Chlorophyll Fluorescence in Leaves of Ficus tikoua Under Arsenic Stress
Yong Wang, Liyuan Chai, Zhihui Yang, Hussani Mubarak & Chongjian Tang
Bulletin of Environmental Contamination and Toxicology ISSN 0007-4861 Volume 97 Number 4 Bull Environ Contam Toxicol (2016) 97:576-581 DOI 10.1007/s00128-016-1905-5
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Author's personal copy Bull Environ Contam Toxicol (2016) 97:576–581 DOI 10.1007/s00128-016-1905-5
Chlorophyll Fluorescence in Leaves of Ficus tikoua Under Arsenic Stress Yong Wang1 • Liyuan Chai1,2 • Zhihui Yang1,2 • Hussani Mubarak1 Chongjian Tang1,2
•
Received: 6 February 2016 / Accepted: 9 August 2016 / Published online: 19 August 2016 Ó Springer Science+Business Media New York 2016
Abstract A greenhouse culture experiment was used to quantify effects of arsenic (As) stress on the growth and photochemical efficiency of Ficus tikoua (F. tikoua). Results showed growth of F. tikoua leaves was significantly inhibited at As concentrations higher than 80 lmol/ L in solution. Root arsenic concentration was significantly higher than that in stem and leaf. The 320 and 480 lmol/L As concentrations in solution resulted in significant decreases in maximum quantum efficiency of photosystem II (PSII) (Fv/Fm), variable to initial chlorophyll fluorescence (Fv/Fo), and quantum yield of PSII electron transport (Y(II)) of F. tikoua leaves, whereas significantly higher non-photochemical quenching of fluorescence and photochemical quenching of fluorescence values were found at 160, 320 and 480 lmol/L As concentrations in solution, implying that PSII reaction centers were damaged at high As concentrations and that F. tikoua eliminates excess energy stress on the photochemical apparatus to adapt to As stress. Keywords Arsenic toxicity Arsenic resistant Photosynthesis Photochemistry quenching Arsenic (As) is a metalloid commonly present in metal mining, smelting and burning of municipal wastes. Its chronic toxicity to plant life is well-known, particularly
& Zhihui Yang
[email protected] 1
School of Metallurgical Science and Environment, Central South University, Changsha 410083, China
2
Chinese National Engineering Research Center for Control and Treatment of Heavy Metal Pollution, Changsha 410083, China
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when exposure occurs over prolonged periods (Anawar et al. 2011; Halder et al. 2014). Although As is not essential for plant growth, it can be taken up by roots and then translocated into leaves in many plant species, causing disorders in plant growth by adversely affecting important physiological processes (Meharg and Hartley-Whitaker 2002). At toxic concentrations, As has the capacity to directly induce oxidative damage because of its redox properties (Gunes et al. 2009), interfering with the vital cellular processes of photosynthesis, pigment synthesis and plasma membrane permeability (Wang et al. 2012). Uptake of As leads to concentration-dependent losses of chlorophyll (Chl) and carotenoid pigments and inhibition of Chl synthesis enzymes, including Rubisco, as well as alterations to the photosynthetic apparatus at both the thylakoid and the chloroplast level (Tripathi et al. 2015; Wang et al. 2012). Measurement of Chl fluorescence is a useful technique to differentiate environmental stresses in plant at an early stress stage (Ralph et al. 2005). A Chl fluorescence imaging system is often used to determine the fluorescence quenching that results from both photochemical and nonphotochemical processes (Lichtenthaler et al. 2005b; Ralph et al. 2005). Ficus tikoua (F. tikoua), an endemic species in China belonging to the Moraceae family, has been recognized as a valuable restoration species and a medicinal source (Sirisha et al. 2010; Zhan et al. 2013). During the past decade, research focused on the evaluation of biological activities of F. tikoua (Jiang et al. 2013). Few research studies have investigated the photosynthetic response of F. tikoua to metal/metalloid stress. Moreover, little is known about the response of F. tikoua to As treatment. The objective of this study was to assess the As resistance capacity of F. tikoua to As in preparation for eventual application for the re-vegetation of As-polluted soils.
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Materials and Methods Antimony (Sb) mining in Xikuangshan area, Lengshuijiang city, Hunan province, China, has been conducted for more than 100 years, resulting in severe Sb and As contamination in soils and ecological destruction. Ficus tikoua was one of dominant species well grown in the contaminated environment of Xikuangshan area and was chosen for this present study. Cuttings of F. tikoua from Xikuangshan area (111°290 E, 27°450 N) were grown on water-saturated perlite in a growth chamber in a greenhouse. Cuttings were illuminated with three, 25 W fluorescent lamps with a 14 h/ 10 h photoperiod and a light intensity of 150 lmol m-2s-1 photosynthetically active radiation (PAR). Day/night temperatures of 25°C/18°C were applied, and the humidity was 60 %–80 %. Cuttings were supplied with half-strength Hoagland nutrient solution until all six or seven leaves were completely unfolded. Thereafter, four plants of F.tikoua were carefully transferred to plastic pots filled with perlites. Seven As concentrations (0, 8, 40, 80, 160, 320 and 480 lmol/L in solution) were tested in this study. Arsenic was supplied as NaAsO2. Each pot was supplied with 50 mL of As-containing 1/2 Hoagland solution at 5-day intervals. All conditions were the same as those described above. The Chl content in the fourth leaf from the top of F. tikoua was estimated in vivo for each As concentration with a portable Chl meter (SPAD-502plus, Konica Minolta Optics Inc., Osaka, Japan) (Netto et al. 2005). After 100 days, As-exposed plants were collected and then rinsed three times with deionized water. Roots, stems and leaves were separately collected, dried at 70°C, and weighed. Plant samples were ground and sieved through a 1 mm sieve and digested with HNO3:HClO4 (4:1, v/v). The As concentration in the digested solution was quantified by atomic fluorescence spectrometry (AFS) (Titan AFS-810). The translocation factor (TF) estimating the ability of the plant to translocate the metal from root to stem (TFrs) or from stem to leaf (TFsl) was calculated based on As concentrations in different tissues according to following equation (Yang et al. 2015): TFrs = [As] in stem/[As] in root, and TFsl = [As] in leaf/[As] in stem, where [As] represents As concentration. Chl fluorescence of the fully expanded, fourth leaf from top was measured. All fluorescence measurements were carried out on in darkness for 40 min to obtain equilibrium of the Photosystem II (PSII) redox state, and the induction curve (slow kinetics) for PSII was measured using the MINI-version of the Imaging-PAM fluorometer (Walz, Effeltrich, Germany). For fluorescence measurements, the actinic light was set at 135 lmol m-2s-1 PAR. The irradiance step was 20 s, and the length of the program was
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5 min. All Chl fluorescence parameters, including maximum quantum efficiency of PSII (Fv/Fm); variable to initial chlorophyll fluorescence (Fv/Fo); quantum yield of PSII electron transport (Y(II)); non-photochemical quenching of fluorescence (NPQ); photochemical quenching of fluorescence (qP); and non-photochemical quenching of fluorescence (qN) were calculated (Kramer et al. 2004; Lichtenthaler et al. 2005a). Analysis of variance (ANOVA) was performed using SPSS statistical software. A p value of \0.05 denoted significance. The Tukey test and subsequent pairwise comparisons were employed to compare significant differences (p \ 0.05) between treatment means. Measured values of NPQ and qN were transformed by square root, and values of qP were transformed by exponent before ANOVA followed by Tukey tests. All data presented are mean values of three replicate experiments.
Results and Discussion The present study investigated the toxicity of As on F. tikoua and the acclimation of this plant to As stress via alteration of the phyto-availability of As. When As was transported from the matrix to the roots and then further to different parts in the shoots, toxicity occurred, affecting various plant processes (Maciaszczyk-Dziubinska et al. 2012). The dry mass of the F. tikoua leaves treated with As concentrations higher than 80 lmol/L in solution was significantly (p \ 0.05) lower than that of the untreated controls (without As application) and 8 lmol/L As in solution (Fig. 1a). Differences in dry stem weight among all As concentrations were not statistically significant. In addition, the dry weight of root significantly decreased at concentrations of 320 lmol/L in solution and 480 lmol/L As in solution compared with 8 lmol/L As in solution. Although branch number and branch length were not significantly different among all concentrations, leaf number was inhibited at As concentrations ranging from 80 to 480 lmol/L As in solution (Fig. 1b, c). The effect of As on plant physiology function is concentration-dependent. Arsenic has a negative effect on plant growth, especially at higher As concentrations where inhibited plant growth was observed (Carbonell-Barrachina et al. 1998). In the present study, when the As concentration was 8 lmol/L in solution, leaf biomass was not significantly decreased compared with the untreated control. However, a considerable negative effect of As on leaf biomass was observed at concentrations ranging from 80 to 480 lmol/L in solution. This suggests that As was toxic to F. tikoua, especially at higher concentrations. Results imply that 80 lmol/L As in solution could be the leaf threshold for As stress.
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Fig. 1 Growth response of F. tikoua under As treatments (0–480 lmol/L in solution). a dry masses; b branch length and number; c leaves number. In individual subfigures, values followed by same uppercase letters are not significantly different at p = 0.05, for treatments; values followed by same lowercase letters are not significantly different at p = 0.05, for treatments; values followed by same uppercase letters are not significantly different at p = 0.05, for treatments. Error bars are standard errors
For As-tolerant and -sensitive species, As accumulates primarily in roots (Mateos-Naranjo et al. 2012; Meharg and Hartley-Whitaker 2002). In the present study, As levels in
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the roots of F. tikoua were significantly (p \ 0.05) higher than in similarly-treated stems and leaves (Table 1). The As concentration in roots varied from 0 to 480 lmol/L in solution. The As concentration in roots treated with 40–320 lmol/L As in solution was significantly higher than that with 480 lmol/L As in solution followed by control and 8 lmol/L As in solution. Arsenic accumulations in stems increased with As treatments from 0 to 480 lmol/L in solution. Arsenic concentration in stems was significantly different among different treatments. For instance, 480 lmol/L As in solution resulted in significantly higher concentrations in stem than 0–160 lmol/L As in solution. The highest As concentration in leaves was found in 320 lmol/L As in solution. Ficus tikoua was able to accumulate up to 793.69 mg/kg of As in roots when plants were exposed to 80 lmol/L As in solution. However, the maximum As accumulation (130.94 mg/kg) in leaves was noted at 320 lmol/L As in solution. The highest As treatment concentration (480 lmol/L in solution) resulted in decreased As accumulation in leaves. Similar result was reported by Singh and Ma (2006). They found the maximum As accumulation (38.4 mg/kg) in Pteris ensiformis fronds at 133 lmol/L in solution rather than 267 lmol/L As in solution after 10-day treatment. The translocation factor (TF) can be used to evaluate a plant’s capacity to translocate metals within its tissues. The TF root to stem (TFrs) values for all As concentrations were less than 1 (Table 1), indicating the limited capacity for F. tikoua to translocate As from root to stem. Moreover, TFrs values for the same As concentrations (except controls) were lower than their corresponding TF stem to leaf (TFsl). Results indicate the root system for F. tikoua transferred less As to stems and stems transferred more As to leaves. The maximum of TFrs appear in the 0 and 480 lmol/L As in solution. On one hand, normal growth condition (control treatment) can be favourable for translocation of elements, consequently resulting in high As translocation. Under high As treatments (480 lmol/L in solution), it is likely that to adapt to high stress, a portion of As could be translocated from root to stem. Similar results were previously reported. For instance, As TF of frond/root in Pteris vittata increased with longer exposure and greater As concentrations (Singh and Ma 2006). In aqueous solution, As TF of stem/root in Micranthermum umbrosum increased with greater As concentrations when As concentration increased from 0.1 to 1.0 lg/mL in solution (Islam et al. 2013). Chlorophyll content is a measure of the ability of a plant to convert photosynthetic energy into biomass, or the measurement of the efficiency of a plant to produce biomass. Thus, it is a measurement of photosynthesis. Indeed, the soil and plant analyzer development (SPAD) value of chlorophyll is an indication of plant stress as metabolism is affected by stress. The variation in the SPAD of F. tikoua
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Table 1 As content in different parts of F. tikoua As (lmol/L)
Root (mg/kg)
0
Stem (mg/kg)
5.17 ± 0.69d A*
Leaf (mg/kg)
TFsl
TFrs
3.19 ± 0.18c B
0.20 ± 0.02d C
0.06 ± 0.01b
0.63 ± 0.07a
8
90.74 ± 9.41d A
9.29 ± 0.41c B
10.48 ± 2.87d B
1.16 ± 0.38a
0.10 ± 0.01c
40
634.83 ± 56.30ab A
33.48 ± 5.60c B
15.45 ± 2.11cd B
0.48 ± 0.08ab
0.05 ± 0.01c
80
793.69 ± 12.41a A
52.67 ± 10.20c B
25.51 ± 5.13cd B
0.49 ± 0.05ab
0.07 ± 0.01c
160
692.92 ± 38.63ab A
60.11 ± 7.97bc B
39.04 ± 10.94c B
0.66 ± 0.20ab
0.09 ± 0.02c
320
622.14 ± 64.96b A
139.01 ± 3.25ab B
130.94 ± 15.66a B
0.96 ± 0.08a
0.23 ± 0.04bc
480
454.85 ± 32.31c A
167.88 ± 38.00a B
100.30 ± 7.26b B
0.65 ± 0.12ab
0.38 ± 0.11a
* Values in each vertical column followed by the same lowercase letter and values in each horizontal line followed by the same capital letter was not significantly different at p = 0.05
was observed at As concentrations of 0–480 lmol/L in solution, but the only statistical difference was observed between 320 lmol/L As in solution and both 0 and 8 lmol/ L As in solution (Fig. 2). Chlorophyll fluorescence parameters showed As had a negative effect on plant photosynthesis, especially at higher concentrations (Fig. 3). The 480 lmol/L As concentration in solution resulted in significant decreases in Fv/Fm and Fv/Fo of F. tikoua, although a difference in Fv/Fm was not obtained for As concentrations of 0, 8, 40, 80 160 and 320 lmol/L in solution (Fig. 3a, b). These results indicated PSII reaction centers were seriously damaged when As concentrations were 480 lmol/L in solution. In addition, Y(II) provides a more realistic impression of the overall leaf photosynthetic condition when the plant is under As stress. A considerable decrease in Y(II) was observed at As concentrations of 320 and 480 lmol/L in solution, which indicated photosynthesis was severely inhibited compared with the control and other As treatments (Fig. 3c). Generally, Fv/Fm was the most frequently applied Chl fluorescence ratio because it is easy and fast to determine. 45
a
40
a
ab
35
ab
SPAD
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ab
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25 20 15 10 5 0 0
8
40
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160
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As (µmol/L) Fig. 2 Mean ± SE values of F. tikoua leaf SPAD under As treatments (0–480 lmol/L in solution). Values followed by same letters are not significantly different at p = 0.05
Moreover, Fv/Fm is a relatively inert ratio, where stressinduced changes are detected late. A much more sensitive ratio, Fv/Fo, has been suggested as an alternative (Lichtenthaler et al. 2005a; Pereira et al. 2000). When plants are exposed to metal/metalloid stresses, decline in Fv/Fm and Fv/Fo indicates a disturbance in or damage to the photosynthetic apparatus (Li et al. 2015; Wang et al. 2012). Change in PSII efficiency due to metal excess were observed in different plant species and were dependent upon the time of exposure to metals and their content in leaf tissue (Li et al. 2015; Pereira et al. 2000; Ralph et al. 2005). Present results indicate the efficiency of the photosynthetic apparatus and the size and number of active photosynthetic centers were not inhibited or decreased at As concentrations of 8–160 lmol/L in solution, but were significantly inhibited and decreased at 320 and 480 lmol/ L As in solution. Decreases in Fv/Fm and Y(II) indicated the photoactivation of PSII was inhibited by As toxicity, which resulted from the destruction of antennae pigments, and the limitation of primary ‘stable’ electron acceptor of PSII (QA) reoxidation due to partial blockade of electron transport from PSII to PSI (Li et al. 2015; Mallick and Mohn 2003). Photon energy captured by a chlorophyll molecule can drive photosynthesis (photochemical quenching, qP), be emitted as fluorescence, or be converted to heat (nonphotochemical quenching, qN and NPQ) (Kramer et al. 2004; Ralph and Gademann 2005). qN and NPQ are both measures of the energy flow into heat. In this study NPQ and qN values varied among different As treatments, but no significant difference was observed in 8–80, 80–320 and 160–480 lmol/L As in solution, respectively. However, compared to the control, only a significant high NPQ value was found at 480 lmol/L As in solution, and there was no significant difference of qN among all As treatments. The increased qN and NPQ values of F. tikoua imply the plant eliminates excess energy due to As stress on the photochemical apparatus through heat dissipation as a means of stress adaptation. Moreover, the qP values at higher As
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Author's personal copy 580 Fig. 3 Mean ± SE effect of As on fluorescence quenching parameters. Values followed by same letters are not significantly different at p = 0.05
Bull Environ Contam Toxicol (2016) 97:576–581
(A) 0.7
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concentrations (320 and 480 lmol/L in solution) were less than those at other As concentrations, indicating greater inhibition of photosynthesis was found at high As concentrations. Considerable heterogeneity of Y(II) and Fv/Fm was present in As stressed leaves. Similarly, the heterogeneity of Y(II) was demonstrated in leaves of Populus x euramericana when subjected to zinc-induced stress (Sighicelli and Guarneri 2014). High photosynthetic activity, measured as Y(II), Fv/Fm and qP over veins and neighboring areas, was observed (Calatayud et al. 2006). In contrast, non-photochemical quenching parameters showed a spatial variation in F. tikoua leaves. Therefore, a spatial heterogeneity in qN and NPQ values could be expected. Photorespiration most likely acts as a type of buffer to maintain the spatial heterogeneities of Chl fluorescence (Massacci et al. 2008). Ficus tikoua can alleviate damage to photosynthetic mechanistic processes to a certain extent
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by regulating dissipation of excessive excitation energy into harmless heat at low As concentrations, but these defense mechanisms are destroyed by 320 and 480 lmol/L As concentrations in solution. Arsenic affected the growth of F. tikoua through its effects on photosynthesis. Ficus tikoua shows a certain tolerance to As concentrations less than 320 lmol/L in solution. The As tolerance mechanism contributes to the capacity of F. tikoua to accumulate As in its roots and thus hinder the transport of As to stems and leaves. Photosynthesis in F. tikoua can be inhibited by high ([320 lmol/L in solution) As concentrations. Ficus tikoua eliminates excess energy stress on the photochemical apparatus though heat dissipation to adapt to As stress. In former metal mining areas, especially those in developing countries, ecological restoration can be a challenge. The screening process for metal resistant plants is crucial in order to implement such restoration. Current study results
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suggest F. tikoua can tolerate certain As concentrations and may be used in ecological restoration of former mining areas. Acknowledgments This work was supported by Science and Technology Program for Public Wellbeing, China (2012GS430203-1) and The Key Project of Science and Technology of Hunan Province, China (2012FJ1010).
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