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Effects of gibberellin A3 on growth and microcystin production in Microcystis aeruginosa (cyanophyta)
ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 1691—1697
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Effects of gibberellin A3 on growth and microcystin production in Microcystis aeruginosa (cyanophyta) Xiaojie Pana,b, Fengyi Changa,b, Lijuan Kanga,b, Yongding Liua,, Genbao Lia, Dunhai Lia a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 7 South Donghu Road, Wuhan 430072, Hubei Province, PR China b Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China Received 25 April 2007; received in revised form 11 August 2007; accepted 15 August 2007
KEYWORDS Gibberellin A3; Growth; Microcystin; Microcystis aeruginosa; Pollutant
Summary Environmental factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A3 (GA3) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA3 on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001–25 mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA3 treatment. GA3 increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA3 was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-linked immunosorbent assay (ELISA) increased in GA3 treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA3 increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin. & 2007 Elsevier GmbH. All rights reserved.
Abbreviations: DW, dry weight; ELISA, enzyme-linked immunosorbent assay; GA3, gibberellin A3; M. aeruginosa, Microcystis aeruginosa; MC-LR, microcystin-LR. Corresponding author. Tel./fax: +86 27 68780371. E-mail addresses: [email protected] (X. Pan), [email protected] (F. Chang), [email protected] (L. Kang), [email protected] (Y. Liu), [email protected] (G. Li), [email protected] (D. Li). 0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2007.08.012
ARTICLE IN PRESS 1692
Introduction Algae blooms, and cyanobacterial blooms, in particular, have been observed in eutrophic fresh waters all over the world. Recent studies show that cyanobacterial blooms can also occur in oligotrophic (Pernthaler et al., 1998) and mesotrophic lakes (Pernthaler et al., 2004). Among these bloomforming algae, approximately half can produce toxins (Sivonen and Jones, 1999) which can pose a great threat to the environment and to public health. Microcystins, a type of cyclic heptapeptide hepatotoxins (Rinehart et al., 1994), are the main types of toxins produced by cyanobacteria. Microcystins can be produced by some species of Microcystis, Anabaena, Nostoc, Planktothrix, Lyngbya, Aphanocapsa and Synechocystis. Of these, Microcystis aeruginosa (M. aeruginosa) is the most common microcystin-producing cyanobacteria (Long et al., 2001). Because of the hazards posed toward humans and the environment by microcystins, many studies have been conducted to investigate the factors that influence the growth and microcystin production of cyanobacteria. Many environmental factors, including temperature, pH, light intensity, UV irradiation and nutrients (especially N and P), have been shown to affect microcystin production and Microcystis growth (Kotak et al., 2000; Sivonen and Jones, 1999). Trace elements such as Fe and Zn have also demonstrated to have a positive influence on Microcystis growth and microcystin production (Lukac and Aegerter, 1993). However, all of the above studies have been performed with various mineral matters and physical factors. In contrast, little is known about the effects of organic anthropogenic pollution such as vegetable hormones. Only a few studies have shown the influence of vegetable hormones on phytoplankton, particularly cyanobacteria. Moreover, these studies have primarily focused on the beneficial effects of vegetable hormones. Little attention has been paid to their environmental hazards. Gibberellin is a type of diterpenoid compound and vegetable hormone that is commonly used in fertilization. Among many isomeric compounds of gibberellins, gibberellin A3 (GA3) is the most widely used in agriculture and horticulture. In China, GA3 has been used to fertilize about 26.7 h m2 of land every year for the last 10 years, and this figure is increasing with the development of crop and plant cultivation. In addition, GA3 has chemical stability, a property that allows it to locate at one site in high concentration. In this study, we aimed to test whether GA3 has an effect on the growth and
X. Pan et al. microcystin production of M. aeruginosa strain, the common bloom-forming cyanobacteria. We wished to evaluate the effects of vegetable hormones on bodies of water, and thus draw attention to their overuse.
Materials and methods Cyanobacterial strain and cultivation The microcystin-producing strain M. aeruginosa PCC7806 used in our experiment was originally collected by the Pasteur Culture Collection of Cyanobacteria in France and kindly provided by the Freshwater Algae Culture Collection of the Chinese Academy of Sciences. This strain grows as single cells without any mucilaginous envelope. Microcystin-LR (MC-LR) and cyanopeptolin depsipeptides are the main components of noxious secondary metabolites produced during growth and reproduction (Martin et al., 1993). M. aeruginosa PCC7806 strains at exponential growth phase were used as the inocula. The growth medium of all cultures was BG11 (Stanier et al., 1971). The GA3 (Sigma) concentrations of treatment groups were 0.001, 0.1, 10 and 25 mg/L, and the control group was without GA3. Inocula of every group were grown in batch culture in 500-mL Erlenmeyer flasks containing 300 mL of liquid medium with three replicates. The experiments were conducted at 25711 under light intensity of 60 mmol photons/m2 s with a 12/12 h light/dark cycle. In order to reduce any effect caused by minor difference in photon irradiance, the flasks were shaken manually three times each day and rearranged randomly. Sampling and analysis Samples were taken from the cultures every 4 d, 1 h before the light period of the day, for 28 d. For determination of dry weights (DWs), samples were dried at 60 1C for 4 d to constant weight. Cells were counted microscopically using the procedure of Humphries and Widjaja (1979). The protein concentration was determined by the Coomassie Brilliant Blue G-250 Lowry method (Bradford, 1976) using bovine serum albumin (Sigma) for calibration. The water-soluble carbohydrate content was determined by the phenol anthrone method (Kochert, 1978) using standard solutions of sucrose. Chlorophyll a was measured spectrophotometrically at 665 and 649 nm after extraction with 95% ethanol (Sartory and Grobbelar, 1986). Phycocyanobilin was analyzed according to Bennett and Bogorad’s method (1973) and evaluated with PC ¼ A615–0.474*A652/5.34 (mg/mL). Cell-free culture filtrates, collected at the time of sampling, were used to assay dissolved N and P in the medium. The nitrate was determined after persulfate oxidation (D’Elia et al., 1977), and orthophosphate was determined by the phoshomolybdate method (Murphy and Riley, 1962).
ARTICLE IN PRESS Gibberellin effects on microcystin production in Microcystis Microcystin extraction and quantification by ELISA Samples from days 8, 16 and 24 were taken for toxin analysis. The cells of 30 mL cultures were harvested and stored at 20 1C until analysis. The toxins were extracted in water by sonication and freeze-thawed, centrifuged and concentrated on C18 cartridges, which were then washed with 20% and 60% methanol prior to elution with methanol. The eluted components were evaporated to dry and then resuspended in 2 mL superpure water. Twenty microlitre supernatant of each sample was used in the enzyme-linked immunosorbent assay (ELISA) analyses. Anti-immune complex ELISA analyses was performed according to the procedures described by An and Carmichael (1994). MC-LR in supernatants was determined using microcystin diagnostic kits which were kindly provided by the State Key Laboratory of Algae Resources and Toxicology, Institute of Hydrobiology, Chinese Academy of Sciences, China. Statistical analysis Values were expressed as means7SE and analyzed using the Statistical Package for the Social Sciences, version 11.5 for Windows (SPSS, Chicago, IL, USA). Oneway analysis of variance followed by the Student–Newman–Keuls’ test was applied to examine whether there were any significant differences between the treatments and the control. Probability values were set to 0.05 for difference of low significance or to 0.01 for highly significant differences. Spearman correlation coefficients were chosen to evaluate the correlation of microcystin content with growth and other cellular metabolism products.
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weight (mg DW/mL) increased gradually in the 0.001–10 mg/L range of GA3 treatments (Figure 1B). DW also increased significantly in both the 10 and 25 mg/L treatment groups compared to the control, but there was no difference between the two treatments.
Pigment and intracellular inclusion Cellular chlorophyll a and phycocyanin, the most important components in the course of algae photosynthesis, underwent similar changes (Figure 2A and C). The two pigments in 10 and 25 mg/L treatment groups were greater than that of the control after day 8. The other two treatments were also greater than the control in the amount of pigments, but only some culture phases were statistically significant. The two kinds of intracellular inclusions, cellular-soluble protein and watersoluble carbohydrate content, underwent different changes (Figure 2B and D). The contents of watersoluble carbohydrate decreased with increase in GA3 concentration (Figure 2B). The contents of soluble protein increased with GA3 concentration and the increase was very notable in 10 and 25 mg/ L treatments (Figure 2D). Moreover, we observed that the amounts of chlorophyll a, phycocyanin and soluble protein present gradually increased with time in each experimental group, similar to the changes observed in DW and cell number. Watersoluble carbohydrates showed a different pattern of change. In each group, it increased gradually with time until 24 d of culture and then slightly decreased.
Results Nutrient uptake Growth of experimental strains
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On the whole, the concentrations of nitrogen and phosphorus in culture medium decreased gradually with time (Figure 3). The concentration of nitrogen in culture medium decreased during the first 28 d and decreased more substantially with the increase 1.2
A Dry weight (µg/ml)
Cell number (×106cells/ml)
The growth of M. aeruginosa, estimated by DW and cell number, is shown in Figure 1. Cell numbers increased markedly with the increase of GA3 concentration after 20 d culture (Figure 1A). Dry
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Figure 1. Effect of GA3 on cell number (A) and dry weight (B) of M. aeruginosa with different concentrations: (–’–) control; (–K–) 0.001 mg/L; (–m–) 0.1 mg/L; (–.–) 10 mg/L and (–~–) 25 mg/L. Values are given as means7SE (n ¼ 3).
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X. Pan et al.
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Figure 2. Effect of GA3 on chlorophyll a (A), carbohydrate (B), phycocyanin (C) and protein (D) of M. aeruginosa with different concentrations: (&) control; (O) 0.001 mg/L; (N) 0.1 mg/L; ( ) 10 mg/L and (L) 25 mg/L. Values are given as means7SE (n ¼ 3). *Low significant differences at Po0.05. **Significant differences at Po0.01 (highly significant).
5
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Figure 3. Effect of GA3 on absorption of nitrogen (A) and phosphorus (B) of M. aeruginosa with different concentrations: (–’–) control; (–K–) 0.001 mg/L; (–m–) 0.1 mg/L; (–.–) 10 mg/L and (–~–) 25 mg/L. Values are given as means of three replicates.
of GA3 (Figure 3A). However, phosphorus in culture medium decreased rapidly in the culture prophase (0–12 d) and then the concentration was maintained at a low level after 12 d (Figure 3B). Further, it was also observed that GA3 had no effect on the phosphorus concentration in culture medium.
no statistical difference at days 8 and 16. At 24 d, however, the microcystin content in the 10 and 25 mg/L GA3 treatments was notably greater than in the control (Po0.05, Figure 4). Moreover, the microcystin content corresponded to GA3 concentrations in the telophase, viz. 24 d culture.
Microcystin content
Discussion
The microcystin present in cells (sampling from prophase, metaphase, telophase of culture) increased with time. The microcystin content between the control and the four treatments showed
Cell number and DW are generally used as growth indicators for single-cell algae. In this study, the growth curves indicated by cell number increased with GA3 concentrations from 0.001 to 25 mg/L.
ARTICLE IN PRESS Gibberellin effects on microcystin production in Microcystis
Microcystin content (ng/g DW)
3000 *
2500 * 2000 1500 1000 500 0
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24
Figure 4. Effect of GA3 on microcystin production of M. aeruginosa with different concentrations: (&) control; (O) 0.001 mg/L; (N) 0.1 mg/L; ( ) 10 mg/L and (L) 25 mg/L. Values are given as means7SE (n ¼ 3). *Significant differences at Po0.05 (low significance).
However, when the concentrations of GA3 were above 10 mg/L, the DWs showed no differences among different GA3 treatments. Previous reports found that GA3 can significantly accelerate the cell growth rate of plants, and the mechanism how it does so has been illuminated (Hedden and Phillips, 2000). However, the function and mechanism of this plant hormone to algal growth remains unclear. Studies examining relationships between GA3 and the growth rate of algae have reported that GA3 can increase cell growth rate in species such as Cocconeis scutellum, Navicula mollis, Amphoraa coffeaeformis, Navichla corymbosa, spirulina platens and Skeletonema costatum in concentrations of 0.5–1.25 mg/L, but it inhibits the growth rate beyond these concentrations (Li et al., 2002; Shi et al., 2004; Yang et al., 2000). In contrast, other studies have shown that GA3 had no effect on growth and sporulation of Chlorella fusca (Bendana and Fried, 1967; Evans and Sorokin, 1971). Our results show that GA3 does stimulate M. aeruginosa growth at an optimal concentration of 25 mg/L, which is much higher than previous studies. This may be attributed to the different culture conditions and the different test algae. Moreover, our results show that GA3 can enhance the absorbance ability of M. aeruginosa to nitrogen in culture medium, but has no effect on phosphorus absorption. Those results indicate that GA3 is likely to increase the growth of M. aeruginosa by regulating its nutrient absorbing ability. Pigments are important for photosynthesis and the growth of photoautotrophy. Some studies have reported that phytohormones can promote the contents of algae pigments, for example 2,4-D. It can stimulate the pigment synthesis of Chlamydo-
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monas reinhardtii and Scenedesmus quadricauda at low concentrations (Wong, 2000; Wong and Chang, 1988). However, the effect of GA3 on algae pigments has undergone little investigation. Our results show that GA3 at concentrations of 10 and 25 mg/L significantly increases chlorophyll a and phycocyanin, which corresponds to increased growth. The results indicate that GA3 can increase pigments, enhancing the photosynthesis, and thus promoting the growth of M. aeruginosa. Watersoluble proteins and carbohydrates are the basic intracellular inclusions in metabolism. The results from the present study suggest that GA3 can increase the protein accumulation in algal cells from 0.001 to 25 mg/L, but reduce carbohydrate accumulation. This conclusion that the change of protein is similar to the growth is the same as that of a previous study (Lyck, 2004). However, the result of interest is that carbohydrates decrease with the increase of growth under GA3 treatment, conflicting with the study of Lyck (2004). This is likely a consequence of the phytoplankton’s attempts to cope with a shortage of substance and energy when it grows rapidly under GA3 treatment. Biomass indicators such as biovolume, cell number, DW and chlorophyll a have often been shown in other studies to reflect true changes in microcystin content. Additionally, the linear relations between growth rate and microcystin content have been frequently reported (Long et al., 2001). In our study, we confirm that the toxin content is positively correlated with growth, and that the correlation coefficients of microcystin with DW and cell number are g ¼ 0.680** and 0.825**, respectively. We also observe a positive correlation (g ¼ 0.830, po0.01) between chlorophyll a and microcystin in our study, which is consistent with a previous study (Shi et al., 1995). Microcystins are embedded in the thylakoid membrane (Shi et al., 1995), and thus the content of microcystin increases accordingly at the time when GA3 stimulates the cell growth and pigment contents. The process of microcystin synthesis expends energy provided by ATP (Bickel and Lyck, 2001), the production of which consumes a large quantity of carbohydrates. In addition, the rapid growth under GA3 treatment also expends carbohydrates, as noted above. As a result, microcystin increased significantly, but carbohydrate content decreased after the GA3 treatment. It has been reported that both nitrogen and phosphorus affect the growth and microcystin production of microcystis, but the reaction of different strains to these nutrients can vary to a significant extent (Ve´zie et al., 2002). As noted above, phosphorus in the culture medium can be absorbed rapidly to maintain at a low level,
ARTICLE IN PRESS 1696 and there is no difference between GA3 treatments and the control. Correspondingly, nitrogen has a more potent effect on microcystin production of M. aeruginosa in response to GA3. In conclusion, GA3 has a positive effect on the growth of and microcystin production of M. aeruginosa PCC7806 strain. We suggest that GA3 increases the growth of M. aeruginosa by stimulating its absorbance of nitrogen and ability to utilize carbohydrates, accordingly it increases cellular chlorophyll a and phycocyanin and, finally, induces accumulation of protein and microcystin. Further studies are also required to elucidate the mechanism by which GA3 regulates the growth and microcystin production of cyanobacteria at a molecular level.
Acknowledgments The authors are especially grateful to Dr. Lirong Song and Dr. Nanqin Gan (Institute of Hydrobiology, Chinese Academy of Sciences, China) for their kind help with measuring microcystin. The present investigation was financially supported by Grants 2002CB412300 and 2005AA60101504 from the State Ministry of Science and Technology and by KZCX2YW-426 and KSCX2-1-10 from the Chinese Academy of Sciences.
References An J, Carmichael WW. Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins. Toxicon 1994;32:1495–507. Bendana FE, Fried M. Stimulatory effects of gibberellins on the growth of Chlorella pyrenoidosa. Life Sci 1967; 6:1023–33. Bennett A, Bogorad L. Complementary chromatic adaptation in a filamentous blue-green alga. J Cell Biol 1973;58:419–35. Bickel H, Lyck S. Importance of energy charge for microcystin production. In: Chorus I, editor. Cyanotoxins. Berlin: Springer; 2001. p. 133–41. Bradford MM. A rapid and sensitive method for the quantitation of microgran quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248–54. D’Elia CF, Steudler PA, Corwin N. Determination of total nitrogen in aqueous samples using persulfate digestion. Limnol Oceanogr 1977;22:760–4. Evans WK, Sorokin C. Studies of the effect of gibberellic acid on algal growth. Life Sci 1971;10:1227–35. Hedden P, Phillips AL. Gibberellin metabolism: new insights revealed by the genes. Trends Plant Sci 2000; 5:523–9.
X. Pan et al. Humphries SE, Widjaja F. A simple method for separating cells of Microcystis aeruginosa for counting. Br Phycol J 1979;14:313–6. Kochert G. Carbohydrate determination by phenolsulfuric acid methods. In: Hellebust JA, Craigie JS, editors. Handbook for phycological methods: physiological and biochemical methods. London: Cambridge University Press; 1978. p. 95–7. Kotak BG, Lam AKY, Prepas EE, Hrudey SE. Role of chemical and physical variables in regulating microcystin-LR concentration in phytoplankton of eutrophic lakes. Can J Fish Aquat Sci 2000;57:1584–93. Li YJ, Liu SF, Li M. Influences of three plant growth hormones on growth of Amphora coffeae formis and Navichla corymbosa (in Chinese). J Fishery Sci China 2002;9:18–22. Long BM, Jones GJ, Orr PT. Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate. Appl Environ Microbiol 2001;67: 278–83. Lukac M, Aegerter R. Influence of trace metals on growth and toxin production of Microcystis aeruginosa. Toxicon 1993;31:293–305. Lyck S. Simultaneous changes in cell quotas of microcystin, chlorophyll a, protein and carbohydrate during different growth phases of a batch culture experiment with Microcystis aeruginosa. J Plankton Res 2004;26: 727–36. Martin C, Oberer L, Ino T, Ko ¨nig WA, Busch M, Weckesser J. Cyanopeptolins, new depsipeptides derived from the cyanobacterium Microcystis aeruginosa PCC 7806. J Antibiot 1993;46:1550–6. Murphy J, Riley JP. A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 1962;27:31–6. Pernthaler J, Glo ¨ckner FO, Unterholzner S, Alfreider A, Psenner R, Amann R. Seasonal community and population dynamics of pelagic bacteria and archaea in a high mountain lake. Appl Environ Microbiol 1998;64:4299–306. Pernthaler J, Zollner E, Warnecke F, Ju ¨rgens K. Bloom of filamentous bacteria in a mesotrophic Lake: identity and potential controlling mechanism. Appl Environ Microbiol 2004;70:6272–81. Rinehart KL, Namikoshi M, Choi BW. Structure and biosynthesis of toxins from blue-green algae (cyanobacteria). J Appl Phycol 1994;6:159–76. Sartory DP, Grobbelar JU. Extraction of chlorophylla from freshwater phytoplankton for spectrophotometric analysis. Hydrobiologia 1986;114:117–87. Shi CY, Cai WR, Gan XH, Wang BF, Zhao LX, Tang XY. Effects of six plant growth regulators on the growth of Spirulina platensis (in Chinese). J Anhui Agric Univ 2004;31:26–9. Shi L, Charmichael WW, Miller I. Immuno-gold localization of hepatotoxins in cyanobacterial cells. Arch Microbiol 1995;163:373–84. Sivonen K, Jones G. Cyanobacterial toxins. In: Chorus I, Bartram J, editors. Toxic cyanobacteria in water: a guide to their public health consequences, monitoring
ARTICLE IN PRESS Gibberellin effects on microcystin production in Microcystis and management. London: E & FN Spon; 1999. p. 41–111. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G. Purification and properties of unicellular blue-green algae (order Chroococales). Bacteriol Rev 1971;35:171–205. Ve´zie C, Rapala J, Vaitomaa J, Seitsonen J, Sivonen K. Effect of nitrogen and phosphorus on growth of toxic and nontoxic Microcystis strains and on intracellular microcystin concentrations. Microb Ecol 2002;43: 443–54.
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Wong PK. Effects of 2,4-D, glyphosate and paraquat on growth, photosynthesis and chlorophyll-a synthesis of Scenedesmus quadricauda Berb 614. Chemosphere 2000;41:177–82. Wong PK, Chang L. The effects of 2, 4-D herbicide and organophosphorus insecticides on growth, photosynthesis, and chlorophyll a synthesis of Chlamydomonas reinhardtii (mt +). Environ Pollut 1988;55:179–89. Yang QY, Shi QQ, Chen BL, Wu SG. Study on advancing the growth rate of Pavlova viridis Tseng with plant hormone (in Chinese). J Fujian Teach Univ (Nat Sci) 2000;16:80–3.
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Effects of gibberellin A3 on growth and microcystin production in Microcystis aeruginosa (cyanophyta) Xiaojie Pana,b, Fengyi Changa,b, Lijuan Kanga,b, Yongding Liua,, Genbao Lia, Dunhai Lia a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 7 South Donghu Road, Wuhan 430072, Hubei Province, PR China b Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China Received 25 April 2007; received in revised form 11 August 2007; accepted 15 August 2007
KEYWORDS Gibberellin A3; Growth; Microcystin; Microcystis aeruginosa; Pollutant
Summary Environmental factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A3 (GA3) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA3 on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001–25 mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA3 treatment. GA3 increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA3 was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-linked immunosorbent assay (ELISA) increased in GA3 treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA3 increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin. & 2007 Elsevier GmbH. All rights reserved.
Abbreviations: DW, dry weight; ELISA, enzyme-linked immunosorbent assay; GA3, gibberellin A3; M. aeruginosa, Microcystis aeruginosa; MC-LR, microcystin-LR. Corresponding author. Tel./fax: +86 27 68780371. E-mail addresses: [email protected] (X. Pan), [email protected] (F. Chang), [email protected] (L. Kang), [email protected] (Y. Liu), [email protected] (G. Li), [email protected] (D. Li). 0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2007.08.012
ARTICLE IN PRESS 1692
Introduction Algae blooms, and cyanobacterial blooms, in particular, have been observed in eutrophic fresh waters all over the world. Recent studies show that cyanobacterial blooms can also occur in oligotrophic (Pernthaler et al., 1998) and mesotrophic lakes (Pernthaler et al., 2004). Among these bloomforming algae, approximately half can produce toxins (Sivonen and Jones, 1999) which can pose a great threat to the environment and to public health. Microcystins, a type of cyclic heptapeptide hepatotoxins (Rinehart et al., 1994), are the main types of toxins produced by cyanobacteria. Microcystins can be produced by some species of Microcystis, Anabaena, Nostoc, Planktothrix, Lyngbya, Aphanocapsa and Synechocystis. Of these, Microcystis aeruginosa (M. aeruginosa) is the most common microcystin-producing cyanobacteria (Long et al., 2001). Because of the hazards posed toward humans and the environment by microcystins, many studies have been conducted to investigate the factors that influence the growth and microcystin production of cyanobacteria. Many environmental factors, including temperature, pH, light intensity, UV irradiation and nutrients (especially N and P), have been shown to affect microcystin production and Microcystis growth (Kotak et al., 2000; Sivonen and Jones, 1999). Trace elements such as Fe and Zn have also demonstrated to have a positive influence on Microcystis growth and microcystin production (Lukac and Aegerter, 1993). However, all of the above studies have been performed with various mineral matters and physical factors. In contrast, little is known about the effects of organic anthropogenic pollution such as vegetable hormones. Only a few studies have shown the influence of vegetable hormones on phytoplankton, particularly cyanobacteria. Moreover, these studies have primarily focused on the beneficial effects of vegetable hormones. Little attention has been paid to their environmental hazards. Gibberellin is a type of diterpenoid compound and vegetable hormone that is commonly used in fertilization. Among many isomeric compounds of gibberellins, gibberellin A3 (GA3) is the most widely used in agriculture and horticulture. In China, GA3 has been used to fertilize about 26.7 h m2 of land every year for the last 10 years, and this figure is increasing with the development of crop and plant cultivation. In addition, GA3 has chemical stability, a property that allows it to locate at one site in high concentration. In this study, we aimed to test whether GA3 has an effect on the growth and
X. Pan et al. microcystin production of M. aeruginosa strain, the common bloom-forming cyanobacteria. We wished to evaluate the effects of vegetable hormones on bodies of water, and thus draw attention to their overuse.
Materials and methods Cyanobacterial strain and cultivation The microcystin-producing strain M. aeruginosa PCC7806 used in our experiment was originally collected by the Pasteur Culture Collection of Cyanobacteria in France and kindly provided by the Freshwater Algae Culture Collection of the Chinese Academy of Sciences. This strain grows as single cells without any mucilaginous envelope. Microcystin-LR (MC-LR) and cyanopeptolin depsipeptides are the main components of noxious secondary metabolites produced during growth and reproduction (Martin et al., 1993). M. aeruginosa PCC7806 strains at exponential growth phase were used as the inocula. The growth medium of all cultures was BG11 (Stanier et al., 1971). The GA3 (Sigma) concentrations of treatment groups were 0.001, 0.1, 10 and 25 mg/L, and the control group was without GA3. Inocula of every group were grown in batch culture in 500-mL Erlenmeyer flasks containing 300 mL of liquid medium with three replicates. The experiments were conducted at 25711 under light intensity of 60 mmol photons/m2 s with a 12/12 h light/dark cycle. In order to reduce any effect caused by minor difference in photon irradiance, the flasks were shaken manually three times each day and rearranged randomly. Sampling and analysis Samples were taken from the cultures every 4 d, 1 h before the light period of the day, for 28 d. For determination of dry weights (DWs), samples were dried at 60 1C for 4 d to constant weight. Cells were counted microscopically using the procedure of Humphries and Widjaja (1979). The protein concentration was determined by the Coomassie Brilliant Blue G-250 Lowry method (Bradford, 1976) using bovine serum albumin (Sigma) for calibration. The water-soluble carbohydrate content was determined by the phenol anthrone method (Kochert, 1978) using standard solutions of sucrose. Chlorophyll a was measured spectrophotometrically at 665 and 649 nm after extraction with 95% ethanol (Sartory and Grobbelar, 1986). Phycocyanobilin was analyzed according to Bennett and Bogorad’s method (1973) and evaluated with PC ¼ A615–0.474*A652/5.34 (mg/mL). Cell-free culture filtrates, collected at the time of sampling, were used to assay dissolved N and P in the medium. The nitrate was determined after persulfate oxidation (D’Elia et al., 1977), and orthophosphate was determined by the phoshomolybdate method (Murphy and Riley, 1962).
ARTICLE IN PRESS Gibberellin effects on microcystin production in Microcystis Microcystin extraction and quantification by ELISA Samples from days 8, 16 and 24 were taken for toxin analysis. The cells of 30 mL cultures were harvested and stored at 20 1C until analysis. The toxins were extracted in water by sonication and freeze-thawed, centrifuged and concentrated on C18 cartridges, which were then washed with 20% and 60% methanol prior to elution with methanol. The eluted components were evaporated to dry and then resuspended in 2 mL superpure water. Twenty microlitre supernatant of each sample was used in the enzyme-linked immunosorbent assay (ELISA) analyses. Anti-immune complex ELISA analyses was performed according to the procedures described by An and Carmichael (1994). MC-LR in supernatants was determined using microcystin diagnostic kits which were kindly provided by the State Key Laboratory of Algae Resources and Toxicology, Institute of Hydrobiology, Chinese Academy of Sciences, China. Statistical analysis Values were expressed as means7SE and analyzed using the Statistical Package for the Social Sciences, version 11.5 for Windows (SPSS, Chicago, IL, USA). Oneway analysis of variance followed by the Student–Newman–Keuls’ test was applied to examine whether there were any significant differences between the treatments and the control. Probability values were set to 0.05 for difference of low significance or to 0.01 for highly significant differences. Spearman correlation coefficients were chosen to evaluate the correlation of microcystin content with growth and other cellular metabolism products.
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weight (mg DW/mL) increased gradually in the 0.001–10 mg/L range of GA3 treatments (Figure 1B). DW also increased significantly in both the 10 and 25 mg/L treatment groups compared to the control, but there was no difference between the two treatments.
Pigment and intracellular inclusion Cellular chlorophyll a and phycocyanin, the most important components in the course of algae photosynthesis, underwent similar changes (Figure 2A and C). The two pigments in 10 and 25 mg/L treatment groups were greater than that of the control after day 8. The other two treatments were also greater than the control in the amount of pigments, but only some culture phases were statistically significant. The two kinds of intracellular inclusions, cellular-soluble protein and watersoluble carbohydrate content, underwent different changes (Figure 2B and D). The contents of watersoluble carbohydrate decreased with increase in GA3 concentration (Figure 2B). The contents of soluble protein increased with GA3 concentration and the increase was very notable in 10 and 25 mg/ L treatments (Figure 2D). Moreover, we observed that the amounts of chlorophyll a, phycocyanin and soluble protein present gradually increased with time in each experimental group, similar to the changes observed in DW and cell number. Watersoluble carbohydrates showed a different pattern of change. In each group, it increased gradually with time until 24 d of culture and then slightly decreased.
Results Nutrient uptake Growth of experimental strains
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On the whole, the concentrations of nitrogen and phosphorus in culture medium decreased gradually with time (Figure 3). The concentration of nitrogen in culture medium decreased during the first 28 d and decreased more substantially with the increase 1.2
A Dry weight (µg/ml)
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The growth of M. aeruginosa, estimated by DW and cell number, is shown in Figure 1. Cell numbers increased markedly with the increase of GA3 concentration after 20 d culture (Figure 1A). Dry
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Figure 1. Effect of GA3 on cell number (A) and dry weight (B) of M. aeruginosa with different concentrations: (–’–) control; (–K–) 0.001 mg/L; (–m–) 0.1 mg/L; (–.–) 10 mg/L and (–~–) 25 mg/L. Values are given as means7SE (n ¼ 3).
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Figure 2. Effect of GA3 on chlorophyll a (A), carbohydrate (B), phycocyanin (C) and protein (D) of M. aeruginosa with different concentrations: (&) control; (O) 0.001 mg/L; (N) 0.1 mg/L; ( ) 10 mg/L and (L) 25 mg/L. Values are given as means7SE (n ¼ 3). *Low significant differences at Po0.05. **Significant differences at Po0.01 (highly significant).
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Figure 3. Effect of GA3 on absorption of nitrogen (A) and phosphorus (B) of M. aeruginosa with different concentrations: (–’–) control; (–K–) 0.001 mg/L; (–m–) 0.1 mg/L; (–.–) 10 mg/L and (–~–) 25 mg/L. Values are given as means of three replicates.
of GA3 (Figure 3A). However, phosphorus in culture medium decreased rapidly in the culture prophase (0–12 d) and then the concentration was maintained at a low level after 12 d (Figure 3B). Further, it was also observed that GA3 had no effect on the phosphorus concentration in culture medium.
no statistical difference at days 8 and 16. At 24 d, however, the microcystin content in the 10 and 25 mg/L GA3 treatments was notably greater than in the control (Po0.05, Figure 4). Moreover, the microcystin content corresponded to GA3 concentrations in the telophase, viz. 24 d culture.
Microcystin content
Discussion
The microcystin present in cells (sampling from prophase, metaphase, telophase of culture) increased with time. The microcystin content between the control and the four treatments showed
Cell number and DW are generally used as growth indicators for single-cell algae. In this study, the growth curves indicated by cell number increased with GA3 concentrations from 0.001 to 25 mg/L.
ARTICLE IN PRESS Gibberellin effects on microcystin production in Microcystis
Microcystin content (ng/g DW)
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Figure 4. Effect of GA3 on microcystin production of M. aeruginosa with different concentrations: (&) control; (O) 0.001 mg/L; (N) 0.1 mg/L; ( ) 10 mg/L and (L) 25 mg/L. Values are given as means7SE (n ¼ 3). *Significant differences at Po0.05 (low significance).
However, when the concentrations of GA3 were above 10 mg/L, the DWs showed no differences among different GA3 treatments. Previous reports found that GA3 can significantly accelerate the cell growth rate of plants, and the mechanism how it does so has been illuminated (Hedden and Phillips, 2000). However, the function and mechanism of this plant hormone to algal growth remains unclear. Studies examining relationships between GA3 and the growth rate of algae have reported that GA3 can increase cell growth rate in species such as Cocconeis scutellum, Navicula mollis, Amphoraa coffeaeformis, Navichla corymbosa, spirulina platens and Skeletonema costatum in concentrations of 0.5–1.25 mg/L, but it inhibits the growth rate beyond these concentrations (Li et al., 2002; Shi et al., 2004; Yang et al., 2000). In contrast, other studies have shown that GA3 had no effect on growth and sporulation of Chlorella fusca (Bendana and Fried, 1967; Evans and Sorokin, 1971). Our results show that GA3 does stimulate M. aeruginosa growth at an optimal concentration of 25 mg/L, which is much higher than previous studies. This may be attributed to the different culture conditions and the different test algae. Moreover, our results show that GA3 can enhance the absorbance ability of M. aeruginosa to nitrogen in culture medium, but has no effect on phosphorus absorption. Those results indicate that GA3 is likely to increase the growth of M. aeruginosa by regulating its nutrient absorbing ability. Pigments are important for photosynthesis and the growth of photoautotrophy. Some studies have reported that phytohormones can promote the contents of algae pigments, for example 2,4-D. It can stimulate the pigment synthesis of Chlamydo-
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monas reinhardtii and Scenedesmus quadricauda at low concentrations (Wong, 2000; Wong and Chang, 1988). However, the effect of GA3 on algae pigments has undergone little investigation. Our results show that GA3 at concentrations of 10 and 25 mg/L significantly increases chlorophyll a and phycocyanin, which corresponds to increased growth. The results indicate that GA3 can increase pigments, enhancing the photosynthesis, and thus promoting the growth of M. aeruginosa. Watersoluble proteins and carbohydrates are the basic intracellular inclusions in metabolism. The results from the present study suggest that GA3 can increase the protein accumulation in algal cells from 0.001 to 25 mg/L, but reduce carbohydrate accumulation. This conclusion that the change of protein is similar to the growth is the same as that of a previous study (Lyck, 2004). However, the result of interest is that carbohydrates decrease with the increase of growth under GA3 treatment, conflicting with the study of Lyck (2004). This is likely a consequence of the phytoplankton’s attempts to cope with a shortage of substance and energy when it grows rapidly under GA3 treatment. Biomass indicators such as biovolume, cell number, DW and chlorophyll a have often been shown in other studies to reflect true changes in microcystin content. Additionally, the linear relations between growth rate and microcystin content have been frequently reported (Long et al., 2001). In our study, we confirm that the toxin content is positively correlated with growth, and that the correlation coefficients of microcystin with DW and cell number are g ¼ 0.680** and 0.825**, respectively. We also observe a positive correlation (g ¼ 0.830, po0.01) between chlorophyll a and microcystin in our study, which is consistent with a previous study (Shi et al., 1995). Microcystins are embedded in the thylakoid membrane (Shi et al., 1995), and thus the content of microcystin increases accordingly at the time when GA3 stimulates the cell growth and pigment contents. The process of microcystin synthesis expends energy provided by ATP (Bickel and Lyck, 2001), the production of which consumes a large quantity of carbohydrates. In addition, the rapid growth under GA3 treatment also expends carbohydrates, as noted above. As a result, microcystin increased significantly, but carbohydrate content decreased after the GA3 treatment. It has been reported that both nitrogen and phosphorus affect the growth and microcystin production of microcystis, but the reaction of different strains to these nutrients can vary to a significant extent (Ve´zie et al., 2002). As noted above, phosphorus in the culture medium can be absorbed rapidly to maintain at a low level,
ARTICLE IN PRESS 1696 and there is no difference between GA3 treatments and the control. Correspondingly, nitrogen has a more potent effect on microcystin production of M. aeruginosa in response to GA3. In conclusion, GA3 has a positive effect on the growth of and microcystin production of M. aeruginosa PCC7806 strain. We suggest that GA3 increases the growth of M. aeruginosa by stimulating its absorbance of nitrogen and ability to utilize carbohydrates, accordingly it increases cellular chlorophyll a and phycocyanin and, finally, induces accumulation of protein and microcystin. Further studies are also required to elucidate the mechanism by which GA3 regulates the growth and microcystin production of cyanobacteria at a molecular level.
Acknowledgments The authors are especially grateful to Dr. Lirong Song and Dr. Nanqin Gan (Institute of Hydrobiology, Chinese Academy of Sciences, China) for their kind help with measuring microcystin. The present investigation was financially supported by Grants 2002CB412300 and 2005AA60101504 from the State Ministry of Science and Technology and by KZCX2YW-426 and KSCX2-1-10 from the Chinese Academy of Sciences.
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