Journal of Plant Nutrition, 28: 2211–2220, 2005 Copyright © Taylor & Francis Inc. ISSN: 0190-4167 print / 1532-4087 online DOI: 10.1080/01904160500324782
Cadmium Effects on Sunflower Growth and Photosynthesis Helena Azevedo, Clara Gomes Gl´oria Pinto, Jose Fernandes, Susana Loureiro, and Concei¸ca˜ o Santos Department of Biology, University of Aveiro, Aveiro Portugal
ABSTRACT The objective of this work was to evaluate the effects of cadmium (Cd) exposure on sunflower (Helianthus annuus L.) plant growth, chlorophyll content, and fluorescence. Sunflower plants were exposed to different concentrations of Cd (0, 5, 50, and 500 µM) for 21 d. Growth parameters (organ length, fresh and dry weights) were determined and results compared with two parameters associated with photosynthesis degradation: chlorophyll content and fluorescence (an easy and non-destructive method). Exposure to Cd significantly decreased growth by decreasing shoot and root lengths and their fresh and dry weight. Cadmium also decreased significantly chlorophyll content and fluorescence efficiency in all treatments. Chlorophyll a (chl a) and chl b contents showed a significant correlation with chlorophyll fluorescence (Fv /Fm ratio). The EC50 values showed that the roots’ length was the most sensitive endpoint in this study, followed by the roots’ and shoots’ weight endpoints. Also, chl b showed higher sensitivity to Cd contamination than chl a. These data show that in complement to growth parameters, the use of photosynthetic parameters provides helpful information on plant response to Cd exposure. Keywords: cadmium, chlorophyll, growth parameters, photosynthesis, sunflower
INTRODUCTION Cadmium (Cd) in soil due to industrial pollution, the use of some commercial fertilizers, or contamination from bedrock (Larsson et al., 1998) is a growing problem. Cadmium may reduce plant growth through inhibition of chlorophyll synthesis (Stobart et al., 1985) and light harvesting chl a/chl b protein complex II
Received 24 September 2003; accepted 15 July 2005. Address correspondence to Concei¸ca˜ o Santos, Department of Biology, University of Aveiro, Aveiro 3810, Portugal. E-mail:
[email protected] 2211
2212
H. Azevedo et al.
(Krupa, 1987), thus affecting photosynthesis and carbohydrate and nitrogen metabolisms (Greger and Bertell, 1992; Zhang et al., 2003). Other effects of Cd on plant cell respiration and enzyme activities (Lindberg and Wingstrand, 1985), transpiration rates (Haag-Kerver et al., 1999), protein patterns (Rauser, 1987), and gene expression (Marrs and Walbot, 1997) have been reported. However, in general most of these studies have used methods are destructive and time consuming to perform, and often require highly trained technicians. Therefore, the question remains as to whether parameters exist that can be used easily, non-destructively, and with high reproducibility in rapid and/or field “diagnosis” of Cd toxicity in plants. The use of chlorophyll content and fluorescence has already been shown to be highly reliable and fairly sensitive to Cd stress in cucumis (Cucumis sativus L.) chloroplasts (Zhang et al., 2003); to other abiotic stresses such as KCl in sunflower (Helianthus annuus L.) plants (Santos et al., 2001); to osmotic stress (Brito et al., 2003); and in comparative studies of nutrient deficiencies (Ouzounidov et al., 2003). On the other hand, physiological parameters used as senescence markers may be useful in selecting Cd-tolerant plants and in assaying genetic traits. This report evaluates the possibility of using non-destructive and easy-toassess parameters, namely; chlorophyll content and fluorescence, in the rapid and preliminary diagnosis of metal toxicity induced in plants as a complement to classic and more time-consuming methods such as those related to plant-growth evaluation.
MATERIALS AND METHODS Plant Material and Growth Imbibed seeds of sunflower (Helianthus annuus L. cv. ‘SH222,’ Sclepal, Portugal) were germinated in Long Ashton medium (Meidner, 1984) at 25 ± 1◦ C with a photoperiod of 16 h. Six-day-old seedlings were measured, weighed, and placed in pots with aerated Long Ashton medium containing, 0, 5, 50, or 500 µM Cd supplemented as Cd(NO3 )2 (Sigma). Plants were grown at 24 ± 2◦ C with a photoperiod of 16 h. Light was supplied by Osram 36W lamps with a total intensity of 480 ± 10 µmol/m2 /s. Plants were collected after 0, 8, 15, and 21 d of culture to determine growth (shoot and root lengths as well as fresh and dry weights) and photosynthetic parameters (chlorophyll content and fluorescence). Samples were collected 2 h after light-period initiation.
Chlorophyll Concentration and Fluorescence Parameters in Plants Chlorophyll basal fluorescence (F0 ) and maximum fluorescence (Fm ) were monitored in expanded leaves using a Plant Efficiency Analyzer (Hansatech
Cadmium Effects on Photosynthesis and Growth
2213
Instruments Ltd., UK) by illuminating them with a peak wavelength of 650 nm and a saturating light intensity of 3000 µmol m−2 s−1 . Variable fluorescence (Fv = Fm − F0 ) and the ratio Fv /Fm were calculated. Chlorophyll content was determined according to Arnon (1949) from the same expanded leaves used in fluorescence determination.
Statistical Analysis For all parameters, values are given as mean ± standard deviation (SD) as calculated from data from two independent experiments (one in January and the second in May). In each experiment, for each growth parameter, at least 50 plants were analyzed per exposure condition. For photosynthetic parameters, at least 20 individuals were analyzed per exposure condition. The comparison between the responses to Cd exposures was made using a one-way ANOVA test, followed by a Tukey test when data were statistically different (p < 0.05). If data were not normally distributed and data transformation did not correct for normality, a Kruskal-Wallis one-way analysis of variance on ranks was then performed (Zar, 1996). The EC50 values (the molar concentration of Cd, which produces 50% of the maximum possible response of a particular parameter) were calculated using a four-parameter logistic curve, such as the Hill function. Correlations between endpoints data were also assessed. All statistical procedures were performed using the software package SigmaStat (SPSS, 1995, SigmaStat for Windows version 2.03, Chicago, IL).
RESULTS AND DISCUSSION In these experiments Cd induced senescence characteristics in sunflower plants, including general reduction of growth, reduction of leaf number and size, chlorosis, and the appearance of necrotic spots in leaves. Increases in Cd concentration in the culture medium significantly decreased plant length (one-way ANOVA, F3,8 = 85.82, p < 0.05) (Figure 1 a, b) and shoot and root dry weights (Kruskal-Wallis one-way ANOVA, H = 9.97 df = 3, p < 0.05 and one-way ANOVA, F3,8 = 129.46, p < 0.05, respectively) (Figure 1 e-f; p < 0.05), with symptoms more evident at the end of the experiment. The decreases in root and shoot lengths and in weights are in accordance with the effects of Cd in other plant species such as cucumber (Zhang et al., 2003). With time, the increase of Cd increased the ratios shootLength rootlength , shootFW rootFW , and shootDW /rootDW . At day 21, all plants exposed to 500 µM Cd were dead, and therefore all assays were performed at day 15. For this period, shoot and root fresh and dry weights decreased significantly in stressed plants, with more significant effects evident in 500 µM treated plants (p < 0.05).
2214
H. Azevedo et al.
Figure 1. Growth of sunflower plants exposed to different concentrations of cadmium during 21 d: (a) shoot length; (b) root length; (c) shoot fresh weight; (d) root fresh weight; (e) shoot dry weight; (f) root dry weight. Vertical lines (SD). Means from two independent experiments. In each experiment, at least 50 plant per treatment were used. Control 0 µM ( ); 5 µM ( ); 50 µM ( ); 500 µM ( ). Different letters indicate significantly different means (one-way ANOVA, P < 0.05). Note: At day 21, values of plants under 500 µM stress are not expressed due to the death of plants.
The reduction of plant growth induced by some heavy metals has already been determined in other species (Arnon, 1949; Cieslinski et al., 1996; Gomes et al., 2000; Prasad and Hagemeyer, 1999), although no such comprehensive study was done on the trend response of these three growth parameters (length and fresh and dry weight) in shoots and roots in response to Cd exposure. Simultaneously, all Cd treatments also decreased significantly chl a levels (one-way ANOVA, F3,8 = 329.59, p < 0.05), chl b levels (one-way ANOVA, F3,8 = 138.72, p < 0.05) (Figure 2 a–c), and fluorescence (for F0 , oneway ANOVA, F3,8 = 454.57, p < 0.05; for Fm , one-way ANOVA, F3,8 = 17957.10, p < 0.05; for Fv , one-way ANOVA, F3,8 = 6066.87, p < 0.05) (Figure 3 a–d). These characteristics affect plant photosynthetic efficiency, and thus could be at least partly responsible, for the decrease in plant biomass production and plant growth. Plants exposed to Cd showed general chlorosis symptoms and, in plants exposed to higher concentrations, most of the shoots became precociously necrotic. Chlorophyll a and chl b contents decreased in leaves exposed to Cd, and this decrease was accentuated with time
Cadmium Effects on Photosynthesis and Growth
2215
Figure 2. Chlorophyll content of sunflower leaves of plants exposed to different concentrations of cadmium during 21 d: (a) chlorophyll a; (b) chlorophyll b; (c) ratio chlorophyll a/chlorophyll b. Vertical lines (SD). Means from two independent experiments. In each experiment, at least 50 plants per treatment were used. Control 0 µM ( ); 5 µM ( ); 50 µM ( ); 500 µM ( ). Different letters indicate significantly different means (one-way ANOVA, P < 0.05). Note: At day 21, values of plants under 500 µM stress are not expressed due to the death of plants.
(Figure 2 a, b). By the end of the treatment it was evident that higher Cd concentrations induced higher degradation of chl b. This degradation led to an increase in chl a/chl b ratio in stressed plants that was also more evident at the end of treatment (Figure 2 c). This result can be explained by the fact that the first step in chl b degradation involves its conversion to chl a (Fang et al., 1998), mostly in moderately stressed leaves, while under severe stress both degradation and reduction of synthesis may occur. Quirino et al. (2000) already reported that when leaf senescence begins, chloroplasts are one of the first sites in cells to be affected. The destruction of chloroplasts by Cd was also reported for cucumber by Zhang et al. (2003), who found decreases in chlorophyll and of the antioxidant defense system of chloroplasts (i.e., decreases in antioxidative enzymes). Associated with the decrease in chlorophyll content, chlorophyll fluorescence was also affected by Cd stress. The F0 showed a significant decrease, mostly in severe stress conditions, such as under 50 and 500 µM (Tukey test, p < 0.05) (Figure 3 a). Also, Fm decreased during the treatment in all stress situations (Tukey test, p < 0.05), while it remained stable during the first week in 5 µM-stressed leaves (Figure 3 b). In turn, this decrease of Fm influenced the decrease of Fv (Figure 3 c). The ratio Fv /Fm decreased significantly in Cd treated leaves, and this effect was more evident under the 50 and 500 µM treatments (Figure 3 d). Chlorophyll a and chl b contents showed a significant correlation with chlorophyll fluorescence: The Fv /Fm ratio showed a significant correlation with both chl a (n = 12, r2 = 0.986, p < 0.05) and chl b (n = 12, r2 = 0.909, p < 0.05). The F0 represents the minimal fluorescence yield, and occurs when all reaction centers are in an active, “open” state (Krause and Weis, 1991). The stability
2216
H. Azevedo et al.
Figure 3. Chlorophyll fluorescence of sunflower leaves of plants exposed to different concentrations of cadmium during 21 d: (a) basal fluorescence (F0 ); (b) maximum fluorescence (Fm ); (c) variable fluorescence (Fv ); (d) Fv /Fm ratio. Vertical lines (SD). Means from two independent experiments. In each experiment, at least 50 plants per treatment were used. Control 0 µM ( ); 5 µM ( ); 50 µM ( ); 500 µM ( ). Different letters indicate significantly different means (one-way ANOVA, P < 0.05). Note: At day 21, values of plants under 500 µM stress are not expressed due to the death of plants.
of F0 found for the lowest Cd concentration indicates that this low concentration of Cd caused no significant changes in the reaction centers. However, the variations found for higher Cd concentrations indicate losses of energy transference from pigments to the reaction center, probably due to damage of the LHC center associated with the photosystem II (PSII). Changes in Fv /Fm indicate variations in the photochemical efficiency of the PSII, while the value found for control plants (near 0.8) is within the values typical of healthy plants, independent of species. A reduction of this ratio in Cd-stressed plants is due to a reduction
Cadmium Effects on Photosynthesis and Growth
2217
in Fm and reflects an increase energy in dissipation due to a destruction of the photosynthetic apparatus. Previous studies have show that some stresses induced changes in the photosynthetic apparatus (Stobart et al., 1985; Krupa, 1987; Bhorer and Dorffling, 1993; Lutts et al., 1996) and membrane permeability properties of chloroplasts. Dissociation of the light-harvesting antennae from the PSII core and destruction of the PSII reaction center have been postulated for rice (Oryza sativa L.), NaCl-stressed leaves (Lutts et al., 1996), drought-stressed cotton (Gossypium hirsutum L.) (Genty et al., 1987), and salt-stressed sunflower (Santos et al., 2001) plants. In sunflower cells, our data show that the photosynthetic apparatus, and particularly PSII, is sensitive to Cd stress. The observed decreases in chlorophyll content and fluorescence in response to Cd exposure may be the result of chlorophyll degradation and/or synthesis deficiency, together with a decrease in thylakoid membrane integrity. Several additional data point to this conclusion: (1) a decrease in the content of magnesium (Mg) (Azevedo et al., 2005a), mineral that is essential for chlorophyll synthesis, in the same Cd-stressed plants that suffered a decrease of chlorophyll content; (2) an increase in membrane permeability and lipid peroxidation, probably due to changes in membrane composition (Azevedo et al., 2005b); and (3) a reduction in other nutrients such as calcium (Ca) (Azevedo et al., 2005a)—important for membrane integrity, cell walls, or as secondary messenger, among other functions—iron (Fe); or manganese (Mn) (Azevedo et al., 2005a), minerals that are essential in chlorophyll synthesis and/or photosynthetic electron transport. Regarding the EC50 values obtained from the endpoints data (Table 1), the roots’ length was the most sensitive endpoint in this study, followed by the Table 1 EC50 values (µM Cd) for all the parameters analysed in Cd-treated sunflower plants (shoots and roots) for 15 d EC50 value (µM)
r2
p
Shoots Length Fresh Weight Dry Weight Chlorophyll a Chlorophyll b Fv/Fm
380.08 11.02 32.72 181.31 31.27 296.01
0.970 0.965 0.948 0.986 0.969 0.988
