Changes in Photosystem II Activity and Leaf Reflectance Features of Several Subtropical Woody Plants...

Journal of Integrative Plant Biology 2006, 48 (11): 1274−1286

Received 9 Nov. 2005 Accepted 2 Mar. 2006

Supported by the National Natural Science Foundation of China (30470282).

*Author for correspondence. Tel: +86 (0)20 3725 2995; Fax: +86 (0)20 3725

2831; E-mail: <[email protected]>.

© 2006 Institute of Botany, the Chinese Academy of Sciences

Changes in Photosystem II Activity and Leaf ReflectanceFeatures of Several Subtropical Woody Plants Under

Simulated SO2 Treatment

Nan Liu1, 3, Chang-Lian Peng1, 2*, Zhi-Fang Lin1, Gui-Zhu Lin1, Ling-Ling Zhang1, 3

and Xiao-Ping Pan1

(1. South China Botanical Garden, the Chinese Academy of Sciences, Guangdong Key Laboratory of Digital BotanicalGarden, Guangzhou 510650, China;

2. College of Life Sciences, South China Normal University, Guangzhou 510631, China;3. Graduate University of the Chinese Academy of Sciences, Beijing 100049, China)

Abstract

The effects of simulated SO2 treatment on the photosynthetic apparatus were investigated in five subtropi-cal forest plants, namely Pinus massoniana Lamb., Schima superba Gardn. et Champ., Castanopsis fissa(Champ. ex Benth.) Rehd. et Wils., Acmena acuminatissima (Bl.) Merr et Perry, and Cryptocarya concinnaHance. After leaf sections had been immersed in 0, 20, 50, and 100 mmol/L NaHSO3 for 20 h, total chlorophyll(Chl) content, Chl a/b, maximal photochemical efficiency, and the photochemical quantum yields of photo-system II of all five woody plants were reduced to different degrees, whereas lutein content (Chl base) wasincreased. Two protective mechanisms, namely the xanthophyll cycle (de-epoxidation) and an anti-oxidantsystem (1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacity), showed differences in the degree ofmodulation under simulated SO2 treatment. Compared with control (distilled water treatment), the revisednormalized difference vegetation index, a leaf reflectance index, was lowered with increasing concentra-tions of NaHSO3. Cryptocarya concinna, a dominant species in the late succession stage of subtropicalforests in South China, exhibited less sensitivity to NaHSO3. Conversely, Pinus massoniana, the pioneerheliophyte species, was most susceptible to NaHSO3 treatment. It is suggested that SO2 pollution mayaccelerate the succession of subtropical forest.

Key words: community succession; light intensity; NaHSO3; subtropical forest.

Liu N, Peng CL, Lin ZF, Lin GZ, Zhang LL, Pan XP (2006). Changes in photosystem II activity and leaf reflectance features ofseveral subtropical woody plants under simulated SO2 treatment. J Integr Plant Biol 48(11), 1274−1286.

doi: 10.1111/j.1672-9072.2006.00351.x; available online at www.blackwell-synergy.com, www.jipb.net

Sulfur dioxide (SO2), a primary product of the combustion ofsulfur-containing fossil fuels, was formerly the most importantair pollutant, although the introduction of legislation in recentyears, at least in the developed world, has led to very sub-stantial reductions in emissions (Mansfield 1999). In any case,

long-distance transboundary transport at elevated altitudes inthe atmosphere from industrialized countries contributes to in-creased levels of this pollutant in other regions. As the world’slargest consumer of primary commercial energy and emitter ofSO2, as well as the second largest producer of hard coal,China faces greater risks of acid pollution than any other de-veloping country. The emission of SO2 in China in 2004 was22.549 million tons; in addition, acid rain pollution is becomingmore severe (State Environmental Protection Administration ofChina 2005). It is likely that rain water acidity in China is due tomore local effects than long-range transport and below-cloudscavenging of SO4

2– aerosols and SO2 is the primary mechanism

Changes of Plants Under Simulated SO2 Treatment 1275

creating acid rain (Zhao et al. 1988).The effects of SO2 on vegetation and agriculture, as well as

its role in the formation of acid rain, remains controversial. Ithas been reported that atmospheric SO2 may cross the plas-malemma and enter the symplasm either in the form of sulfate,mainly by active carrier-mediated transport, or in the form ofSO2 by diffusion of sulfate (Rennenberg and Polle 1994). Somestudies have reported that one of the consequences of SO2

treatment is the swelling of thylakoids (Schiffgens-Gruber andLütz 1992; Gonzales et al. 1993). The chloroplast is one of themain targets of SO2 or its degradation products generated inaqueous solution, resulting in an impairment of chloroplast func-tionality through a loss of net CO2 assimilation, a decline in thephotosynthetic electron transport rate, and inhibition of darkreactions of photosynthesis (Veljovic-Jovanovic et al. 1993;Okpodu et al. 1996). Changes were found mainly at the level ofphotosystem (PS) II in young spruce trees following SO2

exposure, particularly affecting the structure of the D1 protein(Lütz et al. 1992).

It is known that SO2 can react with water to yield bisulfite.Within chloroplasts, bisulfite will be further converted to sulfitebecause the pH of the stroma is 8.8 (Kurkdjian and Guern 1989).Reactions initiated by light and mediated by the photosyntheticelectron transport chain lead to the formation of superoxide,hygroxyl radicals, and hydrogen peroxide. Membrane lipids andproteins are the targets of these oxygen radicals. Membranelipids, with unsaturated fatty acids, are easily involved in thefree radical chain reaction, resulting in the decomposition oflipids and oxidation of proteins. Carotenoids, including β-caro-tene (β-Car), lutein, violaxanthin (V), neoxanthin (N), and lowconcentrations of antheraxanthin (A) and zeaxanthin (Z), canoperate as anti-oxidants, minimizing the overoxidation of mem-brane lipids (Frank and Cogdell 1995). Stimulation of the heat-dissipative process, which involves activation of the xantho-phyll cycle, has been reported to be a consequence of severaltypes of stress, including SO2 pollution (Veljovic-Jovanovic etal. 1993; Demmig-Adams and Adams 1996). To counteract thetoxicity of reactive oxygen species, plants have developed ahighly efficient anti-oxidant enzymic defense system, mainlyinvolving superoxide dismutase (SOD), ascorbate peroxidase(APX), catalase (CAT), peroxidase (POD), and glutathione re-ductase (GR), to increase tolerance to different stress factor.This has been researched extensively in recent years (Logginiet al. 1999; Jiang et al. 2002).

The information obtained from a leaf visible/near infraredspectrum is very high, because it provides a concise but veryrich summary of the overall biochemical composition of the leaf(Forley et al. 1998). Many spectral indices of the leaf reflec-tance in the range 400–800 nm can be used to evaluate watercontent, photosynthetic characteristics, oxidative stress, andanti-oxidant protection (Peñuelas and Filella 1998; Peñuelas etal. 2004). There is also a relationship between the foliar

spectra and the chlorophyll (Chl) content and Chla/b ratio (Linand Ehleringer 1982).

Most data reported in literature concern fumigations withvarious concentrations of SO2, along with other pollutants, foreither short (Sandhu et al. 1992; Veljovic-Jovanovic et al. 1993)or long (Garcia et al. 1998; Ranieri et al. 1999) periods.However, little information is available on the effects of thepermeation of NaHSO3 on biochemical and physiological re-sponses of forest plants for an understanding of the effect ofthe intermediate of SO2 metabolism on the forest ecosystem (Jiet al. 2005). To our knowledge, there are no reports using foliarreflectance to analyze the damage caused by SO2 to plants.

South China is located in a south subtropical area with rapidindustrial development and is one of the main acid rain-pollutedareas. The present study focuses on the responses of severaldominant plant species in forest succession grown under dif-ferent light intensities (Figure 1) to simulated SO2 treatment atSouth Botanical Garden. Differences in the physiological re-sponses and protection strategies towards the environmentalconditions may be of major importance for the overall physi-ological performance of the plant and may contribute to com-petitive differences among species.

Results

Chlorophyll and xanthophyll cycle components

The total Chl content of plant leaves decreased gradually withincreasing light intensity and was further lowered by 100 mmol/L NaHSO3 (Table 1). Compared with the other four speciesinvestigated in the present study (Pinus massoniana, Schimasuperba, Castanopsis fissa, and Acmena acuminatissima),

Figure 1. Diurnal changes in natural light intensity in August at theexperimental site in the South China Botanical Garden, Guangzhou,China.

1276 Journal of Integrative Plant Biology Vol. 48 No. 11 2006

Cryptocarya concinna was less affected by NaHSO3 with15.9%, 1.8%, and 5.0% decrements in total Chl content forplants grown at 12%, 32%, and 100% of natural light intensity,respectively. In contrast, the total Chl content in P. massonianadeclined most significantly (28.6%–41.8%) after treatment, in-dicating that this is the most sensitive species to simulated SO2

treatment among the five woody plants investigated.The Chl a/b ratio was higher under 100% natural light inten-

sity than under 32% or 12% natural light intensity (Table 1). TheChl a/b ratios declined after NaHSO3 treatment, except in C.concinna under 100% illumination, which indicated Chl a wasmore sensitive to NaHSO3 than Chl b. The Chl a/b ratios in P.massoniana decreased more significantly than did the Chl a/bratios in the other four species studied. After immersion in 100mmol/L NaHSO3, the Chl a/b ratios of P. massoniana decreasedby 20.3%, 29.1%, and 35.8% under 12%, 32%, and 100% ofnatural light intensity, respectively.

Lutein is the most aboundant component of carotenoids. Asshown in Figure 2A, the lutein content (on Chl base) increasedin NaHSO3-treated leaves, but there were obvious differencesamong the five species under different light intensities. In P.massoniana and C. concinna grown under lower light intensity,lutein content was higher than that in plants grown under 100%natural light intensity. However, in the case of S. superba andC. fissa, the opposite was observed. Thus, compared with100% natural light, lutein was found to be changed most(53.69% by 100 mmol/L NaHSO3) in P. massoniana grown

under 12% natural light and changed least (6.64%) in C. fissa.Except for A. acuminatissima, the changing patterns of the

de-epoxidation state (DES), part of the xanthophyll cycle re-lated to photoprotection, at various concentrations of NaHSO3

were similar to the variations observed in lutein. The DES wasimproved by elevated NaHSO3 concentrations, significantly soin S. superba and C. fissa. (Figure 2B).

The ratio of lutein to the xanthophyll cycle pool size (V+A+Z)also increased with NaHSO3 concentration and the most sig-nificant change was observed in P. massoniana under a 12%natural light intensity (increased 2.04-fold; Figure 2C). Incontrast, this ratio in C. concinna under a 32% light intensityand S. superba under 100% light intensity was increased atlow (< 50 mmol/L), but decreased at high (100 mmol/L), con-centrations of NaHSO3. These results demonstrate that the pro-tective functions of the xanthophyll cycle and lutein dissipationwere accelerated by NaHSO3 treatment.

Reflectance of the leaf upper surface

Reflectance of the leaf upper surface to incident light is theresult of photochemical properties and their relationships withenvironmental factors. Leaf reflectance increased from 400 to554 nm, reaching a maximum at 554 nm (green wavelength),then decreased, reaching a minimum at 677 nm (red wavelength),and, thereafter, increased sharply from 680 nm (Figure 3),which is similar to reports for leaf reflectance in pecan leaves

Table 1. Changes in chlorophyll (Chl) content and the Chl a/b ratio in Pinus massoniana, Schima superba, Castanopsis fissa, Acmenaacuminatissima, and Cryptocarya concinna according to long-term light gradient and short-term NaHSO3 treatment

SpeciesRelative light Total Chl (µg/cm2) Chl a/b ratiointensity (%) 0 mmol/L NaHSO3 100 mmol/L NaHSO3 0 mmol/L NaHSO3 100 mmol/L NaHSO3

P. massoniana 100 0.34±0.04a* 0.20±0.02a* 4.09±0.01a 3.26±0.01a32 0.49±0.07a* 0.35±0.02b* 3.58±0.01b 2.54±0.01b12 0.73±0.04b* 0.45±0.03c* 3.44±0.01b 2.21±0.02b

S. superba 100 18.62±0.87a 15.08±0.33a 3.94±0.01a 3.16±0.01a32 28.19±1.50b 24.99±1.80b 3.71±0.01a 2.42±0.01b12 37.87±0.60c 35.65±0.35c 3.59±0.01a 3.02±0.01a

C. fissa 100 15.53±0.66a 12.10±1.34a 4.30±0.03a 3.34±0.02a32 22.32±1.94b 15.83±1.67b 3.97±0.01b 3.11±0.02a12 32.21±1.65c 24.79±1.24c 3.69±0.01b 3.07±0.01a

A. acuminatissima 100 14.78±1.39a 11.73±0.08a 3.28±0.04a 2.69±0.01a32 23.58±0.30b 20.15±0.77b 3.18±0.01a 2.45±0.01b12 25.82±0.28b 24.99±1.16b 3.04±0.01a 2.77±0.01a

C. concinna 100 2.64±0.10a 2.22±0.05a 3.71±0.11a 3.84±0.08a32 7.78±0.33b 7.64±0.23b 3.44±0.12a 3.09±0.01b12 7.62±0.26b 7.24±0.25b 3.25±0.03b 3.04±0.04b

Data are the mean ± SEM. Within each row, mean values with different letters are significantly different at P<0.05.*Chlorophyll content is expressed as mg/g.


Figure 2. Xanthophyll cycle characters for Pinus massoniana, Schima superba, Castanopsis fissa, Acmena acuminatissima, and Cryptocaryaconcinna after growth at a long-term light gradient and following short-term treatment with NaHSO3 solution.

(A) Lutein content.(B) De-epoxidation state (DES).(C) Lutein/violaxanthin + antheraxanthin + zeaxanthin (V+A+Z) ratio for Pinus massoniana, Schima superba, Castanopsis fissa, Acmenaacuminatissima, and Cryptocarya concinna after growth at a long-term light gradient and following short-term treatment with NaHSO3 solution.( ), 100% natural light intensity; ( ), 32% natural light intensity; ( ), 12% natural light intensity. Data are the mean ± SEM (n=3).


Figure 3. Leaf reflectance spectra of Pinus massoniana, Schima superba, Castanopsis fissa, Acmena acuminatissima, and Cryptocaryaconcinna altered by long-term light gradient and short-term treatment with NaHSO3 solution.

(A,D,G,J,M). Plants grown at 100% light intensity.(B,E,H,I,N). Plants grown at 32% light intensity.(C,F,I,L,O). Plants grown at 12% light intensity. 1, 2, 3, and 4, treatment with 0, 20, 50, and 100 mmol/L NaHSO3, respectively.Each curve is the mean of three or four individual experiments.


(Qi et al. 2003). The ref lectance of C. f issa and A.acuminatissima was higher than that of other species and theleaf reflectance for P. massoniana and S. superba was thelowest among the five species studied. Leaves treated withdistilled water showed the lowest reflectance over the entirespectrum and reflectance increased with increasing concen-trations of NaHSO3. The reflectance of leaves under a 12%natural light intensity was smaller than at 32% or 100% naturallight, and displayed little sensitivity to changes in NaHSO3

concentration. Most leaf reflectance curves were lowest forleaves treated with distilled water and grown under 12% natu-ral light intensity. The leaf reflectance of A. acuminatissimaunder 100% natural l ight increased with the NaHSO3

concentration. However, there was an Einstein shift of thepeak to 583 nm in the reflectance curve and another two newpeaks at 528 and 627 nm.

In the present study, we observed that NaHSO3 had a signifi-cant effect on reflectance at 554 nm. Obvious increases in leafref lectance were found for P. massoniana and A.acuminatissima grown under 100% natural light (71% and 41%,respectively). Reflectance at 554 nm for S. superba and C.fissa grown under a 32% light intensity showed less changefollowing NaHSO3 treatment, with increments of only 22% and21%, respectively. However, NaHSO3 decreased the reflec-tance of C. concinna leaves grown under 100% natural light.The decrease in reflectance at 554 nm associated with a re-duction in the intensity of the light under which plants weregrown may be due to the higher absorption of light by thehigher Chl content at a lower light intensity (Table 1).

The Chl NDI is a normalized difference vegetation index(Gitelson et al. 1994). With the exception of C. concinna, thelowest reflectance value was observed at 100% natural lightillumination (Table 3). The Chl NDI was increased followingNaHSO3 treatment in all woody plants tested, of which P.massoniana changed the least. Using analysis of variance(ANOVA), it was found that l ight intensities, NaHSO3

concentration, and plant species significantly affected valuesof Chl NDI (P < 0.001) and combined factors, namely light inten-sity × plant species, also significantly affected Chl NDI (P <0.001). The other two interactions, namely light intensity ×NaHSO3 concentration and species × NaHSO3 concentration,had no significant effect on Chl NDI (P=0.060 and 0.473,respectively).

Chlorophyll fluorescence

The maximum efficiency of PSII photochemistry (Fv/Fm, whereFm is the maximum fluorescence yield and Fv is variablefluorescence) and the effective photochemical quantum yieldsof PSII (ΦPSII) of plant leaves were higher at lower light intensi-ties (12% and 32% natural light intensity) than at 100% naturallight intensity. After treatment with NaHSO3, both Fv/Fm and

ΦPSII decreased with increasing concentrations of NaHSO3 (from0 to 100 mmol/L) in P. massoniana, S. superba, and C. fissa.However, Fv/Fm and ΦPSII first increased at a low concentration(20 mmol/L) of NaHSO3, then decreased at higher concentra-tions (from 20 to 100 mmol/L NaHSO3) in A. acuminatissimaand C. concinna (Figure 4). The results indicate that A.acuminatissima and C. concinna seem to have a greater toler-ance to simulated SO2 treatment. For plants grown at differentlight intensities, the two Chl fluorescence parameters differedbetween plant species at 20–50 mmol/L NaHSO3 but the differ-ences were reduced at the high concentration of NaHSO3.

Anti-oxidant capacity (1,1-diphenyl-2-picrylhydrazylradical-scavenging capacity)

Scavenging of the 1,1-diphenyl-2-picrylhydrazyl free radical(DPPH·) is a rapid, simple, sensitive, and practical assay forthe evaluation of the anti-oxidant capacity of plants (Peng et al.2000). Plants grown under 100% natural light had higher anti-oxidant capacities compared with those grown under lowerlight intensities (Figure 5). These differences were much moresignificant for P. massoniana and C. concinna, which indi-cates that higher irradiation by sunlight stimulates anti-oxidantenzymes and anti-oxidants and, consequently, improves theplants’ scavenging capacity of free radicals. With increasingconcentrations of NaHSO3, the anti-oxidant capacity of all fiveplants increased at first, but then declined. After treatment with20 mmol/L NaHSO3, the DPPH·-scavenging capacity was im-proved by 229%, 121%, 76%, 87%, and 100% for P.massoniana, S. superba, C. fissa, A. acuminatissima, and C.concinna, respectively, compared with control. The DPPH·-scavenging capacity at 50 mmol/L NaHSO3 was close to that ofsamples treated with 20 mmol/L NaHSO3. Further increases inthe concentration of NaHSO3 resulted in decreases in the anti-oxidant capacity. The results demonstrate that leaves wereinduced to produce harmful free radicals by low concentra-tions of NaHSO3, which led to enhanced anti-oxidant capacity.However, when higher NaHSO3 concentrations were used,the plant anti-oxidant systems may have been damaged byexcessive free radicals, which would have contributed to thereduction in anti-oxidant function.

Discussion

When SO2 enters the leaf by diffusing through the stomata, itmust be dissolved in extracellular water before it can have anyeffect on cellular function. Thus, the use of buffered H2SO3 toexpose cells to different concentrations of SO2 is arguablyrealistic (Taylor et al. 1981). It has been reported the SO2/HSO3

–

may cause injury in a certain position, restrain the Calvin cycle,and affect the electron transport rate (Rennenberg 1991;


Figure 4. Maximum efficiency of PSII photochemistry (Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence)and effective photochemical quantum yields of photosystem II (ΦPSII) of leaves from Pinus massoniana, Schima superba, Castanopsis fissa,Acmena acuminatissima, and Cryptocarya concinna after growth at a long-term light gradient and following short-term treatment with NaHSO3

solution.

( ), 100% natural light intensity; ( ), 32% natural light intensity; ( ), 12% natural light intensity. Data are the mean ± SEM (n=6).

Veljovic-Jovanovic et al. 1993). Chronic SO2 exposure affectsphotosynthesis in forest plants, depending on the exposuredose and the species analyzed (Garcai et al. 1998). In the

present study, after NaHSO3 treatment, total Chl content, Chl a/b, Fv/Fm, and ΦPSII were reduced to different extents, whereasan increase in lutein content based on Chl was detected in the


Chl b and most carotenoids (including lutein) are the main com-ponents of the light-harvesting protein complex. Reductions inthe Chl a/b ratio and elevations of lutein content indicate thatthe stability of Chl b, lutein, and the related light-harvestingantenna pigments complex was better than that of Chl a, which

Table 2. Reflectance rate (%) at 554 nm of leaves from Pinus massoniana, Schima superba, Castanopsis fissa, Acmena acuminatissima, andCryptocarya concinna according to long-term light gradient and short-term NaHSO3 treatment

Species Relative light intensity (%) NaHSO3 concentration (mmol/L)

0 20 50 100P. massoniana 100 9.35a 13.99b 15.67b 16.05b

32 10.96a 10.08a 10.42a 10.17a12 9.54a 8.87a 8.55a 11.06a

S. superba 100 11.37a 10.44a 10.87a 11.43a32 10.29a 10.01a 10.46a 12.58b12 8.47a 8.81a 10.53a 8.71a

C. fissa 100 22.63a 23.29a 23.50a 22.48a32 13.26a 14.31a 15.17b 16.08b12 12.90a 12.70a 12.79a 13.87a

A. acuminatissima 100 16.60a 18.51a 19.62b 23.53b32 13.34a 14.70a 17.19b 15.17b12 14.64a 15.05a 13.90a 12.65a

C. concinna 100 19.81a 18.09b 18.38b 18.39b32 11.60a 12.41b 14.54b 15.44b12 11.17a 11.60a 11.87a 12.35b

Within each row, mean values with different letters are significantly different at P<0.05.

five plants examined. A reduction in the Chl a/b ratio indicatesthat the ratio of the reaction center pigments to light-harvestingpigments in the photosystem on the thylakoid membrane in chlo-roplasts was altered by simulated SO2 treatment. Chlorophyll ais the main pigment in the photosystem reaction center, whereas

Table 3. Changes in Chl NDI for Pinus massoniana, Schima superba, Castanopsis fissa, Acmena acuminatissima, and Cryptocarya concinnaaccording to long-term light gradient and short-term NaHSO3 treatment

Species Relative light intensity (%) NaHSO3 concentration (mmol/L)

0 20 50 100P. massoniana 100 0.517 6a 0.528 0a 0.520 8a 0.510 8a

32 0.621 0a 0.653 4a 0.573 5a 0.452 1b12 0.679 3a 0.668 5a 0.551 3b 0.512 5b

S. superba 100 0.587 7a 0.435 0b 0.482 4b 0.445 9b32 0.640 7a 0.643 0a 0.550 9b 0.544 6b12 0.672 5a 0.600 9a 0.616 3a 0.619 4a

C. fissa 100 0.438 1a 0.385 3b 0.365 1b 0.307 2b32 0.628 7a 0.596 5a 0.455 6b 0.345 2b12 0.505 6a 0.521 2a 0.595 4a 0.451 0b

A. acuminatissima 100 0.425 7a 0.408 5a 0.267 9b 0.236 6b32 0.467 4a 0.435 6a 0.334 8b 0.267 8b12 0.589 0a 0.476 6b 0.443 0b 0.441 1b

C. concinna 100 0.592 4a 0.707 6b 0.585 6a 0.617 2a32 0.577 2a 0.531 6a 0.432 5b 0.301 0b12 0.631 4a 0.593 2a 0.533 2b 0.497 2b

Within each row, mean values with different letters are significantly different at P<0.05.


means that the reaction center was more vulnerable tosimulated SO2 treatment than the light-harvesting antennasystem. Decreases in Fv/Fm and ΦPSII indicate that part of thePSII photochemistry and effective quantum yield of photochemi-cal energy conversion in PSII were inactivated by NaHSO3.Among the five woody plants investigated herein, significantchanges in Chl a/b, lutein content, Fv/Fm, and ΦPSII were foundfor P. massoniana. However, there was little change in theseparameters at low concentrations of NaHSO3; at higherconcentrations, these parameters were obviously decreasedin A. acuminatissima and C. concinna. Thus, we deduced thatP. massoniana was more sensitive to lower concentrations ofNaHSO3 than A. acuminatissima and C. concinna but the inhi-bition of PSII activities of the latter two species was greaterthan in the former species in the presence of higher concen-trations of NaHSO3.

There is a growing consensus that air pollution, like SO2, andother forms of environmental stress to which plants may besubjected result in the formation of harmful free radicals(superoxide, hydroxyl radical etc.), which are toxic to the bio-logical macromolecules and membrane system (Winner et al.1985; Willekens et al. 1994). Plants resistant to air pollutants(and free radicals) have better defense mechanisms than dosusceptible plants (Bowler et al. 1992). Active oxygen speciescan be detoxified through the coordinated action of anti-oxi-dant enzymes and the anti-oxidants. The anti-oxidant enzymicdefense system mainly includes SOD, APX, CAT, POD, and GR,whereas glutathione and other reductant components may havefunctions in the non-enzymatic defense system. The scaveng-ing capacity against DPPH· of a biological reagent can be usedto represent its anti-oxidant capacity and reflects approximatelythe resistance of the plant anti-oxidant system to extrinsic freeradicals (Larrauri et al. 1998; Peng et al. 2000). In addition to itsfunction in energy dissipation, mutual transformation ofantheraxanthin (A) and zeaxanthin (Z) can counteract the dam-age caused by lipid overoxidation (Yamamoto et al. 1999). Thedominant component of carotenoids, lutein, is considered ananti-oxidant that can participate in the non-radiative energydissipation through the quenching of Chl fluorescence(Mortensen et al. 1997; Niyogi et al. 1997). The activation of theVAZ cycle under the influence of SO2 is not only the result of adecrease in intrathylakoid pH caused by ATP consumption as aconsequence of SO2-inhibited carbon assimilation, but may alsoderive from another, more indirect, acidifying effect of SO2

related to the activation of mechanisms for the detoxification ofSO2-derived toxic molecules (Okpodu et al. 1996). In the presentstudy, after treatment of plants with low concentrations ofNaHSO3, the scavenging capacities against DPPH· and the luteincontent in plant leaf extractions were increased considerably(Figure 5), which proves that the protective functions of resis-tance to photoinhibition and photo-oxidation damage in plant

Figure 5. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH·)-scav-enging capacity (mg/cm2 for Schima superba, Castanopsis fissa,Acmena acuminatissima, and Cryptocarya concinna; mg/g for Pinusmassoniana) after growth at a long-term light gradient and follow-ing short-term treatment with NaHSO3 solution.

( ), 100% natural light intensity; ( ), 32% natural light intensity;( ), 12% natural light intensity. Data are the mean ± SEM (n=4).


cells were aroused and strengthened by NaHSO3. However,when plants were treated with 100 mmol/L NaHSO3, thedestruction of the anti-oxidant system resulted in a decreasein the DPPH·-scavenging capacity of plant leaves. The reduc-tion in the anti-oxidative capacity is consistent with changes inFv/Fm and ΦPSII (Figure 4), which demonstrates that PSII activityis correlated with anti-oxidation in plant cells. Following simu-lated SO2 treatment, the DES level, lutein content, and lutein/(V+A+Z) increased to different extents (Figure 2). At high con-centrations of NaHSO3, DPPH·-scavenging capacities were re-duced with high DES levels, but lutein levels were constant,which may reveal certain differences in the modulation of thesetwo protective mechanisms in response to NaHSO3. In terms ofthe changes in these parameters as they relate to anti-oxidation,P. massoniana was the most sensitive species (except in theDES response), which means that the response of P.massoniana to free radical damage was much greater thanthat of the other broad-leaved species.

For plant foliage, reflectance spectra are the product of com-plex patterns of scattering and absorption by numerous struc-tural and biochemical components. Leaf reflectance measure-ment is a rapid, non-destructive, and efficient index that can beused to gain a deeper insight into environmental stress. Someprevious studies on leaf reflectance of one broad-leaf and twoconifer trees along an elevation gradient has demonstratedrelationships between leaf reflectance, Fv/Fm and Pmax

(Richardson et al. 2001; Richardson and Berlyn 2002). Reflec-tance is dependent not only on the surface properties of theleaf, but also on the internal anatomical structure and biochemi-cal content (Gamon and Surfus 1999), which is supported bythe variations in leaf reflectance observed among the five spe-cies examined in the present study. By eliminating the effectsof leaf age, water content, and other factors in sampling, thechanges in reflectance curves can exhibit the response prop-erties of leaves to light gradient and simulated SO2 treatment.The increase in leaf reflectance with increasing NaHSO3 con-centrations may be related to the decomposition of Chl and areduction of light absorption. In plants grown at 12% naturallight, the leaf reflectance rate was low and less affected byNaHSO3 (Figure 3), which may be due to two possible reasons.First, high Chl content (especially Chl b, the light-harvestingpigment) in shade leaves was beneficial in increasing light ab-sorption and decreasing light reflectance. Second, shade-tol-erant species have more grana stacks in their thylakoids andlarger light-harvesting antenna. So, little damage was causedto the light-harvesting system on the thylakoid membrane byNaHSO3 and the activity of PSII was maintained at a higher levelunder 12% natural light than under 32% and 100% natural light.The Chl NDI was used to characterize the complex spectra andANOVA. The Chl NDI is similar to Carter’s (1994) stress ratioR695/R760, which is known to be sensitive to a wide variety ofstress agents. In the present experiment, responses of Chl NDI

to simulated SO2 treatment were examined. Of all plants tested,only the reflectance of C. concinna at 554 nm under 100%natural light intensity decreased (Table 2) and Chl NDI increased(Table 3) following treatment with NaHSO3, which furtherproves the higher resistance of this plant to simulated SO2

treatment.Light can act as an impeller to promote forest succession by

changing the degree of shade within a community. Photosyn-thesis is one of the most important determining factors for plantgrowth. Adaptations and responses of photosynthesis to envi-ronmental changes offer the plant the capacity to exist anddevelop. Growth in permanent shade is inescapable for thelower leaves of a plant forming a multilayer canopy and it fol-lows that both plastic (within a genotype) and genetic (betweengenotype) differences must exist in the photosynthetic system.A leaf below the light compensation point uses more energy forrespiration than it can fix and is effectively a non-functionalorgan (Fitter and Hay 2002). In contrast, when the energy ab-sorbed is in excess of the amount that can be used for carbonfixation, the photosystems become overexcited and the elec-tron transport chains may become over-reduced. Transfer ofexcitation energy from excited Chl molecules to molecular oxy-gen or the leakage of electrons to oxygen lead to the formationof highly toxic active oxygen species (Foyer and Noctor 2000).In the present study, total Chl content, Chl a/b, and DPPH·-scavenging capacities were changed in response to lightgradients, because the parameters above may be easily influ-enced by light intensity.

Near the Tropic of Cancer, it is almost desert. However, inChina, controlled by the monsoon from the Pacific Ocean, it israiny and humid throughout the year and a subtropical ever-green broad-leaf forest stands alone in the world (Chen et al.1996). The forest developed with the succession process ofconifer forest→coniferous and broad-leaved mixed forest withconifer dominating→coniferous and broad-leaved mixed for-est with heliophyte broad-leaved species dominating→ ever-green broad-leaved forest with heliophyte broad-leaved spe-cies dominating→evergreen broad-leaved forest with shade-tolerant species (Peng et al. 1996). The five woody plants se-lected for investigation in the present study were dominantspecies at different times during the succession process. Study-ing the responses and adaptations of these plant species grownunder different light intensities to simulated SO2 and acid pre-cipitation is useful to determine the effects of human activitieson subtropical forest structures and functions. The results dem-onstrated that P. massoniana, the dominant species during theearly stage of succession, is more sensitive to simulated SO2

treatment compared with the other broad-leaved species, andC. concinna, the species dominant during the late stage ofsuccession, exhibited better tolerance to simulated SO2

treatment. Hence, we deduced that, in subtropical coniferousand broad-leaved mixed forest, acid pollution and low light will


accelerate the decline of P. massoniana and other conifer for-ests and promote the course of succession. In contrary, thedevelopment of the forest climax dominant species C. concinna,which is less sensitive to simulated SO2 treatment, will be stimu-lated by the same conditions.

Materials and Methods

Field site and plant materials

The experimental site was located in the South China BotanicalGarden, Guangzhou, Guangdong Province, China (23°35' N and122°57' E), belonging to the south subtropical monsoon climate.The annual average temperature is 21.4–21.9 °C. The averagetemperature during July and August, the hottest months of theyear, is 28.0–28.7 °C, whereas that in January or February, thecoldest months, averages 12.4–13.5 °C. Average precipitationis 1 623.6–1 899.8 mm and average radiation is 4 367.2–4 597.3MJ/m2 over the entire year. Five typical woody plants, namelyP. massoniana Lamb, S. superba Gardn. and Champ, C. fissaR and W, A. acuminatissima Bi. Merr. and Perry, and C.concinna Hance, represent three succession periods and werechosen for investigation in the present study. Pinus massonianais the pioneer heliophyte species; S. superba and C. fissa aremedium-succession species, whereas A. acuminatissima andC. concinna are climax species in a subtropical wildwood atDinghu Mountain nature reserve. Potted seedlings were grownunder different light intensities (100%, 32% and 12% of naturallight), with regular management of water and fertilizer for 21months. Havelocks were used in setting light gradients and 12potted plants subjected to treatment with each light intensity.Diurnal changes in natural light intensity (Figure 1) were mea-sured using a photosynthetically active radiation (PAR) sensor(Licor, Lincoln, USA).

Sampling and simulated SO2 treatment

Leaves from different individuals (n>3) with a similar positionon plants were taken for in vitro treatments and measurements.Leaf disks (2.0 cm lengths for P. massoniana needles) wereimmerged, adaxial side up, in 20, 50, and 100 mmol/L NaHSO3

(pH 5.3, 5.0, and 4.8, respectively), as well as 0 mmol/L NaHSO3

(distilled water; pH 6.0; control), under 20 µmol/m2 per s illumi-nation for 20 h (25 °C).

Leaf pigment analysis

After treatment with NaHSO3 or water, leaf disks of a knownarea (or weight for P. massoniana needles) were first frozenin liquid nitrogen and then ground in a mortar with the addi-tion of CaCO3, silica, and cold acetone. The mixture was

centrifuged at 11 300g for 10 min at 0–4 °C, with supernatantfluid passed through a 0.45-µm filter. Liquid samples were ana-lyzed using HPLC system (Waters 2695; Waters, Milford, MA,USA) according to the method described by Gilmore andYamamoto (1991). Pigments were eluted at a flow rate of 1.8mL/min. The de-epoxidation state of the xanthophyll cycle wasdefined as DES=(Z+0.5 × A)/(V+A+Z).

Total Chl content

Disks treated with NaHSO3 or water were extracted with 80%acetone. Absorption was measured at 663 and 645 nm usingan ultraviolet (UV)-visible spectrophotometer (Lambda 25, USA).Total Chl content and Chl a/b were calculated according to Linet al. (1984).

Spectral reflectance of the leaf upper surface

Spectral reflectance at wavelengths ranging from 400 to 800nm was measured using a UV spectrophotometer with inte-grated sphere (Lambda 650; Perkin-Elmer, Norwalk, CT, USA).After treatment, all leaf disks were washed with distilled water,wiped, fixed onto filter paper with their lower surface on thepaper, and their upper surface was exposed to a lamp usingthe same filter paper as a blank.

A revised version of the normalized difference vegetationindex, which is well correlated with and sensitive to a widerange of Chl a concentrations, was calculated as Chl NDI=(R750–R705)/(R750+R705) as described by Gitelson et al. (1996).

Chlorophyll fluorescence

Measurements of the maximum efficiency of PSII photochemis-try (Fv/Fm) and effective quantum yield of photochemical en-ergy conversion in PSII (ΦPSII) were made using a portablepulse-modulated fluorimeter (PAM-2100; Walz, Efeltrich,Germany). Leaf disks treated with NaHSO3 were dark adaptedwith leaf clips for 30 min. The Fv/Fm ratio was calculated as(Fm–Fo)/Fm, where Fo is the minimum fluorescence, and ΦPSII

was equal to 1–Fs/Fm', where Fm' is the maximum fluorescenceyield after light adaptation and Fs is steady state fluorescenceyield.

Anti-oxidant capacity (DPPH·-scavenging capacity)

The DPPH· solution has a unique absorption at 525 nm. Thedecline of A525 can be used as an index of organic free radical-scavenging capacity (ORSC) of a plant extract. In this assay,the total reaction volume was 2 mL and DPPH· was dissolved ina little methanol followed by the addition of 50% ethanol to finalconcentration of 120 µmol/L. Then, 0.1 mL plant extract(dissolved in 50% ethanol) was added to 1.9 mL DPPH· and


A525 was measured after 20 min reaction. The percentage ofDPPH·remaining (R) and scavenging ratio (ORSC) werecalculated as follows:

R (%) = ((A–B)/Ao) × 100ORSC (%) = (1–R) × 100

where Ao is DPPH· absorption without leaf extraction (0.1 mLof 50% ethanol+1.9 mL DPPH·), A is DPPH· absorption afterreaction with the sample and B is the absorption of the blank(0.1 mL sample+1.9 mL of 50% ethanol). The DPPH·-scaveng-ing capacity was calculated as ORSC% × DPPH· in reaction(mg)/leaf disk areas (cm2) or sample weight (g), as describedby Peng et al. (2000).

Acknowledgements

The authors are grateful to Shaowei Chen for her help andconstructive suggestions in the experiments on leaf spectraproperties. The authors also thank Jian Liu for his technicalassistance with the HPLC procedure.

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Changes in Photosystem II Activity and Leaf Reflectance Features of Several Subtropical Woody Plants...

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