Responses of Landoltia punctata to cobalt and nickel: Removal, growth, photosynthesis, antioxidant system and starch metabolism

Accepted Manuscript Title: Responses of Landoltia punctata to cobalt and nickel: Removal, growth, photosynthesis, antioxidant system and starch metabolism Authors: Ling Guo, Yanqiang Ding, Yaliang Xu, Zhidan Li, Yanling Jin, Kaize He, Yang Fang, Hai Zhao PII: DOI: Reference:

S0166-445X(17)30178-9 http://dx.doi.org/doi:10.1016/j.aquatox.2017.06.024 AQTOX 4689

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

11-5-2017 15-6-2017 24-6-2017

Please cite this article as: Guo, Ling, Ding, Yanqiang, Xu, Yaliang, Li, Zhidan, Jin, Yanling, He, Kaize, Fang, Yang, Zhao, Hai, Responses of Landoltia punctata to cobalt and nickel: Removal, growth, photosynthesis, antioxidant system and starch metabolism.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2017.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Responses of Landoltia punctata to cobalt and nickel: removal, growth,

photosynthesis,

antioxidant

system

and

starch

metabolism

Ling Guoa,b,c, Yanqiang Ding a,b,c, Yaliang Xua,b,c, Zhidan Li a,b,c, Yanling Jina,b, Kaize Hea,b, Yang Fanga,b,*, Hai Zhaoa,b,*

a

Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology,

Chinese Academy of Sciences, Chengdu 610041, China b

Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China

c

University of Chinese Academy of Sciences, Beijing 100049, China

*Corresponding

author. E-mail addresses: [email protected]

(Y. Fang), [email protected]

(H. Zhao).

HIGHLIGHTS 

The physiological and biochemical responses of Landoltia punctata 0202 after exposure to cobalt and nickel were investigated.



Landoltia punctata was a potential hyperaccumulator of both cobalt and nickel.



A starch content of 53.3% DW could be realized at 5 mg L-1 Co2+.



Cobalt and nickel generally increased the AGPase and SSS activities and decreased the α-amylase activity.



The high metal tolerance of Landoltia punctata was partly derived from the efficient regulation of antioxidant enzymes and the high flavonoid content. 1

ABSTRACT Landoltia punctata has been considered as a potential bioenergy crop due to its high biomass and starch yields in different cultivations. Cobalt and nickel are known to induce starch accumulation in duckweed. We monitored the growth rate, net photosynthesis rate, total chlorophyll content, Rubisco activity, Co2+ and Ni2+ contents, activity of antioxidant enzymes, starch content and activity of related enzymes under various concentrations of cobalt and nickel. The results indicate that Co2+ and Ni2+ (≤ 0.5 mg L-1) can facilitate growth in the beginning. Although the growth rate, net photosynthesis rate, chlorophyll content and Rubisco activity were significantly inhibited at higher concentrations (5 mg L-1), the starch content increased sharply up to 53.3 % dry weight (DW) in L. punctata. These results were attributed to the increase in adenosine diphosphate-glucose pyrophosphorylase (AGPase) and soluble starch synthase (SSS) activities and the decrease in α-amylase activity upon exposure to excess Co2+ and Ni2+. In addition, a substantial increase in the antioxidant enzyme activities and high flavonoid contents in L. punctata may have largely resulted in the metal tolerance. Furthermore, the high Co2+ and Ni2+ contents (2012.9±18.8 and 1997.7±29.2 mg kg-1 DW) in the tissue indicate that L. punctata is a hyperaccumulator. Thus, L. punctata can be considered as a potential candidate for the simultaneous bioremediation of Co2+- and Ni2+-polluted water and high-quality biomass production. Keywords: Cobalt; Nickel; Landoltia punctata; Hyperaccumulator; Antioxidant system; Starch biosynthesis 2

1. Introduction Currently, metal pollution in water and soil is increasingly severe in China. Cobalt and nickel are two of the most prevalent contaminants in water and agricultural soils (Kumar et al., 2016)and readily transfer into crop, such as rice(Nazir et al., 2015), thus posing a considerable threat to human life. Both metals cannot be chemically degraded and are difficult to remove from polluted biota (Salt et al., 1995). Therefore, finding a solution for Co2+ and Ni2+ contamination has received wide scientific attention. Phytoremediation is an applicable environmentally friendly technology for pollution treatment, in which plants are utilized for metal removal (Meagher, 2000; Pilonsmits, 2005; Raskin et al., 1997; Salt et al., 1998). One of the key points in this technology

is

the

selection

of

a

hyperaccumulator

that

can

grow

in

metal-contaminated sites and achieve high accumulation efficiency of the metals (Rani and Juwarkar, 2013). Duckweed, the smallest flowering aquatic monocotyledon, features great adaptability and worldwide distribution (Hillman, 1961). It also exhibits much higher growth rates than most other aquatic or terrestrial plants (Hillman and Culley, 1978; Yin et al., 2015). Because of these characteristics, duckweed species have been utilized in phytotoxicity tests (Jenner and Janssen-Mommen, 1993; Lewis, 1995; Wang and Williams, 1990) and have played a major role in sewage treatment (Oron, 1990). In previous studies, various species have exhibited excellent tolerance to metals, such as Cu, Pb, Zn, Cd and As (Boonyapookana et al., 2002; Qiao et al., 2012; Sasmaz et al., 2015; Seth et al., 2007). In this regard, two papers (Appenroth et al., 3

2010; Sree et al., 2015) have reported the toxicity of Co2+ and Ni2+ to duckweed, and both studies only focused on Lemna and Spirodela species to investigate the accumulation of metals and the starch content in vivo. No information is available regarding the changes in enzymes related to starch biosynthesis upon exposure to Co2+ and Ni2+. According to Liu et al. (Liu et al., 2015), L. punctata accumulated starch efficiently up to 48% DW in a short period of time and was regarded as a high-quality biomass producer. In addition, Zhao et al. (Zhao et al., 2015) reported that L. punctata tolerated and accumulated copper at moderate copper concentrations. Therefore, L. punctata may be a more promising candidate for Co2+ and Ni2+ removal and starch accumulation compared to Lemna and Spirodela species. However, very few studies have reported the starch accumulation in L. punctata under Co2+ and Ni2+ stress and the application of L. punctata for Co2+ and Ni2+ removal. Thus, it is of great value to investigate the physiological and biochemical performance of L. punctata under these conditions. In our study, L. punctata 0202 was chosen to investigate its physiological and biochemical responses to Co2+ and Ni2+. The growth rate, net photosynthesis rate, chlorophyll content, Rubisco activity, Co2+ and Ni2+ contents, activity of antioxidant enzymes, starch content and activity of related enzymes were monitored. This work provides biochemical and physiological insights in L. punctata’s high starch content and metal-hyperaccumulation when exposed to Co2+ and Ni2+. It also evidenced dual benefits of using duckweeds to not only clean-up contaminated water but also produce potentially high quality feedstock for fuels. 4

2. Materials and methods 2.1 Plant materials and cultivation Landoltia punctata 0202, isolated from Sichuan Province, China, was cultivated in 1/5 Hoagland medium (pH 5.0)(Hoagland and Arnon, 1937) in culture containers (23*14*4.5 cm) for one week under a 16 h:8 h (day: night) photoperiod at 25 °C: 15 °C with a light intensity of 130 μM photons m-2 s-1. 2.2 Metal treatments One gram of fresh duckweed was inoculated into 1/5 Hoagland medium in 500 mL beakers (90.0 mm outer diameter *120.0 mm tall), adding Co2+ and Ni2+ to a final concentration of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 mg L-1. According to the results of dry weight, starch content and bioconcentration factor (Fig. S1, S2 and Table S2), 0.05 (Growth-promotion concentration), 0.5 (Emission standard of pollutants) and 5 mg L-1 (High starch-induction concentration) Co2+ and Ni2+ were chosen for further enzyme assays and physiological analyses (Appenroth et al., 2010; Ministry et al., 2010; Sree et al., 2015) . The Co2+ and Ni2+ in this study were derived from CoCl2* 6H2O and NiCl2 *6H2O (purity ≥ 99.5 %, AR, Kelong ltd., Chengdu, China). The same cultivation conditions were implemented as described above. Then, the duckweed was sampled in the beginning of light supply at 0, 1, 2, 3, 5, 7 and 10 d after treatment. Three biological replicates were performed in the experiment. 2.3 Determination of dry weight, growth rate, net photosynthesis rate, total chlorophyll content and ribulose-1, 5-bisphosphate carboxylase (Rubisco) activity 5

Duckweed was surface-dried by centrifugation (100 rpm, 5 min) to remove the free water. To determine the dry weight, samples were dried in an oven at 60 °C overnight. The growth rate was calculated as follows Xiao et al. (2013): Growth rate (g m-2 d-1) = increased dry weight/area/time. The net photosynthesis rate was measured using a photosynthesis system (LI-6400xt, LI-COR, Inc., USA). The total chlorophyll content was estimated using the method described by Arnon (1949). The Rubisco (EC4.1.1.39) activity was tested as described by Sharkey et al. (1991) using a spectrophotometric diagnostic kit (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China). 2.4 Co2+ and Ni2+ accumulation assay To measure the Co2+ and Ni2+ contents in plant tissue, 150 mg dry weight of samples was digested (HNO3:HClO4, 5 mL:1 mL) at 150 °C for 2 h, then the solutions were diluted with deionized water to a final volume of 50 mL. To measure the Co2+ and Ni2+ contents in medium, 2 mL test medium was sampled and filtered, and then the Co2+ and Ni2+ contents were estimated employing Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES 8300, PE, Inc., USA) using Co (GSB G 62021-90(2701), 5% HNO3) and Ni (GSB G 62022-90(2801), 5% HNO3) as standard substances. Working conditions for ICP-OES was as follows: RF power was 1.3 KW; plasma gas flow was 15 L min-1; auxiliary gas flow was 0.2 L min-1; atomizer gas flow was 0.55 L min-1; detection times were 5 s and argon purity ≥ 99.99％.The BFC was calculated as follows: BCF = increased metal in plant tissue (mg kg-1)/metal in initial medium (mg L-1) 6

2.5 Determination of the starch content and activities of enzymes related to starch metabolism The starch content was determined according to the methods described by Xiao et al. (2013) and Liu et al. (2015). Dry duckweed powder (0.03 g) was hydrolyzed with 2.6mL 1.2 M HCl in a boiling water bath for 2 h. Then adjusting the pH to 7.0 with NaOH, adding PbAc to precipitate protein and diluting the solution with deionized water to a final volume of 10 mL. Thereafter, the solution was filtered and treated with a C18 extraction column. Finally, the hydrolysate was analyzed by HPLC (Thermo 2795, Thermo Corp.) with an Evaporative Light Scattering Detector (All-Tech ELSD2000, All-tech., Corp.). The starch content was determined using the total sugar content (starch content = glucose content × 0.909). Fresh duckweed tissue (0.5 g) was homogenized in 5mL precooled enzyme extracting solution (100 mM Tricine-NaOH (pH 8.0), 8mM MgCl2, 2mM EDTA, 50 mM 2-mercaptoethanol, 12.5% (v/v) glycerol, and 5% (w/v) insoluble polyvinylpyrrolidone-40) (Nakamura et al., 1989). The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C. Then, the supernatant was used to measure the enzyme’s activity. The activities of AGPase (EC 2.7.7.27) and SSS (EC 2.4.1.21) were analyzed using the methods described by Nakamura et al. (1989). Both enzyme activities were tested from the variation of NADH at 340 nm measured by a microplate reader (Thermo Scientific Varioskan Flash, Thermo Fisher Scientific Inc., USA). The activities of α-amylase (EC 3.2.1.1) and β-amylase (EC 3.2.1.2) were estimated following the method of Liu et al. (2015). 2.6 Analysis of antioxidant enzyme activities 7

Fresh duckweed tissue (0.5 g) was suspended in 5mL precooled PBS buffer (pH 7.0) and homogenized by trituration. The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C. Then, the supernatant was used to measure the enzyme’s activity and total protein content. The activities of antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were determined as described previously using a spectrophotometric diagnostic kit (NanJing JianCheng Bio Institute, Nanjing, China)(Zhong and Cheng, 2016). Total protein content was determined according to the methods described by Bradford (1976) applying bovine serum albumin as a standard. 2.7 Estimation of the flavonoid content The flavonoid content was estimated using the procedure described by Qiao et al. (2011) with some modifications. Samples of 100 mg dry weight were homogenized in 30 mL of 70% (v/v) methanol and then treated in a water bath at 70 °C for 60 min. The solutions were centrifuged at 3000 rpm for 10 min. Thereafter, the supernatant was transferred to a rotary evaporator and concentrated at 70 °C. The dry residue was dissolved in 10 mL of 70% (v/v) methanol. The flavonoid extract was obtained and the flavonoid content was estimated by HPLC (Thermo spectra system AS3000/UV6000 Detector, USA) at 340 nm applying rutin as a standard. 2.8 Statistics All samples were investigated in triplicate. All results are expressed as the mean ± standard error in the tables and figures. The statistical significance (p＜0.05) was analyzed by one-way analysis of variance (ANOVA) using SPSS software (Version 8

15.0, SPSS Inc., Chicago, IL, USA). 3. Results 3.1 L. punctata growth At lower concentrations (≤0.5 mg L-1, Table S1), Co2+ and Ni2+ had a positive effect on the duckweed growth rates during the first 3-5 d but had a slightly negative effect after 7 d. At higher concentrations (≥5 mg L-1), Co2+ and Ni2+ significantly inhibited duckweed growth. The highest growth rate (7.42±0.35 g m-2 d-1) in the control occurred on day 7, but the highest growth rates in the treatment groups occurred on day 10 and day 5, reaching 7.21±0.23 and 8.30±0.53 g m-2 d-1 at 0.05 mg L-1 Co2+ and Ni2+, respectively. In contrast, at ≥5 mg L-1 Co2+ and Ni2+, the dry weight increased significantly slower after 2 d compared to the control (Fig. S1), possibly due to the toxicity of excess metals. 3.2 Photosynthesis, chlorophyll content and Rubisco activity To analyze the impact of Co2+ and Ni2+ on the photosynthetic performance, the net photosynthesis rate, total chlorophyll content and Rubisco activity were measured during the treatment. As shown in Fig. 1a, the chlorophyll content varied significantly with the Co2+ and Ni2+ concentrations, tending to be lower at higher metal concentrations. The total chlorophyll content increased from the original value of 2.17 to 2.46 mg g-1 FW after 1 d and then decreased slightly to 2.13 mg g-1 FW after 10 d in the control. Likewise, the pigment content at 0.05 mg L-1 Co2+ and Ni2+ did not decrease significantly. Nevertheless, there was a sharp decrease in the total chlorophyll content after exposure to 0.5-5 mg L-1 Co2+ and Ni2+. 9

As shown in Fig. 1b, the net photosynthesis rate decreased remarkably with increasing Co2+ and Ni2+ concentrations over time. After 10 d of treatment, the net photosynthesis rate decreased 5.6% and 4.9%, 43.5% and 47.7%, and 66.0% and 70.7% relative to controls at 0.05, 0.5 and 5 mg L-1 Co2+ and Ni2+, respectively. These results indicate that the net photosynthesis rate of L. punctata was inhibited differentially at different Co2+ and Ni2+ concentrations. Rubisco is the first enzyme involved in CO2 fixation during photosynthesis and is also the sink for nitrogen assimilation in higher plants. Thus, the measurement of Rubisco activity is important for plant physiology investigations. As shown in Fig. 1c, the Rubisco activity decreased with increasing Co2+ and Ni2+ concentrations. The Rubisco activity in the control increased from 1.48 to 1.65 U mg-1 protein but decreased gradually thereafter. The Rubisco activity trends at 0.05 mg L-1 Co2+ and Ni2+ were in accordance with those of the control. However, the Rubisco activity continuously decreased at 0.5-5 mg L-1 Co2+ and Ni2+, and the lowest activity was observed at 3 d at 5 mg L-1 and at 10 d at 0.5 mg L-1, respectively. Finally, the results showed that no significant difference was observed in the net photosynthesis rate, chlorophyll content or Rubisco activity at the same concentration of Co2+ and Ni2+. 3.3 Co2+ and Ni2+ accumulation To assess the accumulation capacity, the concentrations of Co2+ and Ni2+ in the fronds of L. punctata were measured after treatment for 10 d (Table S2). In this study, the Co2+ and Ni2+ contents in L. punctata increased with increasing original concentrations of Co2+ and Ni2+ in the medium (Table S2). The highest Co2+ and Ni2+ 10

contents in L. punctata reached 2012.9±18.8 and 1997.7±29.2 mg kg-1 DW at 10 mg L-1 (Table S2), respectively. However, the maximum BCFs were observed at 0.5 mg L-1, reaching 402.6±26.1 and 673.0±9.9, respectively, which indicates that L. punctata is a potential hyperaccumulator of Co2+ and Ni2+. 3.4 Starch accumulation and the activities of enzymes involved in starch metabolism In addition to Co2+ and Ni2+ accumulation, L. punctata was identified as having great potential for starch accumulation. In this study, the starch content was measured from 0 to 10 d (Fig. 2 and S2). The results showed that starch biosynthesis responded sensitively to high levels of Co2+ and Ni2+. At the same time, Co2+ appeared to be more effective at starch induction than Ni2+ at the same concentration. The starch content increased proportionally with the cultivation time, without lag time. The highest starch content reached 53.3% DW at 5 mg L-1 Co2+ at 10 d (Fig. 2 and S2). To investigate the mechanism of high starch accumulation in the presence of Co2+ and Ni2+, the activities of four enzymes involved in starch metabolism were analyzed. The biosynthesis of starch involves several key enzymes, including AGPase, SSS, and sucrose synthase (SuSy, EC 2.4.1.13). Among these, AGPase was regarded as the rate-limiting enzyme in the starch biosynthesis metabolism pathway (Streb and Zeeman, 2012). As shown in Fig. 3a, the AGPase activity increased more significantly at higher concentrations (0.5-5 mg L-1) than at lower concentrations (0-0.05 mg L-1) after 1 d. The peaks in AGPase activity were found mainly after 1 d and 2 d at 0.5 and 5 mg L-1, respectively, and after 7 d and 10 d at 0 and 0.05 mg L-1, 11

respectively. After 3 d, the AGPase activity reached a relatively stable level. SSS is another key enzyme involved in starch synthesis. As illustrated in Fig. 3b, the SSS activity increased sharply from 4.49 to 12.73 and 9.38 U mg-1 protein at 5 mg L-1 Co2+ and Ni2+ after 1 d, respectively. However, there was no significant difference observed between the low concentration treatment groups and the control, indicating that Co2+ and Ni2+ did not substantially affect the SSS activity at low concentrations. The degradation of starch in higher plants involves two enzymes, α-amylase and β-amylase, which both were tested (Fig. 3c, 3d). The α-amylase activity increased significantly over time at 0-0.5 mg L-1 Co2+ and Ni2+ but decreased sharply after 10 d at 5 mg L-1. The β-amylase activity increased with increasing Co2+ and Ni2+ concentrations, and the peak activities reached 0.62 and 0.41 U mg-1 protein after 10 d of treatment, respectively. 3.5 Activities of antioxidant enzymes A concentration-dependent increase in the activities of the antioxidant enzymes was evident after Co2+ and Ni2+ treatment. The SOD activity increased significantly with increasing Co2+ and Ni2+ concentrations between 0 and 0.5 mg L-1 but decreased at 5 mg L-1 (Fig. 4a). Similar trends were observed in both the POD and CAT activities (Fig. 4b and 4c). The results showed a positive effect between the Co2+ and Ni2+ concentration and antioxidant enzyme activity at lower concentrations (0-0.5 mg L-1). However, higher Co2+ and Ni2+ concentrations (5 mg L-1) significantly inhibited the antioxidant enzyme activities. 3.6 Flavonoid content 12

To analyze the physiological response to metal stress, the flavonoid content was measured at 0, 3 and 10 d. As shown in Fig. 5, the total flavonoid content continuously increased with time at 0-0.5 mg L-1. In contrast, the total flavonoid content decreased slightly at 5 mg L-1 Co2+ and Ni2+. 4. Discussion 4.1 Effects of Co2+ and Ni2+ on L. punctata growth, photosynthesis and the antioxidant system Cobalt and nickel have long been known as essential trace metals for plants, but excess cobalt and nickel can exhibit considerable toxicity and deleteriously affect plant growth (Macnicol and Beckett, 1985; Seregin et al., 2003). In this study, both metals exhibited similar effects on the growth of L. punctata. At low concentrations (≤ 0.5 mg L-1), the growth rate was promoted by Co2+ and Ni2+ in the beginning (Table S1), but at high concentrations (≥ 5 mg L-1), the growth rate was significantly inhibited. The highest growth rate (8.30±0.53 g m-2 d-1) was observed on day 5 at 0.05 mg L-1 Ni2+ (Table S1). This suggests that Ni2+ can promote duckweed growth more efficiently than Co2+ at low concentrations. However, high concentrations of Co2+ and Ni2+ (≥ 5 mg L-1) may inhibit photosynthesis physiologically and biochemically and thus decrease the yield of L. punctata. Excess Co2+ and Ni2+ can obviously inhibit plant photosynthesis because both metals can deteriorate the chloroplast structure (Heath and D'Allura, 1997; Hermle et al., 2007), decrease the chlorophyll content (Seregin and Kozhevnikova, 2006), inhibit Rubisco activity (Ahmad and Ashraf, 2011) and lower the Hill reaction (Gopal 13

et al., 2003). Thus, the net photosynthesis rate, chlorophyll content and Rubisco activity can be used as indicators to reflect Co2+ and Ni2+ stress. In this study, the chlorophyll content decreased with increasing metal concentrations over time. At high concentration (≥ 5 mg L-1), the color of the fronds quickly varied from green to yellow, and foliar chlorosis appeared after 2 or 3 d of treatment, followed by necrosis. Both of these processes are symptoms of Co2+ and Ni2+ toxicity (Gajewska et al., 2006). Similarly, the Rubisco activity was concentration-dependently inhibited in the present study. As the concentrations of Co2+ and Ni2+ increased in the plant, the Rubisco activity was inhibited significantly (48.8% and 51.1% decrease, respectively), thereby decreasing the photosynthesis. The analysis of the net photosynthesis rate showed that the variation trends of the net photosynthesis rate were in accordance with the changes in the chlorophyll content and Rubisco activity. All these changes were responsible for the yield reduction. The variation of antioxidant enzymes, such as SOD, POD, CAT and others, is considered an indicator of metal exposure. Cobalt and nickel can serve as oxidants at certain concentrations due to the induction of ROS production in plants. Plants primarily defend themselves from oxidative attacks by activating antioxidant enzymes. In this study, L. punctata sensitively responded to Co2+ and Ni2+, and the antioxidant enzymes were activated immediately. The increased activities of antioxidant enzymes protected cells from excessive oxidative stress. Thus, L. punctata acclimated to the new conditions and grew normally. However, when exposed to high concentrations of metals, the activities of the antioxidant enzymes were inhibited and thus failed to 14

sufficiently scavenge excess ROS, resulting in cell damage and growth inhibition. Flavonoids are a group of important compounds in higher plants. As shown in a previous study, the flavonoid content ranged from 0.5 to 1.5% dry weight in a majority of plants (Jovanovic et al., 1994), while a higher flavonoid content (>2%) was found in duckweed (Fig. 5). Flavonoids can alleviate the oxidative toxicity and chelate metals (Hall, 2002). Therefore, they have been considered as protectants against metals (Keilig and Ludwigmüller, 2009). In this study, the increase of 21% and 17% in the flavonoid content observed at 0.5 mg L-1 Co2+ and Ni2+, respectively, showed that flavonoid synthesis can be positively induced by Co2+ and Ni2+, especially when the concentrations of Co2+ and Ni2+ are very low. These results are in complete agreement with the results of a previous study (Aziz et al., 2007). Although the flavonoid content decreased slightly at 5 mg L-1 Co2+ and Ni2+, the lowest content (2.17% DW) was still higher than that found in most plants. Presumably, duckweed can utilize flavonoids as non-enzymatic means to defend itself, and thus, the high flavonoid content in L. punctata may partly contribute to Co2+ and Ni2+ tolerance. 4.2 Absorption capacity of L. punctata for Co2+ and Ni2+ The results from the metal analysis in duckweed confirmed the accumulation of cobalt and nickel in duckweed and a corresponding decrease in metals in the medium. In general, plants are defined as hyperaccumulators when the Co2+ and Ni2+ contents in their tissues exceeds 1000 mg kg-1 DW (Zayed et al., 1998). In this study, L. punctata accumulated Co2+ and Ni2+ at up to 2012.9±18.8 and 1997.7±29.2 mg kg-1 DW in vivo, respectively, at 10 mg L-1 Co2+ and Ni2+, which was a value similar to 15

that observed in a previous study (Zayed et al., 1998). Thus, the results suggest that L. punctata is a hyperaccumulator of Co2+ and Ni2+. However, taking into consideration the original metal concentration in the medium, the BCF may sometimes be a better indicator for identifying hyperaccumulators. In the present study, the BCFs did not increase as expected. The highest BCF values (402.6±26.1 and 673.0±9.9) were observed when both concentrations of Co2+ and Ni2+ were 0.5 mg L-1, the highest removal rate reached approximately 58.63% and 56.23% and the peak percentage of recovery reached 30.24% and 44.16%, respectively. However, Khellaf and Zerdaoui (2010) have reported that the BCF values of L. gibba for Ni2+ were very low (BCF≤100). According to the BCF criteria described by Zayed et al. (1998), L. punctata is considered a better accumulator of Ni2+ relative to L. gibba. Furthermore, in China, the average concentrations of Co2+ and Ni2+ in freshwater ranged from 0.2 to 0.5 μg L-1，but higher concentrations of 2.0~4.0 mg L-1 have been detected in Co2+ and Ni2+-polluted site (Luo et al., 2010; Sun and Xiao, 2012). Based on these results, we can conclude that L. punctata can tolerate moderate Co2+ and Ni2+ exposure and be utilized in phytoremediation in nature. 4.3 Effect of Co2+ and Ni2+ on starch biosynthesis and accumulation in L. punctata Starch biosynthesis and accumulation is an important and complicated metabolic process in duckweed and is regulated by several factors endogenously and exogenously. Appenroth et al. (2010) and Sree et al. (2015) have reported that starch accumulation in S. polyrhiza and L. minor was strikingly affected by Co2+ and Ni2+. In 16

addition, the positive effects of Se and Cr on starch accumulation have been reported by Zhong et al. (2016) and Appenroth et al. (2003). These authors analyzed the starch content and monitored the starch granules in the chloroplast under different cultivations. In this study, we analyzed the changes in starch content and key enzyme activities related to starch biosynthesis at different concentrations of Co2+ and Ni2+. The results showed that starch accumulated efficiently at high concentrations of Co2+ and Ni2+ and the highest starch content exceeded 50% of the dry weight, which was approximately 3.5 and 2.7 times higher compared to control, respectively. As shown in Fig. 2, the starch content was not apparently different between Co2+ and Ni2+ at 0.05 mg L-1, but interestingly, the starch content at 0.5 mg L-1 Co2+ was much higher than that at the same concentration of Ni2+. These results indicate that both cobalt and nickel induced starch accumulation in L. punctata and cobalt affected starch accumulation more efficiently relative to nickel. In addition, it was found that the starch content increased dramatically,

whereas

the dry weight

increased

inconspicuously in the initial stage. This result indicates that carbon metabolism may be disordered, which is in good agreement with a previous study (Sree et al., 2015). Both Severi (2001) and Sree et al. (2015) inferred that the impaired metabolic utilization of photosynthesis may cause starch accumulation. This inference was verified at the enzymatic level in this study. The results related to the enzymes involved in starch biosynthesis confirmed high starch accumulation. The activity of AGPase and SSS increased sharply without lag time after metal treatment (Fig. 3a, 3b), which directly caused the over-accumulation of starch granules in the 17

chloroplasts. Furthermore, the high starch accumulation in duckweed may also derive from the inhibition of starch degradation. Researchers have reported that excess Ni may restrict the starch digestion involved in the activity of enzymes, including α- and β-amylases in rice seedlings (Ahmad and Ashraf, 2011). In this study, the α-amylase activity decreased with increasing Co2+ and Ni2+, but conversely, the β-amylase activity significantly increased. This result was probably due to the abnormal starch accumulation that was detrimental to duckweed. Consequently, the increased activity of starch biosynthetic enzymes and the decreased activity of α-amylase resulted in the high starch accumulation. However, it should be noted that this study only examined the variation in starch metabolism enzymes affected by Co2+ and Ni2+. The results do not reveal the detailed molecular regulation mechanism of starch accumulation after exposure to Co2+ and Ni2+. In a subsequent study, we will uncover the molecular mechanism of starch accumulation affected by Co2+ and Ni2+ by applying RNA-seq, bisulfite-seq and qRT-PCR. 5. Conclusions In this study, we offer new insight into the effects of Co2+ and Ni2+ on L. punctata. Under Co2+ and Ni2+ stress, the growth rate, net photosynthesis rate, chlorophyll content and Rubisco activity were significantly affected at high concentrations. Meanwhile, starch was efficiently accumulated to a high level in a short period of time. The increase in AGPase and SSS activities and decrease in α-amylase activity directly resulted in the high starch accumulation. In addition, the metal analysis results revealed that L. punctata is a potential hyperaccumulator of 18

Co2+ and Ni2+. Its metal tolerance may be related to the efficient antioxidant enzymes and high content of flavonoids in L. punctata. In conclusion, L. punctata is a potential candidate for the simultaneous bioremediation of Co2+- and Ni2+-polluted water and production of high-quality biomass. ACKNOWLEDGMENTS This work was supported by the National Key Technology R&D Program of China (No. 2015BAD15B01); Projects of International Cooperation of the Ministry of Science and Technology of China (No. 2014DFA30680); Science and Technology Service Network Initiative (No. KFJ-EW-STS-121); Science & Technology Program of Sichuan Province (No. 2016SZ0070; No.2017NZ0018; No.2017HH0077); Funds for Advanced Manufacturing Innovation Education of De yang, Chinese Academy of Sciences (No. YC-2015-QC01); Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences (No. KLEAMCAS201501; No. KLCAS-2014-02); Environmental Protection Program of Yunnan Province (2014BI008).

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Fig. 1. The change in the total chlorophyll content, net photosynthesis rate (Pn) and Rubisco activity of L. punctata at different Co2+ and Ni2+ concentrations (0.05, 0.5 and 5 mg L-1). A: the total chlorophyll content; B: Pn; C: Rubisco activity; The values represent the mean ± standard error (n=3).

Fig. 2. The change in the starch content of L. punctata at different Co2+ and Ni2+ concentrations (0.05, 0.5 and 5 mg L-1). The values represent the mean ± standard error (n=3). 23

Fig. 3. The AGPase, SSS, α-amylase and β-amylase activities of L. punctata at different Co2+ and Ni2+ concentrations (0.05, 0.5 and 5 mg L-1): (A) AGPase activity, (B) SSS activity, (C) α-amylase activity and (D) β-amylase activity. The values represent the mean ± standard error (n=3). * p < 0.05 as compared to control at the given time point.

Fig. 4. The changes in the antioxidant enzyme activity of L. punctata at different Co2+ and Ni2+ 24

concentrations (0.05, 0.5 and 5 mg L-1): (A) SOD, (B) POD, and (C) CAT. The values represent the mean ± standard error (n=3).

Fig. 5. The change in the flavonoid content of L. punctata at different Co2+ and Ni2+ concentrations (0.05, 0.5 and 5 mg L-1). The values represent the mean ± standard error (n=3). * p < 0.05 as compared to control at the given time point.

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