Environ Sci Pollut Res DOI 10.1007/s11356-014-4020-3
RESEARCH ARTICLE
Comparative proteomics exploring the molecular mechanism of eutrophic water purification using water hyacinth (Eichhornia crassipes) Xiong Li & Houcheng Xi & Xudong Sun & Yunqiang Yang & Shihai Yang & Yanli Zhou & Xinmao Zhou & Yongping Yang
Received: 23 September 2014 / Accepted: 16 December 2014 # Springer-Verlag Berlin Heidelberg 2015
Abstract Eutrophication is a serious threat to ecosystem stability and use of water resources worldwide. Accordingly, physical, chemical, and biological technologies have been developed to treat eutrophic water. Phytoremediation has attracted a great deal of attention, and water hyacinth (Eichhornia crassipes) is regarded as one of the best plants for purification of eutrophic water. Previous studies have shown that water hyacinths remove nitrogen (N) and phosphorus (P) via diverse processes and that they can inhibit the growth of algae. However, the molecular mechanisms
responsible for these processes, especially the role of proteins, are unknown. In this study, we applied a proteomics approach to investigate the protein dynamics of water hyacinth under three eutrophication levels. The results suggested that proteins with various functions, including response to stress, N and P metabolic pathways, synthesis and secretion, photosynthesis, biosynthesis, and energy metabolism, were involved in regulating water hyacinth to endure the excess-nutrient environment, remove N and P, and inhibit algal growth. The results help us understand the mechanism of purification of eutrophic water by water hyacinth and supply a theoretical basis for improving techniques for phytoremediation of polluted water.
Responsible editor: Thomas Braunbeck Xiong Li and Houcheng Xi contributed equally to this study. X. Li : X. Sun : Y. Yang : S. Yang : Y. Zhou : Y. Yang (*)
Keywords Water hyacinth . Eutrophication . Phytoremediation . Nitrogen . Phosphorus . Proteomics
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China e-mail:
[email protected]
Introduction
X. Li : X. Sun : Y. Yang : S. Yang : Y. Zhou : Y. Yang China Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China X. Li : Y. Zhou University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China H. Xi Guiyang Botanical Garden of Medicinal Plants, Guiyang 550002, People’s Republic of China S. Yang Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China X. Zhou Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China
Eutrophication of rivers, lakes, and marine ecosystems is a universal problem primarily caused by excessive input of nitrogen (N) and phosphorus (P) (Conley et al. 2009). Eutrophication leads to changes in community composition and algal outbreaks (Monteagudo et al. 2012), resulting in decreased dissolved oxygen levels and water quality, fish death, and biodiversity loss (Carpenter et al. 1998; Smith et al. 1999). Because eutrophication is a serious threat to ecosystem stability and use of water resources, it is necessary to control eutrophication and remediate systems that it has affected. There are two methods of controlling eutrophication, (1) decreasing the trophic level and (2) controlling the growth of algae (Qi and Liu 2004). Researchers have developed a variety of control strategies based on these methods, including physical, chemical, and biological technologies (Bernhardt and Clasen 1992; Eunpu 1973; Grobler and Toerien 1986;
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Grundy 1971; Oglesby and Edmondso 1966). Physical and chemical measures usually require a high level of human and financial resources and commonly exert adverse effects on the environment; therefore, biological technologies have received increased attention (Quan et al. 2003). An essential part of the bioremediation technology is phytoremediation, which has attracted the most attention among available methods. Previous studies have indicated that many plant species have good efficiency for the purification of eutrophic water (Ge et al. 1999; Li and Hu 2004; Tong et al. 2003; Xu et al. 2012). Despite being an invasive plant in most of the world (Zhang et al. 2010a), the water hyacinth (Eichhornia crassipes) has been widely applied in ecological restoration engineering. Indeed, the water hyacinth is regarded as one of the best plants for purification of eutrophic water (Hu et al. 2009; Wang et al. 2012; Zhang et al. 2010b) owing to its strong tolerance of sewage, well-developed root system, and rapid growth and reproduction (Wang et al. 2012; Zhang et al. 2010b). The methods and processes through which water hyacinth improves eutrophic water have been thoroughly investigated, and there has been great progress in its application to remediation projects (Chen et al. 2012; Malik 2007; Wang et al. 2012). Many studies have reported that the water hyacinth removes N and P via direct absorption (Ge et al. 1999; Liu et al. 2011a, b; Lou et al. 2005; Tong et al. 2003; Zhang et al. 2010b), and its absorption ability has been shown to differ with changes in the level of eutrophication (Ge et al. 1999; Zhang et al. 2010b). Additionally, some studies have shown that high levels of N or P are removed through activities of rhizospheric microorganisms including ammonification, nitrification, and denitrification (Reddy and Debusk 1987; Santos and Oliveira 1987; Xu et al. 1999). The water hyacinth can also adsorb and precipitate N and P via dense roots (Liu et al. 2002) and inhibit the growth of algae via competition or allelopathic effects (Shanab et al. 2010; Soltan and Rashed 2003). Although many studies have demonstrated the various patterns and effects of water hyacinth during purification of eutrophic water, few have focused on the molecular mechanisms of these activities. Plant life activities and metabolism, such as absorption, synthesis, transformation, and secretion, cannot occur without the participation and regulation of proteins and genes. In addition, most pollution components in eutrophic water, especially N and P, are the fundamental constituents of proteins and nucleic acids (Conley et al. 2009). Thus, proteins must play important roles in the processes of phytoremediation during eutrophication repair. Comparative proteomics is commonly applied to investigate possible relationships between protein abundance and plant growth, development, stress acclimation, or other life activities (Deswal et al. 2014; Ghosh and Xu 2014; Salunke et al. 2014). In the present study, we conducted comparative proteomics to investigate and analyze the expression patterns of proteins in water hyacinth leaves
cultured in water adjusted to four different degrees of eutrophication. Specifically, we attempted to understand the molecular mechanism responsible for the interaction between the water hyacinth and the eutrophic environment at the protein level to improve phytoremediation of systems that have been subjected to eutrophication.
Materials and methods Materials and experimental design Water hyacinth plantlets were collected from Lake Dianchi in May 2013 during the clonal reproduction period. Samples were collected from the same parent so that they would have a similar genetic background. The plantlets were acclimated in a greenhouse (sunlight; 25–28 °C/18–20 °C, day/night) using Hoagland’s nutrient solution (HNS) containing 0.21 mg/L N and 0.031 mg/L P (Hothem et al. 2003) as the planting water. The eutrophication level was determined based on the total N (NO3-N, NH3-N) and total P (PO4-P) (Liu et al. 2011b; Xu et al. 2012; Zhang et al. 2010b). After 30 days of acclimation, plantlets with similar growth and health potential were divided equally into four groups to culture at different trophic levels: (1) group I, original HNS; (2) group II, HNS (N and P loss)+ 5 mg/L N (NH4NO3) and 0.5 mg/L P (KH2PO4); (3) group III, HNS (N and P loss)+20 mg/L N (NH4NO3) and 2 mg/L P (KH2PO4); and (4) group IV, HNS (N and P loss)+50 mg/L N (NH4NO3) and 5 mg/L P (KH2PO4). An isometric plantlet of water hyacinth (about 53 g fresh weight) was placed into one water box (30 cm×20 cm×20 cm) containing 10 L water and subjected to one of the aforementioned trophic levels. After 15 days cultivation, the plantlets were collected for subsequent measurement and analysis. There were three replicates for each water sample. Morphology and biomass measurement The morphology of the plantlets was evaluated based on photographs taken before and after cultivation. The total fresh weight, number of leaves and roots, and length of roots of individual plantlets were measured after 15 days of cultivation and compared with the values from before culture. Observation of allelopathic effect Well-grown Microcystis aeruginosa were equally cultivated in equal volumes of water from the four groups mentioned above. The water from before and after cultivation of the water hyacinth was used to cultivate the algae simultaneously. After 15 days, the absorbance of the algae liquid at 663 nm was determined as the growth value (Liu et al. 2005). Three replicates were performed for each water sample.
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Photosynthesis After being cultured for 15 days, the net photosynthetic rate of leaves was measured using a portable photosynthesis system (Li-6400, Li-Cor Inc., Lincoln, NE, USA). During the measurements, the water vapor pressure deficit was set to about 1.0 kPa and the atmospheric CO 2 concentration was 400 μmol mol−1. The leaf was illuminated by either a quartz halogen light source or a red light-emitting diode (Li-6400-02, Li-Cor Inc.) under a light intensity of 1,000 μmol photons m−2 s−1. Antioxidant enzyme activity assays The activities of catalase (CAT; EC1.11.1.6) and glutathione reductase (GR; EC1.8.1.7) were determined as previously described (DeKosky et al. 2004; Li et al. 2010, 2014a, b). Determination of nitrate reductase activity The activity of nitrate reductase (NR; 1.6.6.1-3) was determined according to a previously described in vitro method (Chen and Zhang 1980; Radin 1973; Schrader et al. 1968). Briefly, about 0.5 g of fresh leaves was homogenized with 2 mL extraction solution (25 mM phosphate buffer, pH 8.8, 1 mM EDTA, 10 mM cysteine) using a mortar and pestle. The homogenates were then filtered through two layers of gauze and centrifuged at 4 °C and 4,000×g for 20 min. Next, 0.2-mL aliquots of the supernatants were mixed with 0.5 mL KNO3 (0.1 M) and 0.3 mL NADH (2 mg ml−1) and placed at 25 °C for 30 min. The liquids were subsequently mixed with 2 mL sulfanilamide (10 g/L) and 2 mL α-naphthylamine (2 g/L) and allowed to stand for 15 min. The absorbance of the supernatant at 520 nm was then determined, after which the NO2− content was calculated from a standard curve based on the reaction of NaNO2 with sulfanilamide and α-naphthylamine. The NR activity was expressed as μmol NO2− h−1 g−1 FW (fresh weight). Measurement of protein content The protein extraction was performed using TRIzol reagent as previously described (Young and Truman 2012). Briefly, approximately 1 g of fresh leaves was cleaved with 5 mL TRIzol for 5 min. Next, 1 mL chloroform was added and the mixture was allowed to stand at −20 °C for 5 min. Following centrifugation at 4 °C and 12,000×g for 10 min, the supernatants were removed. The lower phases were then mixed with isometric isopropanol and allowed to stand at −20 °C for 2 h. Next, the mixtures were centrifuged at 4 °C and 12,000×g for 10 min, after which the supernatants were removed. The precipitates were then washed three times with isopropanol and dried at room temperature, after which they were dissolved in lysate (7 M urea, 2 M thiourea, 4 % (w/v), CHAPS
(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate)) and 60 mM DTT for 1 h with intermittent shaking. Finally, the protein concentration (mg g−1 FW) was determined based on comparison of the absorbance at 595 nm according to a standard curve (Yang et al. 2009). Two-dimensional electrophoresis A total of 1,200 μg extracted proteins from each sample were used for two-dimensional electrophoresis (2-DE) as previously described (Li et al. 2014a, b) employing gel strips with a pH gradient of 4 to 7 (Immobiline DryStrip, pH 4–7 NL, 17 cm; BioRad, Hercules, CA, USA). Spot digestion and protein identification for mass spectrometry analyses Protein spot digestion and identification were performed as previously described (Li et al. 2014a, b) with the MOWSE threshold set above 40 (P
