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Effects of high light and temperature on Microcystis aeruginosa cell growth and β-cyclocitral emission
Ecotoxicology and Environmental Safety 192 (2020) 110313
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Effects of high light and temperature on Microcystis aeruginosa cell growth and β-cyclocitral emission
T
Tiefeng Zhenga,1, Min Zhoua,1, Lin Yangb,1, Yan Wangb, Yaya Wangb, Yiyu Menga, Jialu Liua, Zhaojiang Zuoa,∗ a b
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, 311300, China Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin, 300387, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Cyanobacteria β-Cyclocitral Environmental stressors High light High temperature
Cyanobacteria always massively grow and even occur blooms in summer, with releasing amount of β-cyclocitral. To uncover the effects of summer high irradiance and temperature on cyanobacterial growth and β-cyclocitral emission, the cell growth, reactive oxygen species (ROS) levels, photosynthetic pigment content, chlorophyll fluorescence and β-cyclocitral emission were investigated in Microcystis aeruginosa under high light and temperature. Compared to the control under 50 μmol m−2·s−1, the cell growth was promoted under 100 μmol m−2·s−1, but inhibited under 500 and 1000 μmol m−2·s−1. The inhibition was also detected under high temperature at 30 and 35 °C in contrast to the control at 25 °C. Under high light and high temperature, M. aeruginosa increased ROS levels and reduced photosynthetic pigment content and photosystem II (PSII) efficiency, which resulted in the inhibition on cell growth. With increasing the light intensity and temperature, 1O2 levels gradually increased, while β-carotene content gradually decreased by quenching 1O2, with increasing βcyclocitral emission. In summer, high irradiance and temperature not benefited the growth of cyanobacteria, but the emission of β-cyclocitral derived from β-carotene quenching 1O2 may offset the disadvantages by poisoning other algae.
1. Introduction Water eutrophication promotes massive cyanobacterial growth and even blooms, leading to the emission of an abundance of volatile organic compounds (VOCs) through secondary metabolism (Zuo, 2019). These VOCs not only deteriorate water quality by increasing unpleasant odor (Yang et al., 2008; Zhang et al., 2010), but also inhibit other algal growth, resulting in cyanobacteria becoming the dominant species (Xu et al., 2017; Zuo et al., 2018a, b). Among cyanobacterial VOCs, terpenoids is a main type, which are mainly synthesized via methylerythritol-4-phosphate pathway (MEP) for isoprene and monoterpenes, and via mevalonate pathway (MVA) for sesquiterpenes (Zuo, 2019). Moreover, there is another kind of terpenoids, which is formed through degradation of carotenoids, including βcyclocitral, α-ionone and β-ionone. These compounds contribute to the unpleasant water odor (Ma et al., 2013), and are released from several cyanobacteria species, such as Microcysits, Cyanidium caldarium, Synura uvella, and Planktothrix rubescens (Watson et al., 2016; Xu et al., 2017; Zuo et al., 2018a). During cyanobacterial blooms in summer, these
compounds were detected up to several hundred ng per liter water, e.g., up to 284.3 ng L−1 of β-cyclocitral and 185.0 ng L−1 of β-ionone in Lake Taihu in China (Ma et al., 2013), and up to 538.12 ng L−1 of βcyclocitral and 50.44 ng L−1 of β-ionone in Western Lake Chaohu in China (Jiang et al., 2016). Besides causing water odor, these compounds have inhibitory effects on other algae. When C. pyrenoidosa cells were exposed to β-cyclocitral, α-ionone and β-ionone, the cell growth was inhibited significantly (Ikawa et al., 2001). Meanwhile, β-cyclocitral at 0.1–0.5 mg ml−1 can cause Nitzschia palea cell rupture (Chang et al., 2011), and at high concentration even caused cyanobacteria cell lysis and changed the water color from green to blue (Harada et al., 2009; Arii et al., 2015). Recently, we found that β-cyclocitral can kill C. reinhardtii by inducing programmed cell death (Sun et al., 2019). Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidation of different double bonds in carotenoids to form various products (Havaux, 2013). In Nostoc sp. PCC 7120, three CCD homologs (NSCs) were identified, of which two NSCs showed cleavage function on β-carotene at the C9=C10 double bonds, with producing β-ionone (Marasco et al., 2006; Scherzinger et al., 2006). In
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Corresponding author. E-mail address: [email protected] (Z. Zuo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ecoenv.2020.110313 Received 3 November 2019; Received in revised form 4 February 2020; Accepted 6 February 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.
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emission were investigated after 48 h.
37 species of cyanobacteria, a total of 61 putative ccd sequences have been identified, which were possible involving in the formation of βionone but not β-cyclocitral, suggesting that there is no ccd gene serving β-cyclocitral formation in cyanobacteria (Cui et al., 2012). Besides the dioxygenase cleavage, carotenoids can be degraded by reactive oxygen species (ROS). Singlet oxygen (1O2) is one of the most harmful ROS, which is generated by the transfer of excitation energy from triplet excited chlorophyll (3Chl*) to O2, with the decline of photosynthetic efficiency and over-reduction of the electron transport chain (KriegerLiszkay, 2005; Munné-Bosch et al., 2013). In plants, the oxidation of βcarotene at C7=C8 double bonds by 1O2 results in the formation of βcyclocitral (Ramel et al., 2012; Havaux, 2013), indicating that β-cyclocitral formation relates with photosynthetic electron production and transport. Compared to the terpenoids from MEP and MVA pathways and carotenoid enzymatic cleavage, there were very limited reports about β-cyclocitral formation in cyanobacteria, although the compound can destroy water quality and poison other algae. In previous studies, we found that the emission of β-cyclocitral from M. aeruginosa and M. flos-aquae increased when the cells were kept under nitrogen (N) or phosphorus (P) deficiency condition (Xu et al., 2017; Ye et al., 2018; Zuo et al., 2018a, b). That was consistent with previous study about M. aeruginosa cells increasing β-cyclocitral emission after exhausting the nitrate nutrient (Hasegawa et al., 2012). In exposure to longtime sunshine and high ion concentration, M. aeruginosa cells promoted the emission of VOCs, including β-cyclocitral (Walsh et al., 1998). In addition, heat and wounding stresses promoting β-cyclocitral emission from foliose lichen (García-Plazaola et al., 2017). These results suggest that environmental stressors can influence β-cyclocitral production and emission. In eutrophicated waters, cyanobacteria always massively grow and even occur blooms in summer, with releasing amount of β-cyclocitral (Ma et al., 2013; Jiang et al., 2016), indicating that high irradiance and temperature should promote cyanobacterial cell growth. However, some opposite phenomena have been reported against the promotion (Bañares-España et al., 2013; Lürling et al., 2013; Mowe et al., 2015; Xu and Juneau, 2016). To uncover the effects of the two environmental stressors on cyanobacterial cell growth and β-cyclocitral emission, we investigated M. aeruginosa cell growth, 1O2 and total ROS levels, Chl and carotenoid (Car) content, Chl fluorescence, and β-cyclocitral emission under high light and temperature conditions, and proposed the probable relationship among summer conditions, cell growth and βcyclocitral emission.
2.2. Measurement of ROS levels For analysis of 1O2, M. aeruginosa cells from 10 ml cultures were collected by centrifugation and washed 3 times with 10 mM phosphate buffer (PBS) at pH7.4. Then, the alga cells were resuspended in 200 μl PBS, and added into 1O2 fluorescent probe R to incubate in darkness for 3 h at 37 °C according to the instruction of 1O2 detection kit. The fluorescence intensity of probe R was analyzed by using a flowsight (Merck millipore, FlowSight® Imaging Flow Cytometer, Germany) with excitation wavelength at 488 nm and emission wavelength at 526 nm. Similar to the operational process of 1O2 analysis, the total ROS levels were analyzed by using ROS detection kit. The probe 20,70-dichlorofluorescein diacetate (DCFH-DA) was added into the resuspended alga cells. After incubation in darkness for 20 min at 37 °C following the kit instruction, the fluorescence intensity of 20,70-dichlorofluorescein that derived from ROS oxidizing DCFH was analyzed with excitation wavelength at 488 nm and emission wavelength at 530 nm. 2.3. Determination of photosynthetic pigment content Three ml M. aeruginosa cultures were centrifuged at 6000 rpm for 8 min, and the cell pellets were used to extract photosynthetic pigments by using 3 ml 80% acetone. The content of Chl and Car were determined following our previous method (Zuo et al., 2018b). β-Carotene and zeaxanthin were analyzed by using high performance liquid chromatography (HPLC) (Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column (25 cm × 4.6 mm inner diameter, 5 μm), according to the method of Rao et al. (2006). The mobile phase consisted of a mixture of dichloromethane: acetonitrile: methanol (20: 70: 10, v/v/v), with a flow rate of 1.0 ml min−1 and column temperature at 25 °C. The injection volume was 10 μL of 80% acetone extracts. The β-carotene and zeaxanthin were monitored at 450 nm, and their content was quantified using the curve of corresponding standards. 2.4. Analysis of Chl fluorescence Following our previous methods (Zuo et al., 2018c), the Chl fluorescence from about 1 × 107 M. aeruginosa cells was measured by using YZQ-500 non-modulation Chl fluorescence analyzer (YZQ Technology Co., Beijing, China), and Chl fluorescence parameters maximum quantum yield of photosystem II (PSII) photochemistry (ϕPO), quantum yield for electron transport at t = 0 (ϕEo), and maximum quantum yield of nonphotochemical de-excitation (ϕDO) were estimated according to the method of Rohácek (2002).
2. Materials and methods 2.1. M. aeruginosa cultures treated with high light and temperature M. aeruginosa FACHB-912 was grown in liquid BG11 medium, and kept at a regime with 16 h light (50 μmol m−2·s−1)/8 h dark at 25 °C. When the cell density reached to the mid-logarithmic phase, they were collected by centrifugation and used for high light and temperature treatments. Under sterile condition, the cells were harvested and resuspended in fresh BG11 medium to give a final density of 3 × 107 cells·ml−1. According to the light intensity (about 100–1000 μmol m−2·s−1) for Microcystis colony formation (Zhang et al., 2014) and summer high temperature at Lake Taihu water surface (more than 30 °C) (Jiang et al., 2009), the light intensity and temperature were set. For high light treatment, the cells were treated with 100, 500 and 1000 μmol m−2·s−1 light intensity, respectively, at 25 °C. For high temperature treatment, they were treated with 30/25 °C, 35/ 30 °C and 40/30 °C (day/night), respectively, with light intensity at 50 μmol m−2·s−1. The cells kept in the normal condition of 16 h light (50 μmol m−2·s−1)/8 h dark at 25 °C were considered as the control. After 24 and 48 h, the cell density was measured by using a blood cell counting plate (25 × 16 hemocytometer). Meanwhile, the ROS levels, photosynthetic pigment content, Chl fluorescence and β-cyclocitral
2.5. Determination of β-cyclocitral emission The VOCs from 300 ml M. aeruginosa cultures kept in a 500 ml conical flask were extracted by using 50 μm Carboxen (CAR)/PDMS fiber (Supelco, Bellefonte, PA, US) for 30 min following the solid phase microextraction (SPME) method. The chemical composition of VOCs was analyzed by using a gas chromatograph-mass spectrometer (GCMS) (Thermo Fisher ISQ, Thermo Fisher Scientific, CA, USA). The GC conditions were described in our previous method (Ye et al., 2018). βCyclocitral was determined by searching NIST Library (NIST 08), and whose emission amount was calculated using its standard, according to the method of Koziel et al. (2017). The numbers of the cells used for VOC analysis was counted to calculate β-cyclocitral emission amount (μmol per 107 cells). 2.6. Calculations and statistical analyses There were at least 4 replicates in each determination. One-way 2
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Fig. 1. Effects of high light (A) and temperature (B) on M. aeruginosa cell growth. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. The whole of cells were killed under 40 °C at 24 h, so the cell density was not determined at 48 h. Means ± SE (n = 4).
salt (Zuo et al., 2014b), acid (Zuo et al., 2012, 2015), etc. In the treatments with high light at 100, 500 and 1000 μmol m−2·s−1, the 1O2 levels in M. aeruginosa cells increased by 2.1%, 18.9% (P < 0.05) and 44.8% (P < 0.05), respectively. In the treatments with high temperature at 30 and 35 °C, the 1O2 levels increased by 64.6% (P < 0.05) and 87.8% (P < 0.05), respectively (Fig. 2). Similar to the 1O2, total ROS levels in M. aeruginosa cells also gradually increased with elevating the light intensity and temperature (Fig. 3). Under the light density at 100 μmol m−2·s−1, M. aeruginosa cells increased the levels of total ROS but not 1O2, indicating that the increased ROS should mainly derived from other ROS species. ROS accumulation is harmful to cells, as they can damage photosynthetic pigments, photosynthetic apparatus, cell membranes, proteins, and even DNA (Tartoura and Youssef, 2011; He et al., 2015; Chen et al., 2019). In the present study, the ROS accumulation in M. aeruginosa cells under high light and temperature may result in oxidative damage, which may be one reason for the inhibition on cell growth (Fig. 1).
ANOVA was performed using Origin 8.0 (Origin Lab, USA) to analyze the differences among the treatments, and the figures were drawn using the same software. 3. Results and discussion 3.1. M. aeruginosacell growth under high light and temperature In contrast to the CK under light intensity at 50 μmol m−2·s−1, no significant difference was detected in M. aeruginosa FACHB-912 cell growth under 100, 500 and 1000 μmol m−2·s−1 at 24 h. At 48 h, the cell growth increased by 5.2% (P < 0.05) under 100 μmol m−2·s−1, but reduced by 5.4% (P < 0.05) and 8.2% (P < 0.05), respectively, under 500 and 1000 μmol m−2·s−1 (Fig. 1A). Similarly, low light intensity promoting and high light intensity inhibiting cell growth have also been detected in other M. aeruginosa strain and cyanobacteria. When M. aeruginosa FACHB-927 cells were kept under the light intensity at 80 μmol m−2·s−1, they showed faster growth than under 35 μmol m−2·s−1 (Yang et al., 2012). When the light intensity was up to 176 μmol m−2·s−1, the growth of M. aeruginosa Ma17D and Ma2M was inhibited markedly (Bañares-España et al., 2013). Under high light condition at 300 μmol m−2·s−1, a declined growth rate was also detected in M. aeruginosa CPCC632 and Synechocystis sp. FACHB898 (Xu and Juneau, 2016). In Lake Mendota, 25 °C was the optimum temperature for Aphanizomenon, Anabaena and Microcystis growth and photosynthesis (Konopka and Brock, 1978). For five tropical Microcystis species, high temperature at 30 °C promoted their growth, but extreme high temperature at 36 °C showed inhibitory effects, especially on M. ichthyoblabe, M. flosa-quae and M. viridis (Mowe et al., 2015). According to the analysis of 62 cyanobacteria species, the mean optimum temperature for their growth is 27.2 °C (Lürling et al., 2013). Similarly, high temperature at 30 and 35 °C significantly (P < 0.05) inhibited M. aeruginosa cell growth, and 40 °C high temperature even killed the cells after 24 h (Fig. 1B). These results indicate that cyanobacteria grow faster in a certain deep water area with low light intensity and temperature rather than on water surface in summer, due to high light and temperature inhibiting their growth by causing ROS production (Figs. 2 and 3) and inhibiting photosynthesis (Fig. 4).
3.3. Decrease of photosynthetic pigment content under high light and temperature Chl and Car are essential photosynthetic pigments, which function in the capture of light and its transduction to biochemical energy. βCarotene is one of Car, whose content reduced by 17.8% (P < 0.05), 24.4% (P < 0.05) and 31.1% (P < 0.05), respectively, under 100, 500 and 1000 μmol m−2·s−1, and reduced by 15.6% (P < 0.05) and 26.7% (P < 0.05), respectively, at 30 and 35 °C. Similarly, the content of zeaxanthin (one of Car), Car and Chl also declined with increasing the light intensity and temperature (Table 1), which may be caused by ROS oxidative damage. Similarly, the reduction has also been detected in Synechocystis sp., other Microcystis species and M. aeruginosa strains under high light and temperature stresses (Davis et al., 2009; BañaresEspaña et al., 2013; Xu and Juneau, 2016), indicating that the cells may decline the light absorption and transduction in the light reactions. 3.4. Decrease of Chl fluorescence under high light and temperature Photosynthesis is the principal process of energy metabolism in cyanobacteria, and high light and temperature can influence the process. ϕPO reflects the maximum quantum yield or efficiency of primary photochemistry, while ϕEo reflects the quantum yield for electron transport (Zhao et al., 2016). They significantly (P < 0.05) reduced under high light at 500 and 1000 μmol m−2·s−1 and high temperature at 30 and 35 °C, suggesting that photochemical flux of photons, excitons, electrons and further metabolic events in M. aeruginosa photosystems declined (Fig. 4). In the light reactions, Chl absorbs light energy and changes to singlet excited states (1Chl*) that can be modulated by a safe
3.2. Increase of ROS levels under high light and temperature ROS are unavoidably generated as byproducts in chloroplasts, mitochondria, peroxisomes and cytoplasm during electron transport as well as substance and energy metabolism (Darehshouri and LützMeindl, 2010; Zuo et al., 2014a). O2−·, 1O2, ·OH and H2O2 are the main species of ROS, which massively accumulate under stressful conditions, such as high light (Hu et al., 2019), high temperature (Zuo et al., 2017), 3
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Fig. 2. Effects of high light and temperature on the 1O2 levels in M. aeruginosa cells. A: The fluorescence intensity of randomly selected cells (Scale bar = 20 nm Ch01 is in the bright field, and Ch02 is the fluorescence intensity). B and C: The fluorescence intensity with respect to the CK as 1 in the high light and temperature treatment, respectively. CK: The control, the cells were kept under 50 μmol m-2·s-1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 5).
Fig. 3. Effects of high light and temperature on the total ROS levels in M. aeruginosa cells. A: The fluorescence intensity of randomly selected cells (Scale bar = 20 nm Ch01 is in the bright field, and Ch02 is the fluorescence intensity). B and C: The fluorescence intensity with respect to the CK as 1 in the high light and temperature treatment, respectively. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 5). 4
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Fig. 4. Effects of high light (A, C and E) and temperature (B, D and F) on the photosynthetic abilities in M. aeruginosa cells. A and B: ϕPO, C and D: ϕEO, E and F: ϕDO. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 4).
high temperature at 30 and 35 °C, the nonphotochemical quenching (ϕDO) increased by 50.2% (P < 0.05), 1.2 folds (P < 0.05), 27.0% (P < 0.05) and 81.6% (P < 0.05), respectively (Fig. 4E and F). This was consistent with previous studies in other Microcystis species and M.
dissipation, nonphotochemical quenching. This dissipation is activated when the absorbed light energy exceeds downstream metabolic activity for ATP synthesis (Ballottari et al., 2013; Magdaong and Blankenship, 2018). In exposure to high light at 500 and 1000 μmol m−2·s−1 and Table 1 Effects of high light and temperature on the content of photosynthetic pigments. β-Carotene (μg·10−8 cells)
Zeaxanthin (μg·10−8 cells)
Carotenoids (μg·10−8 cells)
Chlorophylls (μg·10−8 cells)
CK 100 μmol m−2·s−1 500 μmol m−2·s−1 1000 μmol m−2·s−1
0.45 0.37 0.34 0.31
0.20 0.18 0.14 0.12
1.71 1.49 1.28 0.84
0.03a 0.03b 0.02c 0.02d
15.44 ± 0.11a 12.86 ± 0.34b 7.91 ± 0.13c 3.53 ± 0.27d
CK 30 °C 35 °C
0.45 ± 0.06a 0.38 ± 0.01b 0.33 ± 0.01c
1.71 ± 0.03a 1.28 ± 0.03b 1.08 ± 0.02c
15.44 ± 0.11a 9.94 ± 0.32b 6.51 ± 0.35c
± ± ± ±
0.06a 0.03b 0.01b 0.002c
± ± ± ±
0.02a 0.02a 0.004b 0.001c
0.20 ± 0.02a 0.16 ± 0.02b 0.15 ± 0.003b
± ± ± ±
CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 4). 5
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and 40.1 folds (P < 0.05), respectively, at 30 and 35 °C (Fig. 5B). With the increase of β-cyclocitral emission, the β-carotene content declined gradually (Table 1). In addition, N and P deficiency can also promote βcyclocitral emission from M. aeruginosa and M. flos-aquae cells (Hasegawa et al., 2012; Xu et al., 2017; Ye et al., 2018; Zuo et al., 2018a, b). These results demonstrated that β-cyclocitral formation reflected the protection of β-carotene on photosynthetic apparatus by quenching 1O2 under abiotic stresses. Besides 1O2, β-carotene can also quench other ROS produced under high light and temperature, due to its antioxidant properties (Cerezo et al., 2012), which might also contribute to the emission of β-cyclocitral. That can be supported by the emission of β-cyclocitral under 100 μmol m−2·s−1 light intensity, as M. aeruginosa cells declined β-carotene content and accumulated total ROS but not 1O2. α-Ionone and β-ionone are also carotenoid degradants which are formed by the cleavage of β-carotene by CCDs (carotenoid cleavage dioxygenases) (Cui et al., 2012; Havaux, 2013). In 37 species of cyanobacteria, several ccd genes have been identified, including ccd7 in M. aeruginosa NIES-843 (Cui et al., 2012). In the present study, the two compounds (α-ionone and β-ionone) were not detected, which was consistent with our previous studies using the same strain (M. aeruginosa FACHB-912) under different N and P conditions (Ye et al., 2018; Zuo et al., 2018a), which might result from the strain without releasing the two compounds and/or low CCD7 enzyme activity. During cyanobacterial blooms in summer, cyanobacteria release amount of β-cyclocitral (Ma et al., 2013; Jiang et al., 2016). The compound can poison other algae by inhibiting growth, causing cell rupture, and inducing programmed cell death (Ikawa et al., 2001; Chang et al., 2011; Sun et al., 2019), which is beneficial to cyanobacterial growth by reducing competitors. The benefits may offset the disadvantages of high irradiance and temperature on cell growth (Fig. 1), and promote cyanobacteria becoming the dominant species in eutrophicated waters.
aeruginosa strains (Bañares-España et al., 2013; Mackey et al., 2013; Xu and Juneau, 2016), indicating that the absorbed light energy was dissipated as heat through the xanthophyll cycle (Magdaong and Blankenship, 2018). Under high light and temperature, the reduction in photosynthetic pigments, quantum yield and electron transport in PSII, as well as the increase in dissipation of absorbed light energy can decline the photosynthetic abilities in M. aeruginosa, which may be another reason for the inhibition on cell growth (Fig. 1). Zeaxanthin, violaxanthin, antheraxanthin and lutein are the main Car in the xanthophyll cycle (Jahns and Holzwarth, 2012; Ballottari et al., 2013). Under high light and temperature, the reduction of zeaxanthin content was not beneficial to the dissipation of the absorbed light energy (Table 1). However, the ϕDO gradually increased with elevating the light intensity and temperature, demonstrating that the absorbed light energy must be dissipated due to the gradual reduction of photochemical quenching (ϕPO and ϕEO) (Fig. 4). In that case, the absorbed light energy after nonphotochemical quenching may still be in excess with respect to photochemical quenching, which results in ROS formation by 1O2 production (Ballottari et al., 2013; Magdaong and Blankenship, 2018). In the generation of 1O2, the excitation energy from 3Chl* that is produced from 1Chl* by intersystem crossing transfers to O2 (Krieger-Liszkay, 2005; Munné-Bosch et al., 2013). When M. aeruginosa cells were treated with high light and temperature, the 1O2 levels gradually increased with raising the light intensity and temperature, suggesting that massively absorbed light energy was dissipated trough 1O2 production (Fig. 2).
3.5. Emission of β-cyclocitral under high light and temperature 1 O2 can readily oxidize macromolecules in its immediate surroundings, and has been identified as the principal cause of photooxidative damage in plants (Triantaphylidès et al., 2008). β-Carotene can quench 1O2 and 3Chl* to protect PSII against oxidative damage (Triantaphylidès and Havaux, 2009). The quenching of 1O2 by β-carotene at C7=C8 double bonds results in the formation of β-cyclocitral (Ramel et al., 2012; Havaux, 2013). When M. aeruginosa cells were exposed to longtime sunshine and high ion concentration, they increased the emission of β-cyclocitral (Walsh et al., 1998). Under heat and wounding stresses, the increased β-cyclocitral emission from foliose lichen was detected (García-Plazaola et al., 2017). With raising the light intensity, the emission amount of β-cyclocitral from M. aeruginosa increased gradually, with the increase of 41.8% (P < 0.05), 2.0 folds (P < 0.05) and 3.9 folds (P < 0.05), respectively, at 100, 500 and 1000 μmol m−2·s−1 (Fig. 5A). Similarly, β-cyclocitral emission also gradually increased with elevating the temperature, and the emission amount increased by 4.1 folds (P < 0.05)
4. Conclusion In summer, high irradiance and temperature not benefited the growth of cyanobacteria, but they can tolerate the stress conditions by using β-carotene quenching ROS, mainly 1O2, resulting in amount of βcyclocitral production. The increasing temperature due to climate change can increase the production of β-cyclocitral from cyanobacteria. The emission of β-cyclocitral may aggravate cyanobacterial blooms, as it is beneficial to cyanobacteria dominating water bodies by poisoning other algae.
Fig. 5. Effects of high light (A) and temperature (B) on the emission of β-cyclocitral from M. aeruginosa. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 4). 6
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Declaration of interests
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There is no interest conflict in the paper. CRediT authorship contribution statement Tiefeng Zheng: Investigation, Methodology, Data curation. Min Zhou: Investigation, Data curation. Lin Yang: Investigation, Methodology, Data curation. Yan Wang: Investigation. Yaya Wang: Investigation. Yiyu Meng: Investigation. Jialu Liu: Investigation. Zhaojiang Zuo: Conceptualization, Supervision, Writing - original draft, Writing - review & editing. Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 31870585 and 31300364), the Basic Public Welfare Research Project of Zhejiang Province (No. LGN19C150006), the Natural Science Foundation of Zhejiang Province (No. LY17C160004), the National Students' Innovation and Entrepreneurship Training Program (No. 201910341007, 201810341004), the Student Science and Technology Innovation Activity and New Talent Plan of Zhejiang Province (No. 2019R412006), the Student Research Training Program in Zhejiang A&F University (No. 102–2013200125, KX20180048). References Arii, S., Tsuji, K., Tomita, K., Hasegawa, M., Bober, B., Harada, K.I., 2015. Cyanobacterial blue color formation during lysis under natural conditions. Appl. Environ. Microbiol. 81, 2667–2675. Ballottari, M., Mozzo, M., Girardon, J., Hienerwadel, R., Bass, R., 2013. Chlorophyll triplet quenching and photoprotection in the higher plant monomeric antenna protein Lhcb5. J. Phys. Chem. B 117, 11337–11348. Bañares-España, E., Kromkamp, J.C., López-Rodas, V., Costas, E., Flores-Moya, A., 2013. Photoacclimation of cultured strains of the cyanobacterium Microcystis aeruginosa to high-light and low-light conditions, vol. 83. pp. 700–710. Cerezo, J., Zúñiga, J., Bastida, A., Requena, A., Cerón-Carrasco, J.P., Eriksson, L.A., 2012. Antioxidant properties of β-carotene isomers and their role in ohotosystems: insights from Ab initio simulations. J. Phys. Chem. A 116, 3498–3506. Chang, D.W., Hsieh, M.L., Chen, Y.M., Lin, T.F., Chang, J.S., 2011. Kinetics of cell lysis for Microcystis aeruginosa and Nitzschia palea in the exposure to β-cyclocitral. J. Hazard Mater. 185, 1214–1220. Chen, Y., Weng, Y., Zhou, M., Meng, Y., Liu, J., Yang, L., Zuo, Z., 2019. Linalool- and αterpineol-induced programmed cell death in Chlamydomonas reinhardtii. Ecotox. Environ. Saf. 167, 435–440. Cui, H., Wang, Y., Qin, S., 2012. Genomewide analysis of carotenoid cleavage dioxygenases in unicellular and filamentous cyanobacteria. Comp. Funct. Genom. 2012, 164690. Darehshouri, A., Lütz-Meindl, U., 2010. H2O2 localization in the green alga Micrasterias after salt and osmotic stress by TEM coupled electron energy loss spectroscopy. Protoplasma 239, 49–56. Davis, T.W., Berry, D.L., Boyer, G.L., Gobler, C.J., 2009. The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae 8, 715–725. García-Plazaola, J.I., Portillo-Estrada, M., Fernández-Marín, B., Kännaste, A., Niinemets, Ü., 2017. Emissions of carotenoid cleavage products upon heat shock and mechanical wounding from a foliose lichen. Environ. Exp. Bot. 133, 87–97. Harada, K.I., Ozaki, K., Tsuzuki, S., Kato, H., Hasegawa, M., Kuroda, E.K., Arii, S., Tsuji, K., 2009. Blue color formation of cyanobacteria with β-cyclocitral. J. Chem. Ecol. 35, 1295–1301. Hasegawa, M., Nishizawa, A., Tsuji, K., Kimura, S., Harada, K., 2012. Volatile organic compounds derived from 2-keto-acid decarboxylase in Microcystis aeruginosa. Microb. Environ. 27, 525–528. Havaux, M., 2013. Carotenoid oxidation products as stress signals in plants. Plant J. 79, 597–606. He, L.L., Wang, X., Wu, X.X., Wang, Y.X., Kong, Y.M., Wang, X., Liu, B.M., Liu, B., 2015. Protein damage and reactive oxygen species generation induced by the synergistic effects of ultrasound and methylene blue. Spectrochim. Acta A 134, 361–366. Hu, C., Cui, D., Sun, X., Shi, J., Song, L., Li, Y., Xu, N., 2019. Transcriptomic analysis unveils survival strategies of autotrophic Haematococcus pluvialis against high light stress. Aquaculture 513, 734430. Ikawa, M., Sasner, J.J., Haney, J.F., 2001. Activity of cyanobacterial and algal odor compounds found in lake waters on green alga Chlorella pyrenoidosa growth. Hydrobiologia 443, 19–22. Jahns, P., Holzwarth, A.R., 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. BBA – Bioenergetics 1817, 182–193.
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Zuo, Z., Yang, Y., Xu, Q., Yang, W., Zhao, J., Zhou, L., 2018b. Effects of phosphorus sources on volatile organic compound emissions from Microcystis flos-aquae and their toxic effects on Chlamydomonas reinhardtii. Environ. Geochem. Health 40, 1283–1298. Zuo, Z., Ni, B., Yang, L., 2018c. Production of primary metabolites in Microcystis aeruginosa in regulation of nitrogen limitation. Bioresour. Technol. 270, 588–595. Zuo, Z., Zhu, Y., Bai, Y., Wang, Y., 2012. Acetic acid-induced programmed cell death and release of volatile organic compounds in Chlamydomonas reinhardtii. Plant Physiol. Biochem. 51, 175–184.
production is associated with glycolate metabolism in Chlamydomonas reinhardtii (Chlorophyceae) under salt stress. Phycologia 53, 502–507. Zuo, Z., Chen, Z., Zhu, Y., Bai, Y., Wang, Y., 2014b. Effects of NaCl and Na2CO3 stresses on photosynthetic ability of Chlamydomonas reinhardtii. Biologia 69, 1314–1322. Zuo, Z., Wang, B., Ying, B., Zhou, L., Zhang, R., 2017. Monoterpene emissions contribute to thermotolerance in Cinnamomum camphora. Trees (Berl.) 31, 1759–1771. Zuo, Z., Yang, L., Chen, S., Ye, C., Han, Y., Wang, S., Ma, Y., 2018a. Effects of nitrogen nutrients on the volatile organic compound emissions from Microcystis aeruginosa. Ecotoxicol. Environ. Saf. 161, 214–220.
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Effects of high light and temperature on Microcystis aeruginosa cell growth and β-cyclocitral emission
T
Tiefeng Zhenga,1, Min Zhoua,1, Lin Yangb,1, Yan Wangb, Yaya Wangb, Yiyu Menga, Jialu Liua, Zhaojiang Zuoa,∗ a b
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, 311300, China Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin, 300387, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Cyanobacteria β-Cyclocitral Environmental stressors High light High temperature
Cyanobacteria always massively grow and even occur blooms in summer, with releasing amount of β-cyclocitral. To uncover the effects of summer high irradiance and temperature on cyanobacterial growth and β-cyclocitral emission, the cell growth, reactive oxygen species (ROS) levels, photosynthetic pigment content, chlorophyll fluorescence and β-cyclocitral emission were investigated in Microcystis aeruginosa under high light and temperature. Compared to the control under 50 μmol m−2·s−1, the cell growth was promoted under 100 μmol m−2·s−1, but inhibited under 500 and 1000 μmol m−2·s−1. The inhibition was also detected under high temperature at 30 and 35 °C in contrast to the control at 25 °C. Under high light and high temperature, M. aeruginosa increased ROS levels and reduced photosynthetic pigment content and photosystem II (PSII) efficiency, which resulted in the inhibition on cell growth. With increasing the light intensity and temperature, 1O2 levels gradually increased, while β-carotene content gradually decreased by quenching 1O2, with increasing βcyclocitral emission. In summer, high irradiance and temperature not benefited the growth of cyanobacteria, but the emission of β-cyclocitral derived from β-carotene quenching 1O2 may offset the disadvantages by poisoning other algae.
1. Introduction Water eutrophication promotes massive cyanobacterial growth and even blooms, leading to the emission of an abundance of volatile organic compounds (VOCs) through secondary metabolism (Zuo, 2019). These VOCs not only deteriorate water quality by increasing unpleasant odor (Yang et al., 2008; Zhang et al., 2010), but also inhibit other algal growth, resulting in cyanobacteria becoming the dominant species (Xu et al., 2017; Zuo et al., 2018a, b). Among cyanobacterial VOCs, terpenoids is a main type, which are mainly synthesized via methylerythritol-4-phosphate pathway (MEP) for isoprene and monoterpenes, and via mevalonate pathway (MVA) for sesquiterpenes (Zuo, 2019). Moreover, there is another kind of terpenoids, which is formed through degradation of carotenoids, including βcyclocitral, α-ionone and β-ionone. These compounds contribute to the unpleasant water odor (Ma et al., 2013), and are released from several cyanobacteria species, such as Microcysits, Cyanidium caldarium, Synura uvella, and Planktothrix rubescens (Watson et al., 2016; Xu et al., 2017; Zuo et al., 2018a). During cyanobacterial blooms in summer, these
compounds were detected up to several hundred ng per liter water, e.g., up to 284.3 ng L−1 of β-cyclocitral and 185.0 ng L−1 of β-ionone in Lake Taihu in China (Ma et al., 2013), and up to 538.12 ng L−1 of βcyclocitral and 50.44 ng L−1 of β-ionone in Western Lake Chaohu in China (Jiang et al., 2016). Besides causing water odor, these compounds have inhibitory effects on other algae. When C. pyrenoidosa cells were exposed to β-cyclocitral, α-ionone and β-ionone, the cell growth was inhibited significantly (Ikawa et al., 2001). Meanwhile, β-cyclocitral at 0.1–0.5 mg ml−1 can cause Nitzschia palea cell rupture (Chang et al., 2011), and at high concentration even caused cyanobacteria cell lysis and changed the water color from green to blue (Harada et al., 2009; Arii et al., 2015). Recently, we found that β-cyclocitral can kill C. reinhardtii by inducing programmed cell death (Sun et al., 2019). Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidation of different double bonds in carotenoids to form various products (Havaux, 2013). In Nostoc sp. PCC 7120, three CCD homologs (NSCs) were identified, of which two NSCs showed cleavage function on β-carotene at the C9=C10 double bonds, with producing β-ionone (Marasco et al., 2006; Scherzinger et al., 2006). In
∗
Corresponding author. E-mail address: [email protected] (Z. Zuo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ecoenv.2020.110313 Received 3 November 2019; Received in revised form 4 February 2020; Accepted 6 February 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.
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emission were investigated after 48 h.
37 species of cyanobacteria, a total of 61 putative ccd sequences have been identified, which were possible involving in the formation of βionone but not β-cyclocitral, suggesting that there is no ccd gene serving β-cyclocitral formation in cyanobacteria (Cui et al., 2012). Besides the dioxygenase cleavage, carotenoids can be degraded by reactive oxygen species (ROS). Singlet oxygen (1O2) is one of the most harmful ROS, which is generated by the transfer of excitation energy from triplet excited chlorophyll (3Chl*) to O2, with the decline of photosynthetic efficiency and over-reduction of the electron transport chain (KriegerLiszkay, 2005; Munné-Bosch et al., 2013). In plants, the oxidation of βcarotene at C7=C8 double bonds by 1O2 results in the formation of βcyclocitral (Ramel et al., 2012; Havaux, 2013), indicating that β-cyclocitral formation relates with photosynthetic electron production and transport. Compared to the terpenoids from MEP and MVA pathways and carotenoid enzymatic cleavage, there were very limited reports about β-cyclocitral formation in cyanobacteria, although the compound can destroy water quality and poison other algae. In previous studies, we found that the emission of β-cyclocitral from M. aeruginosa and M. flos-aquae increased when the cells were kept under nitrogen (N) or phosphorus (P) deficiency condition (Xu et al., 2017; Ye et al., 2018; Zuo et al., 2018a, b). That was consistent with previous study about M. aeruginosa cells increasing β-cyclocitral emission after exhausting the nitrate nutrient (Hasegawa et al., 2012). In exposure to longtime sunshine and high ion concentration, M. aeruginosa cells promoted the emission of VOCs, including β-cyclocitral (Walsh et al., 1998). In addition, heat and wounding stresses promoting β-cyclocitral emission from foliose lichen (García-Plazaola et al., 2017). These results suggest that environmental stressors can influence β-cyclocitral production and emission. In eutrophicated waters, cyanobacteria always massively grow and even occur blooms in summer, with releasing amount of β-cyclocitral (Ma et al., 2013; Jiang et al., 2016), indicating that high irradiance and temperature should promote cyanobacterial cell growth. However, some opposite phenomena have been reported against the promotion (Bañares-España et al., 2013; Lürling et al., 2013; Mowe et al., 2015; Xu and Juneau, 2016). To uncover the effects of the two environmental stressors on cyanobacterial cell growth and β-cyclocitral emission, we investigated M. aeruginosa cell growth, 1O2 and total ROS levels, Chl and carotenoid (Car) content, Chl fluorescence, and β-cyclocitral emission under high light and temperature conditions, and proposed the probable relationship among summer conditions, cell growth and βcyclocitral emission.
2.2. Measurement of ROS levels For analysis of 1O2, M. aeruginosa cells from 10 ml cultures were collected by centrifugation and washed 3 times with 10 mM phosphate buffer (PBS) at pH7.4. Then, the alga cells were resuspended in 200 μl PBS, and added into 1O2 fluorescent probe R to incubate in darkness for 3 h at 37 °C according to the instruction of 1O2 detection kit. The fluorescence intensity of probe R was analyzed by using a flowsight (Merck millipore, FlowSight® Imaging Flow Cytometer, Germany) with excitation wavelength at 488 nm and emission wavelength at 526 nm. Similar to the operational process of 1O2 analysis, the total ROS levels were analyzed by using ROS detection kit. The probe 20,70-dichlorofluorescein diacetate (DCFH-DA) was added into the resuspended alga cells. After incubation in darkness for 20 min at 37 °C following the kit instruction, the fluorescence intensity of 20,70-dichlorofluorescein that derived from ROS oxidizing DCFH was analyzed with excitation wavelength at 488 nm and emission wavelength at 530 nm. 2.3. Determination of photosynthetic pigment content Three ml M. aeruginosa cultures were centrifuged at 6000 rpm for 8 min, and the cell pellets were used to extract photosynthetic pigments by using 3 ml 80% acetone. The content of Chl and Car were determined following our previous method (Zuo et al., 2018b). β-Carotene and zeaxanthin were analyzed by using high performance liquid chromatography (HPLC) (Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column (25 cm × 4.6 mm inner diameter, 5 μm), according to the method of Rao et al. (2006). The mobile phase consisted of a mixture of dichloromethane: acetonitrile: methanol (20: 70: 10, v/v/v), with a flow rate of 1.0 ml min−1 and column temperature at 25 °C. The injection volume was 10 μL of 80% acetone extracts. The β-carotene and zeaxanthin were monitored at 450 nm, and their content was quantified using the curve of corresponding standards. 2.4. Analysis of Chl fluorescence Following our previous methods (Zuo et al., 2018c), the Chl fluorescence from about 1 × 107 M. aeruginosa cells was measured by using YZQ-500 non-modulation Chl fluorescence analyzer (YZQ Technology Co., Beijing, China), and Chl fluorescence parameters maximum quantum yield of photosystem II (PSII) photochemistry (ϕPO), quantum yield for electron transport at t = 0 (ϕEo), and maximum quantum yield of nonphotochemical de-excitation (ϕDO) were estimated according to the method of Rohácek (2002).
2. Materials and methods 2.1. M. aeruginosa cultures treated with high light and temperature M. aeruginosa FACHB-912 was grown in liquid BG11 medium, and kept at a regime with 16 h light (50 μmol m−2·s−1)/8 h dark at 25 °C. When the cell density reached to the mid-logarithmic phase, they were collected by centrifugation and used for high light and temperature treatments. Under sterile condition, the cells were harvested and resuspended in fresh BG11 medium to give a final density of 3 × 107 cells·ml−1. According to the light intensity (about 100–1000 μmol m−2·s−1) for Microcystis colony formation (Zhang et al., 2014) and summer high temperature at Lake Taihu water surface (more than 30 °C) (Jiang et al., 2009), the light intensity and temperature were set. For high light treatment, the cells were treated with 100, 500 and 1000 μmol m−2·s−1 light intensity, respectively, at 25 °C. For high temperature treatment, they were treated with 30/25 °C, 35/ 30 °C and 40/30 °C (day/night), respectively, with light intensity at 50 μmol m−2·s−1. The cells kept in the normal condition of 16 h light (50 μmol m−2·s−1)/8 h dark at 25 °C were considered as the control. After 24 and 48 h, the cell density was measured by using a blood cell counting plate (25 × 16 hemocytometer). Meanwhile, the ROS levels, photosynthetic pigment content, Chl fluorescence and β-cyclocitral
2.5. Determination of β-cyclocitral emission The VOCs from 300 ml M. aeruginosa cultures kept in a 500 ml conical flask were extracted by using 50 μm Carboxen (CAR)/PDMS fiber (Supelco, Bellefonte, PA, US) for 30 min following the solid phase microextraction (SPME) method. The chemical composition of VOCs was analyzed by using a gas chromatograph-mass spectrometer (GCMS) (Thermo Fisher ISQ, Thermo Fisher Scientific, CA, USA). The GC conditions were described in our previous method (Ye et al., 2018). βCyclocitral was determined by searching NIST Library (NIST 08), and whose emission amount was calculated using its standard, according to the method of Koziel et al. (2017). The numbers of the cells used for VOC analysis was counted to calculate β-cyclocitral emission amount (μmol per 107 cells). 2.6. Calculations and statistical analyses There were at least 4 replicates in each determination. One-way 2
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Fig. 1. Effects of high light (A) and temperature (B) on M. aeruginosa cell growth. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. The whole of cells were killed under 40 °C at 24 h, so the cell density was not determined at 48 h. Means ± SE (n = 4).
salt (Zuo et al., 2014b), acid (Zuo et al., 2012, 2015), etc. In the treatments with high light at 100, 500 and 1000 μmol m−2·s−1, the 1O2 levels in M. aeruginosa cells increased by 2.1%, 18.9% (P < 0.05) and 44.8% (P < 0.05), respectively. In the treatments with high temperature at 30 and 35 °C, the 1O2 levels increased by 64.6% (P < 0.05) and 87.8% (P < 0.05), respectively (Fig. 2). Similar to the 1O2, total ROS levels in M. aeruginosa cells also gradually increased with elevating the light intensity and temperature (Fig. 3). Under the light density at 100 μmol m−2·s−1, M. aeruginosa cells increased the levels of total ROS but not 1O2, indicating that the increased ROS should mainly derived from other ROS species. ROS accumulation is harmful to cells, as they can damage photosynthetic pigments, photosynthetic apparatus, cell membranes, proteins, and even DNA (Tartoura and Youssef, 2011; He et al., 2015; Chen et al., 2019). In the present study, the ROS accumulation in M. aeruginosa cells under high light and temperature may result in oxidative damage, which may be one reason for the inhibition on cell growth (Fig. 1).
ANOVA was performed using Origin 8.0 (Origin Lab, USA) to analyze the differences among the treatments, and the figures were drawn using the same software. 3. Results and discussion 3.1. M. aeruginosacell growth under high light and temperature In contrast to the CK under light intensity at 50 μmol m−2·s−1, no significant difference was detected in M. aeruginosa FACHB-912 cell growth under 100, 500 and 1000 μmol m−2·s−1 at 24 h. At 48 h, the cell growth increased by 5.2% (P < 0.05) under 100 μmol m−2·s−1, but reduced by 5.4% (P < 0.05) and 8.2% (P < 0.05), respectively, under 500 and 1000 μmol m−2·s−1 (Fig. 1A). Similarly, low light intensity promoting and high light intensity inhibiting cell growth have also been detected in other M. aeruginosa strain and cyanobacteria. When M. aeruginosa FACHB-927 cells were kept under the light intensity at 80 μmol m−2·s−1, they showed faster growth than under 35 μmol m−2·s−1 (Yang et al., 2012). When the light intensity was up to 176 μmol m−2·s−1, the growth of M. aeruginosa Ma17D and Ma2M was inhibited markedly (Bañares-España et al., 2013). Under high light condition at 300 μmol m−2·s−1, a declined growth rate was also detected in M. aeruginosa CPCC632 and Synechocystis sp. FACHB898 (Xu and Juneau, 2016). In Lake Mendota, 25 °C was the optimum temperature for Aphanizomenon, Anabaena and Microcystis growth and photosynthesis (Konopka and Brock, 1978). For five tropical Microcystis species, high temperature at 30 °C promoted their growth, but extreme high temperature at 36 °C showed inhibitory effects, especially on M. ichthyoblabe, M. flosa-quae and M. viridis (Mowe et al., 2015). According to the analysis of 62 cyanobacteria species, the mean optimum temperature for their growth is 27.2 °C (Lürling et al., 2013). Similarly, high temperature at 30 and 35 °C significantly (P < 0.05) inhibited M. aeruginosa cell growth, and 40 °C high temperature even killed the cells after 24 h (Fig. 1B). These results indicate that cyanobacteria grow faster in a certain deep water area with low light intensity and temperature rather than on water surface in summer, due to high light and temperature inhibiting their growth by causing ROS production (Figs. 2 and 3) and inhibiting photosynthesis (Fig. 4).
3.3. Decrease of photosynthetic pigment content under high light and temperature Chl and Car are essential photosynthetic pigments, which function in the capture of light and its transduction to biochemical energy. βCarotene is one of Car, whose content reduced by 17.8% (P < 0.05), 24.4% (P < 0.05) and 31.1% (P < 0.05), respectively, under 100, 500 and 1000 μmol m−2·s−1, and reduced by 15.6% (P < 0.05) and 26.7% (P < 0.05), respectively, at 30 and 35 °C. Similarly, the content of zeaxanthin (one of Car), Car and Chl also declined with increasing the light intensity and temperature (Table 1), which may be caused by ROS oxidative damage. Similarly, the reduction has also been detected in Synechocystis sp., other Microcystis species and M. aeruginosa strains under high light and temperature stresses (Davis et al., 2009; BañaresEspaña et al., 2013; Xu and Juneau, 2016), indicating that the cells may decline the light absorption and transduction in the light reactions. 3.4. Decrease of Chl fluorescence under high light and temperature Photosynthesis is the principal process of energy metabolism in cyanobacteria, and high light and temperature can influence the process. ϕPO reflects the maximum quantum yield or efficiency of primary photochemistry, while ϕEo reflects the quantum yield for electron transport (Zhao et al., 2016). They significantly (P < 0.05) reduced under high light at 500 and 1000 μmol m−2·s−1 and high temperature at 30 and 35 °C, suggesting that photochemical flux of photons, excitons, electrons and further metabolic events in M. aeruginosa photosystems declined (Fig. 4). In the light reactions, Chl absorbs light energy and changes to singlet excited states (1Chl*) that can be modulated by a safe
3.2. Increase of ROS levels under high light and temperature ROS are unavoidably generated as byproducts in chloroplasts, mitochondria, peroxisomes and cytoplasm during electron transport as well as substance and energy metabolism (Darehshouri and LützMeindl, 2010; Zuo et al., 2014a). O2−·, 1O2, ·OH and H2O2 are the main species of ROS, which massively accumulate under stressful conditions, such as high light (Hu et al., 2019), high temperature (Zuo et al., 2017), 3
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Fig. 2. Effects of high light and temperature on the 1O2 levels in M. aeruginosa cells. A: The fluorescence intensity of randomly selected cells (Scale bar = 20 nm Ch01 is in the bright field, and Ch02 is the fluorescence intensity). B and C: The fluorescence intensity with respect to the CK as 1 in the high light and temperature treatment, respectively. CK: The control, the cells were kept under 50 μmol m-2·s-1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 5).
Fig. 3. Effects of high light and temperature on the total ROS levels in M. aeruginosa cells. A: The fluorescence intensity of randomly selected cells (Scale bar = 20 nm Ch01 is in the bright field, and Ch02 is the fluorescence intensity). B and C: The fluorescence intensity with respect to the CK as 1 in the high light and temperature treatment, respectively. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 5). 4
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Fig. 4. Effects of high light (A, C and E) and temperature (B, D and F) on the photosynthetic abilities in M. aeruginosa cells. A and B: ϕPO, C and D: ϕEO, E and F: ϕDO. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 4).
high temperature at 30 and 35 °C, the nonphotochemical quenching (ϕDO) increased by 50.2% (P < 0.05), 1.2 folds (P < 0.05), 27.0% (P < 0.05) and 81.6% (P < 0.05), respectively (Fig. 4E and F). This was consistent with previous studies in other Microcystis species and M.
dissipation, nonphotochemical quenching. This dissipation is activated when the absorbed light energy exceeds downstream metabolic activity for ATP synthesis (Ballottari et al., 2013; Magdaong and Blankenship, 2018). In exposure to high light at 500 and 1000 μmol m−2·s−1 and Table 1 Effects of high light and temperature on the content of photosynthetic pigments. β-Carotene (μg·10−8 cells)
Zeaxanthin (μg·10−8 cells)
Carotenoids (μg·10−8 cells)
Chlorophylls (μg·10−8 cells)
CK 100 μmol m−2·s−1 500 μmol m−2·s−1 1000 μmol m−2·s−1
0.45 0.37 0.34 0.31
0.20 0.18 0.14 0.12
1.71 1.49 1.28 0.84
0.03a 0.03b 0.02c 0.02d
15.44 ± 0.11a 12.86 ± 0.34b 7.91 ± 0.13c 3.53 ± 0.27d
CK 30 °C 35 °C
0.45 ± 0.06a 0.38 ± 0.01b 0.33 ± 0.01c
1.71 ± 0.03a 1.28 ± 0.03b 1.08 ± 0.02c
15.44 ± 0.11a 9.94 ± 0.32b 6.51 ± 0.35c
± ± ± ±
0.06a 0.03b 0.01b 0.002c
± ± ± ±
0.02a 0.02a 0.004b 0.001c
0.20 ± 0.02a 0.16 ± 0.02b 0.15 ± 0.003b
± ± ± ±
CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 4). 5
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and 40.1 folds (P < 0.05), respectively, at 30 and 35 °C (Fig. 5B). With the increase of β-cyclocitral emission, the β-carotene content declined gradually (Table 1). In addition, N and P deficiency can also promote βcyclocitral emission from M. aeruginosa and M. flos-aquae cells (Hasegawa et al., 2012; Xu et al., 2017; Ye et al., 2018; Zuo et al., 2018a, b). These results demonstrated that β-cyclocitral formation reflected the protection of β-carotene on photosynthetic apparatus by quenching 1O2 under abiotic stresses. Besides 1O2, β-carotene can also quench other ROS produced under high light and temperature, due to its antioxidant properties (Cerezo et al., 2012), which might also contribute to the emission of β-cyclocitral. That can be supported by the emission of β-cyclocitral under 100 μmol m−2·s−1 light intensity, as M. aeruginosa cells declined β-carotene content and accumulated total ROS but not 1O2. α-Ionone and β-ionone are also carotenoid degradants which are formed by the cleavage of β-carotene by CCDs (carotenoid cleavage dioxygenases) (Cui et al., 2012; Havaux, 2013). In 37 species of cyanobacteria, several ccd genes have been identified, including ccd7 in M. aeruginosa NIES-843 (Cui et al., 2012). In the present study, the two compounds (α-ionone and β-ionone) were not detected, which was consistent with our previous studies using the same strain (M. aeruginosa FACHB-912) under different N and P conditions (Ye et al., 2018; Zuo et al., 2018a), which might result from the strain without releasing the two compounds and/or low CCD7 enzyme activity. During cyanobacterial blooms in summer, cyanobacteria release amount of β-cyclocitral (Ma et al., 2013; Jiang et al., 2016). The compound can poison other algae by inhibiting growth, causing cell rupture, and inducing programmed cell death (Ikawa et al., 2001; Chang et al., 2011; Sun et al., 2019), which is beneficial to cyanobacterial growth by reducing competitors. The benefits may offset the disadvantages of high irradiance and temperature on cell growth (Fig. 1), and promote cyanobacteria becoming the dominant species in eutrophicated waters.
aeruginosa strains (Bañares-España et al., 2013; Mackey et al., 2013; Xu and Juneau, 2016), indicating that the absorbed light energy was dissipated as heat through the xanthophyll cycle (Magdaong and Blankenship, 2018). Under high light and temperature, the reduction in photosynthetic pigments, quantum yield and electron transport in PSII, as well as the increase in dissipation of absorbed light energy can decline the photosynthetic abilities in M. aeruginosa, which may be another reason for the inhibition on cell growth (Fig. 1). Zeaxanthin, violaxanthin, antheraxanthin and lutein are the main Car in the xanthophyll cycle (Jahns and Holzwarth, 2012; Ballottari et al., 2013). Under high light and temperature, the reduction of zeaxanthin content was not beneficial to the dissipation of the absorbed light energy (Table 1). However, the ϕDO gradually increased with elevating the light intensity and temperature, demonstrating that the absorbed light energy must be dissipated due to the gradual reduction of photochemical quenching (ϕPO and ϕEO) (Fig. 4). In that case, the absorbed light energy after nonphotochemical quenching may still be in excess with respect to photochemical quenching, which results in ROS formation by 1O2 production (Ballottari et al., 2013; Magdaong and Blankenship, 2018). In the generation of 1O2, the excitation energy from 3Chl* that is produced from 1Chl* by intersystem crossing transfers to O2 (Krieger-Liszkay, 2005; Munné-Bosch et al., 2013). When M. aeruginosa cells were treated with high light and temperature, the 1O2 levels gradually increased with raising the light intensity and temperature, suggesting that massively absorbed light energy was dissipated trough 1O2 production (Fig. 2).
3.5. Emission of β-cyclocitral under high light and temperature 1 O2 can readily oxidize macromolecules in its immediate surroundings, and has been identified as the principal cause of photooxidative damage in plants (Triantaphylidès et al., 2008). β-Carotene can quench 1O2 and 3Chl* to protect PSII against oxidative damage (Triantaphylidès and Havaux, 2009). The quenching of 1O2 by β-carotene at C7=C8 double bonds results in the formation of β-cyclocitral (Ramel et al., 2012; Havaux, 2013). When M. aeruginosa cells were exposed to longtime sunshine and high ion concentration, they increased the emission of β-cyclocitral (Walsh et al., 1998). Under heat and wounding stresses, the increased β-cyclocitral emission from foliose lichen was detected (García-Plazaola et al., 2017). With raising the light intensity, the emission amount of β-cyclocitral from M. aeruginosa increased gradually, with the increase of 41.8% (P < 0.05), 2.0 folds (P < 0.05) and 3.9 folds (P < 0.05), respectively, at 100, 500 and 1000 μmol m−2·s−1 (Fig. 5A). Similarly, β-cyclocitral emission also gradually increased with elevating the temperature, and the emission amount increased by 4.1 folds (P < 0.05)
4. Conclusion In summer, high irradiance and temperature not benefited the growth of cyanobacteria, but they can tolerate the stress conditions by using β-carotene quenching ROS, mainly 1O2, resulting in amount of βcyclocitral production. The increasing temperature due to climate change can increase the production of β-cyclocitral from cyanobacteria. The emission of β-cyclocitral may aggravate cyanobacterial blooms, as it is beneficial to cyanobacteria dominating water bodies by poisoning other algae.
Fig. 5. Effects of high light (A) and temperature (B) on the emission of β-cyclocitral from M. aeruginosa. CK: The control, the cells were kept under 50 μmol m−2·s−1 light intensity at 25 °C. Different lowercases indicate the significant difference at P < 0.05. Means ± SE (n = 4). 6
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Declaration of interests
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There is no interest conflict in the paper. CRediT authorship contribution statement Tiefeng Zheng: Investigation, Methodology, Data curation. Min Zhou: Investigation, Data curation. Lin Yang: Investigation, Methodology, Data curation. Yan Wang: Investigation. Yaya Wang: Investigation. Yiyu Meng: Investigation. Jialu Liu: Investigation. Zhaojiang Zuo: Conceptualization, Supervision, Writing - original draft, Writing - review & editing. Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 31870585 and 31300364), the Basic Public Welfare Research Project of Zhejiang Province (No. LGN19C150006), the Natural Science Foundation of Zhejiang Province (No. LY17C160004), the National Students' Innovation and Entrepreneurship Training Program (No. 201910341007, 201810341004), the Student Science and Technology Innovation Activity and New Talent Plan of Zhejiang Province (No. 2019R412006), the Student Research Training Program in Zhejiang A&F University (No. 102–2013200125, KX20180048). References Arii, S., Tsuji, K., Tomita, K., Hasegawa, M., Bober, B., Harada, K.I., 2015. Cyanobacterial blue color formation during lysis under natural conditions. Appl. Environ. Microbiol. 81, 2667–2675. Ballottari, M., Mozzo, M., Girardon, J., Hienerwadel, R., Bass, R., 2013. Chlorophyll triplet quenching and photoprotection in the higher plant monomeric antenna protein Lhcb5. J. Phys. Chem. B 117, 11337–11348. Bañares-España, E., Kromkamp, J.C., López-Rodas, V., Costas, E., Flores-Moya, A., 2013. Photoacclimation of cultured strains of the cyanobacterium Microcystis aeruginosa to high-light and low-light conditions, vol. 83. pp. 700–710. Cerezo, J., Zúñiga, J., Bastida, A., Requena, A., Cerón-Carrasco, J.P., Eriksson, L.A., 2012. Antioxidant properties of β-carotene isomers and their role in ohotosystems: insights from Ab initio simulations. J. Phys. Chem. A 116, 3498–3506. Chang, D.W., Hsieh, M.L., Chen, Y.M., Lin, T.F., Chang, J.S., 2011. Kinetics of cell lysis for Microcystis aeruginosa and Nitzschia palea in the exposure to β-cyclocitral. J. Hazard Mater. 185, 1214–1220. Chen, Y., Weng, Y., Zhou, M., Meng, Y., Liu, J., Yang, L., Zuo, Z., 2019. Linalool- and αterpineol-induced programmed cell death in Chlamydomonas reinhardtii. Ecotox. Environ. Saf. 167, 435–440. Cui, H., Wang, Y., Qin, S., 2012. Genomewide analysis of carotenoid cleavage dioxygenases in unicellular and filamentous cyanobacteria. Comp. Funct. Genom. 2012, 164690. Darehshouri, A., Lütz-Meindl, U., 2010. H2O2 localization in the green alga Micrasterias after salt and osmotic stress by TEM coupled electron energy loss spectroscopy. Protoplasma 239, 49–56. Davis, T.W., Berry, D.L., Boyer, G.L., Gobler, C.J., 2009. The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae 8, 715–725. García-Plazaola, J.I., Portillo-Estrada, M., Fernández-Marín, B., Kännaste, A., Niinemets, Ü., 2017. Emissions of carotenoid cleavage products upon heat shock and mechanical wounding from a foliose lichen. Environ. Exp. Bot. 133, 87–97. Harada, K.I., Ozaki, K., Tsuzuki, S., Kato, H., Hasegawa, M., Kuroda, E.K., Arii, S., Tsuji, K., 2009. Blue color formation of cyanobacteria with β-cyclocitral. J. Chem. Ecol. 35, 1295–1301. Hasegawa, M., Nishizawa, A., Tsuji, K., Kimura, S., Harada, K., 2012. Volatile organic compounds derived from 2-keto-acid decarboxylase in Microcystis aeruginosa. Microb. Environ. 27, 525–528. Havaux, M., 2013. Carotenoid oxidation products as stress signals in plants. Plant J. 79, 597–606. He, L.L., Wang, X., Wu, X.X., Wang, Y.X., Kong, Y.M., Wang, X., Liu, B.M., Liu, B., 2015. Protein damage and reactive oxygen species generation induced by the synergistic effects of ultrasound and methylene blue. Spectrochim. Acta A 134, 361–366. Hu, C., Cui, D., Sun, X., Shi, J., Song, L., Li, Y., Xu, N., 2019. Transcriptomic analysis unveils survival strategies of autotrophic Haematococcus pluvialis against high light stress. Aquaculture 513, 734430. Ikawa, M., Sasner, J.J., Haney, J.F., 2001. Activity of cyanobacterial and algal odor compounds found in lake waters on green alga Chlorella pyrenoidosa growth. Hydrobiologia 443, 19–22. Jahns, P., Holzwarth, A.R., 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. BBA – Bioenergetics 1817, 182–193.
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