treatment with O3 (under controlled environmental conditions) has been assessed in several ways: visible injury, membrane permeability, photosynthetic performance and leaf pigment content. Visible injury has been the criterion used in many intra and interspecies comparisons (He et al., 2007). After 45 days of the treatment, fully expanded leaves showed symptoms similar to ones previously reported elsewhere in natural (Hildebrand et al., 1996) and controlled conditions (Chappelka et al., 2003; Davis and Skelly, 1992). Prior to the presence of visible injury, there was an increase in membrane damage. As reported in other species, exposure to O3 can induce a deleterious effect on function (Guidi et al., 2001), integrity (Francini et al., 2007), conformation (Ranieri et al., 2001) ˙ and transport capacity of membranes (Płazek et al., 2000). Dynamics of gas exchange results strongly altered regardless of the appearance of symptoms: in treated leaves, the consistent decline of photosynthetic activity was associated to partial stomatal closure and store of CO2 in substomatal chamber. This behaviour has been observed by other authors in L. tulipifera (Rebbeck et al., 2004; Rebbeck and Loats, 1997; Tjoelker and Luxmoore, 1992) and in several woody species, like Populus sp. (Guidi et al., 2001), Pinus ponderosa (Andersen, 2003), Picea abies (Wieser et al., 2002) and Ginkgo biloba (He et al., 2009). In addition, Ryang et al. (2009) reported that in yellow poplar exposed to elevated O3 concentration (100–300 ppb for 14 days) the epi-

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dermis around the stomata was swollen and surrounded them like a tower: this morphological change probably also contributed to the low Gw even when the plant shows severe visible injury. However, treated plants showed a greater WUEi than controls. In ozonated air, for the same Amax rate, they showed lower Gw values and therefore they lost less water through transpiration. Thus the stomata functioned well. Farquhar and Sharkey (1982) considered whether stomatal or non-stomatal factors were the main cause of the reduced photosynthetic rate, that can be judged by the changing pattern of both Ci and Ls : in our case, since Amax decreased and Ci increased accompanied by a reduction of Ls , the photosynthetic activity of mesophyll cells rather than stomatal closure was regarded as the critical factor in reducing photosynthetic rate. This indicates that the response of L. tulipifera was not photoprotective down-regulation, but photodamage. Apparent quantum efficiency was also reduced by O3 , showing that yellow poplar was not able to acclimate by self-regulating mechanism, according with that observed by Reichenauer et al. (1997). Actually, there is debate regarding the principal mechanism that induces decrease in photosynthetic rate, with evidence of direct effects of O3 on light and dark reactions of photosynthesis (Power and Ashmore, 2002) or through an indirect stomatal closure effect (Noormets et al., 2001). It is still unclear whether the effects of this pollutant on photosynthetic processes are based on direct oxidative damage within the chloroplast or if they are the result of a signal produced from outside the chloroplast (Wohlgemuth et al., 2002). The loss in photosynthetic capacity in treated plants was correlated to the slow-down of the dark reactions of the Calvin cycle, mainly due to the losses of Rubisco activity. Quantum efficiency, Vcmax and Jmax decreased, indicating that the reduction in CO2 uptake was correlated to a decrease in carboxylation capacity, as also demonstrated by other authors (Guidi et al., 2001; Noormets et al., 2001). The linear relationship between Jmax and Vcmax showed that L. tulipifera was able to optimize resource allocation to preserve a balance between enzymatic (Rubisco) and light-harvesting (chlorophyll) capabilities. The decrease in Rubisco activity may involve several mechanisms, such as an inhibition of protein synthesis, an increased rate of proteolysis or a direct fragmentation by ROS induced by O3 (Degl’Innocenti et al., 2002). In yellow poplar, the decline observed in Rubisco activity induces a lower demand for reducing power and energy (NADPH and ATP) and this may in turn cause a reduction in (i) Fv /Fm ratio, (ii) electron transport rate and (iii) ˚PSII values, determining significant effects of O3 on PSII photochemistry. In treated plants, the reduction in Fv /Fm was indicative of a damage in the efficiency in energy conversion of PSII imputable to a strong increase of F0 during the entire period of exposure. This result is indicative of a photodamage and a chronic photoinhibition. An increase in F0 under O3 stress was already reported (Barnes et al., 1988) and this, coupled with a decrease in Fm , is a possible sign of an inactivation of PSII centres (Reichenauer et al., 1997). A reduction in heat dissipation can also cause an increase in F0 (Demmig-Adams and Adams, 1992). However, the decline of the carboxylation efficiency was shown to be the initial cause of the impairment of photosynthesis. This is because: (i) there was a significant reduction of carboxylation efficiency, but only a slow decrease of Fv /Fm ; (ii) the maximum reduction of Fv /Fm occurred only after carboxylation efficiency had decreased; (iii) reduction in Amax and carboxylation efficiency preceded the reduction of Fm . Some authors suggested that 1/F0 − 1/Fm reflects accurately the PSII functionality (Walters and Horton, 1993). The significant decrease of this parameter in treated plants agrees with the loss of functional PSII complexes, beginning to limit photosynthetic capacity of leaves. The decrease in Fv /Fm was associated with a change in ˚PSII observed in treated plants, indicating that O3 induced an increase in the proportion of closed PSII centers and also a loss of efficiency of excitation trapping by PSII unites (i.e., an impaired

˚exc ). The correlated strong increase in (1 − qP ) during the entire fumigation period is in accord with the idea that the primary target of O3 is the enzymes involved in the Calvin cycle (Bortier et al., 2000). Following Van Buuren et al. (2002), (1 − qP ) can be a reliable measure of the reduction state of the primary quinone acceptor (QA ): higher values of this parameter in treated plants are indicative of a less effective re-oxidation of this electron acceptor, suggesting that some fractions of the PSII traps were closed during actinic illumination. These closed centers, unable to undergo charge separation and to take part in linear electron transport, lead, in turn, to a decline in the quantum yield of PSII. Another important finding is the decrease of qL values in treated plants, indicative of a reduction in open centres exposure, resulting in an increase in the PSII excitation pressure. Similar results were obtained by Guidi and Degl’Innocenti (2008). Light energy absorbed by chlorophyll has to be dissipated in one of these three ways: (i) it can be used to drive photosynthesis (photochemistry); (ii) as heat; (iii) it can be re-emitted as chlorophyll fluorescence (Nielsen and Orcutt, 1996). These three processes are competitive, so that any increase in the efficiency of one will result in a decrease in the yield of the other two (Rohacek and Bartak, 1999). Effects of oxidative stress on photosynthetic process of yellow poplar are well represented by data obtained from the analysis of energy distribution. O3 limits photosynthetic process in treated plants (as indicated by %P which was reduced during the entire fumigation period), distributing the excess of energy into thermal dissipation (%D increase) and in alternative ways (%X increase), according to Calatayud et al. (2001). The importance of xanthophyll cycle-dependent thermal dissipation of absorbed light energy as a photoprotective mechanism has been reported (Demmig-Adams et al., 1996) and this cycle can be considered the dominant component of %D (as shown by a close relationship between this parameter and de-epoxidation index, DEPS). In our study, the action of oxidative stress regarding the activation and pool size of xanthophyll cycle is confirmed by a strong decrease in PSII yield and by an increase in DEPS values in treated plants. Similar results were also reported in poplar clones (differently sensitive to O3 ) exposed to chronic O3 by Ranieri et al. (2000). A general reduction in chlorophyll content was exhibited in treated plants, indicating that there was an evident effect on the chlorophyll binding proteins of the Light-Harvesting Complexes (LHC). Generally, this phenomenon can be interpreted in two ways: damage, when O3 simply initiates chlorophyll breakdown directly or indirectly, or acclimatization to avoid photoinhibition (Mikkelsen et al., 1995). The significant decrease of total chlorophylls and the maintenance of low concentrations of chlorophylls in leaf tissues seems to be a general feature of plants subjected to oxidative stress induced by this pollutant (Calatayud and Barreno, 2001) and, in particular, a consequence of O3 -induced early senescence (Mikkelsen et al., 1995). The absence of chlorophyll synthesis after the fumigation may contribute to a net decline in the chlorophyll content. This result suggests that the reduced plant pigment contents may represent a possible mechanism to protect the PSII from photoinhibition through a reduction of the number of light-harvesting antennae. However, despite the decrease of the total chlorophyll content, the chlorophyll a/b ratio remained unchanged: O3 induced, rather than a reduction of the chlorophyll antenna size, a decline of the number of functioning photosynthetic units, as already reported by Ranieri et al. (2001). At the chloroplast level, an important antioxidant role is played by ␤-carotene and the decrease of this pigment has often been observed in response to O3 (Castagna et al., 2001), deriving by (i) the oxidative degradation operated by oxygen radicals or by (ii) the possible re-organisation of the photosyntethic apparatus induced by the pollutant. The concomitant loss of ␤-carotene and chlorophyll a seems to suggest a major compromising of reaction centers with respect to LHC. Similar conclusions are reported

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by Ranieri et al. (2000) in poplar clones differently sensitive to O3 . Under these circumstances, it is assumed that O3 is an harmful pollutant to L. tulipifera because of: (i) appearance of visible injury and membrane damage; (ii) reduced photosynthetic capacity due to an impairment, initially, of carboxylation efficiency and, then, of PSII photochemistry. On the other hand, it is necessary to underline that the reduction of functional centres along with the activation of the xanthophyll cycle can be a strategy of the plant exposed to several stresses simultaneously in order to attempt to regulate light absorbed energy limiting oxidative damage. Acknowledgments This research was supported by a grant from MIUR (Italy). We gratefully acknowledge Dr. Valentina Picchi for her help with gas exchange measurements. References Andersen, C.P., 2003. Source-sink balance and carbon allocation below ground in plants exposed to ozone. New Phytol. 157, 213–228. Barnes, J.D., Reiling, K., Davison, A.W., Renner, C.J., 1988. Interaction between ozone and winter stress. Environ. Pollut. 53, 235–254. Berry, J.A., Björkman, O., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 31, 491–543. Björkman, O., Demming, B., 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origin. Planta 170, 489–504. Booker, F., Muntifering, R., McGrath, M., Burkey, K., Decoteau, D., Fiscus, E., Manning, W., Krupa, S., Chappelka, A., Grantz, D., 2009. The ozone component of global change: potential effects on agricultural and horticultural plant yield, product quality and interactions with invasive species. J. Integr. Plant Biol. 51, 337–351. Bortier, K., Ceulemans, R., de Temmerman, L., 2000. Effects of ozone exposure on growth and photosynthesis of beech seedlings (Fagus sylvatica). New Phytol. 146, 271–280. Bussotti, F., Ferretti, M., 2009. Visible injury, crown condition, and growth responses of selected Italian forests in relation to ozone exposure. Environ. Pollut. 157, 1427–1437. Butler, W., 1978. Energy distribution in the photochemical apparatus of photosynthesis. Annu. Rev. Plant Phys. 29, 345–378. Calatayud, A., Alvarado, J.W., Barreno, E., 2001. Similar effects of ozone on four cultivars of lettuce in open top chamber during winter. Photosynthetica 40, 195–200. Calatayud, A., Barreno, E., 2001. Chlorophyll a fluorescence, antioxidant enzymes and lipid peroxidation in tomato in response to ozone and benomyl. Environ. Pollut. 115, 283–289. Calatayud, A., Iglesias, D.J., Talòn, M., Barreno, E., 2003. Effects of 2-month ozone exposure in spinach leaves on photosynthesis, antioxidant systems and lipid peroxidation. Plant Physiol. Biochem. 41, 839–845. Calatayud, A., Pomares, F., Barreno, E., 2006. Interactions between nitrose fertilization and ozone in watermelon cultivar Reina de Corazones in open-top chambers. Effects on chlorophyll a fluorescence, lipid peroxidation, and yield. Photosynthetica 44, 93–101. Cascio, C., Schaub, M., Novak, K., Desotgiu, R., Bussotti, F., Strasser, R.J., 2010. Foliar responses to ozone of Fagus sylvatica L. seedlings grown in shaded and in full sunlight conditions. Environ. Exp. Bot. 68, 188–197. Castagna, A., Nali, C., Ciompi, S., Lorenzini, G., Soldatini, G.F., Ranieri, A., 2001. Ozone exposure affects photosynthesis of pumpkin (Cucurbita pepo) plants. New Phytol. 152, 223–229. Chappelka, A.H., Neufeld, H.S., Davison, A.W., Somers, G.L., 2003. Evaluation of ozone injury on foliage of cutleaf coneflower (Rudbeckia laciniata) and crownbeard (Verbesina occidentalis) in Great Smoky Mountains National Park. Environ. Pollut. 125, 53–59. Ciompi, A., Castagna, A., Ranieri, A., Nali, C., Lorenzini, G., Soldatini, G.F., 1997. CO2 assimilation, xanthophyll cycle pigment and PSII efficiency in pumpkin plants as affected by ozone fumigation. Physiol. Plant 101, 881–889. Cooper, O.R., Parrish, D.D., Stohl, A., Trainer, M., Nédélec, P., Thouret, V., Cammas, J.P., Oltmans, S.J., Johnson, B.J., Tarasick, D., Leblanc, T., McDermid, I.S., Jaffe, D., Gao, R., Stith, J., Ryerson, T., Aikin, K., Campos, T., Weinheimer, A., Avery, A., 2010. Increasing springtime ozone mixing ratios in the free troposphere over western North America. Nature 463, 344–348. Davis, D.D., Skelly, J.M., 1992. Foliar sensitivity of eight eastern hardwood species to ozone. Water Air Soil Pollut. 62, 269–277. Degl’Innocenti, E., Guidi, L., Soldatini, G.F., 2002. Characterisation of the photosynthetic response of tobacco leaves to ozone: CO2 assimilation and chlorophyll fluorescence. J. Plant Physiol. 159, 845–853. Demmig-Adams, B., Adams, W.W., 1992. Photoprotection and other responses of plants to high light stress. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 599–626.

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