Melatonin: A multifunctional molecule that triggers defence responses

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Biotechnology and Biological Transformations

Melatonin: A multifunctional molecule that triggers defence responses against high light and nitrogen starvation stress in Haematococcus pluvialis Wei Ding, Yongteng Zhao, Jun-Wei Xu, Peng Zhao, Tao Li, Huixian Ma, Russel J. Reiter, and Xuya Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02178 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Journal of Agricultural and Food Chemistry

Melatonin: A multifunctional molecule that triggers defence responses against high light and nitrogen starvation stress in Haematococcus pluvialis

Wei Ding1, a, Yongteng Zhao1, a, Jun-Wei Xu1, Peng Zhao1, Tao Li1, Huixian Ma2, Russel J. Reiter3, Xuya Yu 1,* 1

Faculty of Life Sciences and Technology, Kunming University of Science and Technology,

Kunming, Yunnan, China 2

School of Foreign Languages, Kunming University, Kunming 650200, China

3

Department of Cellular and Structural Biology, University of Texas Health Science Center, San

Antonio, TX, USA

Correspondence: Xuya Yu, Faculty of Life Sciences and Technology, Kunming University of Science and Technology, Kunming, China. Emails: [email protected] a These authors contributed equally to this work.

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Abstract: Melatonin (MLT), a ubiquitously-distributed small molecule, functions in plant responses to various biotic and abiotic stresses. However, the interactions between melatonin and other important molecules in Haematococcus pluvialis response stresses are largely unknown. In the present study, exogenous melatonin improved H. pluvialis resistance to nitrogen starvation and high light. We concluded that exogenous melatonin treatment prevented the reactive oxygen species (ROS) burst and limited cell damage induced by abiotic stress through activation of antioxidant enzymes and antioxidants. Astaxanthin, a major antioxidant in H. pluvialis cells, exhibited a 2.25-fold increase in content after treatment with melatonin. The maximal astaxanthin content was 32.4 mg g-1. The functional roles of the nitric oxide (NO)- mediated mitogen activated protein kinase (MAPK) signalling pathway and cyclic adenosine monophosphate (cAMP) signalling pathway induced by melatonin were also evaluated. The results clearly indicate that cAMP signalling pathways are positively associated with microalgal astaxanthin biosynthesis. Additionally, the NO-dependent MAPK signalling cascade is activated in response to astaxanthin accumulation induced by melatonin, confirming that MAPK is a target of NO action in physiological processes. This work is the first to use H. pluvialis as in vivo model and documents the influence of melatonin on the physiological response to abiotic stress in this microalgae. Keywords: Haematococcus pluvialis; astaxanthin; melatonin; antioxidant; abiotic stress; reactive oxygen species; signal transduction

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Introduction In recent years, microalgae have attracted considerable global attention as

alternative microbial cell factories based on the accumulation of desired cellular products.1 Astaxanthin is a major product in microalgae. Natural astaxanthin, a xanthophyll carotenoid with strong antioxidant properties, is widely used in nutraceutical and pharmaceutical industries.2,3 Thus, it is important to explore the production and mechanisms of action of natural astaxanthin. Haematococcus pluvialis, a unicellular green alga, accumulates large amounts of astaxanthin, surpassing that of other reported sources, such as Phaffa rhodozyma and Chlorella zofingiensis.4,5 Previous studies have shown that astaxanthin accumulation in H. pluvialis is triggered when cells are exposed to various stresses，such as nutrient stress, high salinity, and a high light intensity.6,7 However, cell growth is severely impeded under such environmental stressful conditions. Astaxanthin accumulation provides multilevel protective mechanisms against oxidative stress.8 In addition to its previously proposed physiological role as a ‘‘sunscreen’’ and the fact it provides antioxidant protection and/or carbon and energy storage, the synthesis and accumulation of astaxanthin have additional protective roles under abiotic stress.9 Thus, there is considerable potential for improving the extent of astaxanthin production by improving the resistance of H. pluvialis under abiotic stress. In H. pluvialis, astaxanthin molecules are esterified with fatty acids and stored in

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triacylglycerol (TAG)-rich cytosolic lipid bodies (LBs). Melatonin, a molecule also present in algae is efficient at increasing the activity of acetyl-CoA carboxylase (ACCase) and malic enzyme (ME), key steps in the lipid biosynthesis pathway, which further promote lipid accumulation in response to photooxidative stress in Monoraphidium.10 Melatonin, a polyphenic signalling molecule, also provides physiological protection against a variety of environmental stresses, including high salt stress, heat or cold temperatures,etc.11 Another attractive aspect of melatonin is its function as an antioxidant, which has clearly been decomented in plant cells, both in invitro and in vivo systems.12,13 Previous studies have revealed that melatonin and its metabolites scavenge multiple reactive oxygen/nitrogen species (ROS/RNS) as well as organic free radicals, either directly or by up-regulating the activity of antioxidant enzymes and content of other antioxidant compounds.14,15 In eukaryotic organisms, including microalgae, the activities of various signalling pathways are turned on or off by external environmental stimuli, with marked variations in the activities of these pathways. Nitric oxide (NO) is both a gaseous free radical, and multipurpose cell-signalling effector that plays multiple roles in varying metabolic processes, such as resistance to biotic and abiotic stresses, hormonal signalling, and development.16 However, two parallel signalling cascades, the MAPK and cAMP signalling pathways, have also been shown to play pivotal roles in various eukaryotic organisms.17,18 Although several studies have described the central roles of NO, MAPK and the cAMP signalling pathway in regulating a wide variety of cellular functions, there is no information regarding the functions of these 4


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signalling cascades in microalga species during stress responses. Chemical genetics is a new field that studies the functional role(s) of a gene(s) using small molecules. The use of small molecules of interest leads to alterations in the functions of particular signal transduction pathways; the results are analogous to classic gene deletion studies.19,20 Owing to the difficulties of generating loss-of function genetic mutations in microalgae or the inefficiency of single gene deletions stemming from the presence of redundant genes, chemical genetics can act as a powerful and straightforward tool to understand the cellular signalling function(s).21 In the present study, a stress model involving nitrogen starvation and high light exposure was combined with melatonin induction for astaxanthin accumulation in H. pluvialis. This well-established model was applied to elucidate the effect of melatonin on stress-specific responses. Herein, chemical genetics with small molecular inhibitors was used to examine the effect of melatonin on nitric oxide mediation of the MAPK and cAMP signalling pathways on the accumulation of astaxanthin in microalga. Briefly, both the cAMP and MAPK signalling pathways have been implicated in astaxanthin biosynthesis in microalgae. Here, we report novel discoveries of key melatonin signalling pathways that are operative in microalgae in response to abiotic stress.

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Materials and Methods Culture and induction of H. pluvialis: The H. pluvialis strain LUGU (18 s

GenBank: KM115647.1) was obtained by filtration from Lake Lugu, a plateau freshwater lake in Yunnan Province, China.22 The strain was maintained and cultivated in Bold’s basal medium (BBM). Green vegetative H. pluvialis cells were cultured in a bubbling column photobioreactor (0.2 m diameter, 0.3 m height, for 3 L) with 2 L of BBM mixed with filtered air at a rate of 0.4 vvm. The light intensity was 30 µmol m−2 s−1, and the growth temperature was 25 ± 1 °C. Algal cells in the late exponential growth phase (approximately 9.0 × 105 cells mL−1) were centrifuged at 3800 × g for 5 min and washed with aseptic water to remove residual nutrients. The pelleted cells were resuspended in nitrogen-deficient BBM and subjected to a high illumination intensity of 150 µmol m−2 s−1 by a white-light fluorescent lamp. The temperature of the cultures was maintained at 28 ± 1 °C, and the initial cell concentration was adjusted to 2.5 × 105 cells mL−1. For the treatment experiments, several induced substrates were added to nitrogen-deficient BBM (Table 1). Algal cells cultured in Basal medium (without treatment) were treated as a control group. Each treatment was managed in three replicates. Samples were harvested at 3-day intervals. Table 1. Concentration of different treatment agents used for the induction of astaxanthin biosynthesis Treatments a

MLT

SNP

IBMX

CPTIO

U0126

Concentration b

10 µM

200 µM

75 µM

0.15 µM

20 µM

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a

melatonin, MLT; Sodium nitroprusside, SNP (NO donor);23 Carboxy-PTIO, cPTIO

(NO scavenger);23 3-isobutyl-1-methylxanthine, IBMX (Chemical fortifier of cAMP signalling);24 U0126, MAPK inhibitor.25 b

The appropriate concentration for each was agent is as indicated in the table. Sampling analysis of the biomass concentration and astaxanthin and lipid

contents: To examine the defence response induced by melatonin in H. pluvialis under nitrogen starvation and photoinduction, we first measured the biomass and content of astaxanthin. 10 mL of a cell suspension was centrifuged at 3800 × g for 5 min; the pellets were washed twice with distilled water, collected in Eppendorf microtubes and dried in a vacuum freeze dryer at −80 °C for 24 h until a constant weight was obtained. The

astaxanthin

content

was measured

by

high

performance

liquid

chromatography (HPLC) with a photodiode array detector (Waters 996, USA) and a reverse-phase C18 column (Waters, 25 cm × 4.6 mm).26 Approximately 10 mg of dry biomass of each sample was thoroughly ground under lipid nitrogen. The powder was then extracted with an acetone–chloroform (1:1, v/v) solution and centrifuged at 12000 × g for 5 min at 4 °C to extract pigment. The process was replicated several times until the algal cells became white in colour. The extracted solution was combined and completely dried in an electric vacuum drying oven at 45 °C. Finally, the dried extraction was dissolved in 1 mL of a methanol–dichloromethane (3:1, v/v) solution, and 20 µl of each sample was injected into the HPLC. The elution gradient consisted of eluent A (dichloromethane: methanol: acetonitrile: water, 5.0:85.0:5.5:4.5, 7



v/v) and eluent B (dichloromethane: methanol: acetonitrile: water, 25.0:28.0:42.5:4.5, v/v) as follows: 0 % of B for 8 min; phase B from 0 % to 100 % for 6 min; phase B at 100 % for 40 min. The flow rate was 1.0 mL min−1. The detection wavelengths used for integration were 476 nm. Astaxanthin (Sigma; St. Louis, USA) were used as standards to calculate the proportion of each of these compounds in the samples. Weighed dried cell samples were used for total lipid extraction using a method described earlier.27 Quantification of the endogenous NO content and ROS in H. pluvialis: Nitric oxide was quantified using diaminofluorescein-FM diacetate (DAF-FM DA) purchased from Beyotime Institute of Biotechnology (Shanghai, China). DAF-FM DA has been extensively used to detect NO content in both plants and animals.28,29 Algal cells (1.0 × 106 cells mL−1) were vibrated with 5 µM DAF-FM DA in 1 ml of HEPES/PBS buffer (pH 7.4) for 20 min at 37 °C, washed three times in fresh buffer and measured with a spectrofluorophotometer (Shimadzu RF-540; Tokyo, Japan) using an excitation wavelength of 495 nm and emission band between 500 and 600 nm. The average fluorescence density of the intracellular areas was measured to identify the NO level. The ROS levels were monitored using 2′,7′-dichlorodihydrofluorescein diacetate (Beyotime; Shanghai, China) as a probe, as previously described.10 Protein extracts and Western blotting: Fresh microalga cells were harvested by centrifugation at 3,500 × g for 10 min. The pellets were suspended in a Tris-buffered saline solution (TBS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, pH 7.5). 8


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Then, algae were collected by centrifugation at 12000 × g for 5 min and the pellets were ground in extraction buffer containing 1 M Tris-HCl (pH 8.0), 0.2 % polyvinyl pyrrolidone, 2.0 % 2-Mercaptoethanol, 1 mM EDTA, 10 mM phosphatase inhibitor complex I and 1 mM PMSF. The cellular homogenate was centrifugation at 12000 × g for 30 min at 4 °C. The total protein content in the supernatant was determined according to Bradford.30 The supernatant was stored at -80 °C. Samples protein were separated by 12 % SDS-PAGE and subsequently transferred to nitrocellulose membranes (Biosharp; Guangzhou, China). The membranes were blocked for 1 h in a TBST solution with 5 % (w/v) fat-free milk powder and then incubated in a primary antibody solution containing the antiserum of a p38 MAPK antibody (rabbit polyclonal serum, Biorbyt; New York, USA) at a 1:1000 dilution. After an overnight incubation at 4 °C, the blots were washed with TBST and subsequently incubated for 2 h with a horseradish peroxidase-conjugated antibody against rabbit immunoglobulin G (Biorbyt; New York, USA, US) at a 1:1000 dilution. Antigen-antibody complexes were visualized by using an enhanced chemiluminescence substrate detection kit (Thermo Fisher Scientific, Waltham, MA). Enzyme and cAMP assay: Superoxide dismutase (SOD) activity was determined by the Total Superoxide Dismutase Assay Kit with WST-8 (Beyotime; Shanghai, China) according to the manufacturer’s recommendations. The principle of the method is based on WST-8. SOD activity was normalized to the protein level.31 Catalase (CAT) and peroxidase (POD) activities were determined by using the Catalase Assay Kit (Beyotime; Shanghai, China) and peroxidase assay kit (Beyotime; 9



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Shanghai, China), respectively.32 The contents of lipid peroxidation products (MDA) was determined according to Heath and Parker.33 cAMP was measured by using the plant cAMP Elisa Assay Kit (Biovision; New York, USA) according to the manufacturer’s instructions. Total

RNA isolation

and

real-time

quantitative

PCR

(qRT-PCR)

amplification: Total RNA was extracted using TriZol reagent (Invitrogen; Shanghai, China). After an RNase-free DNase I treatment, 1 µg of total RNA was reverse-transcribed with the RT-PCR kit (TaKaRa; Shanghai, China) according to the manufacturer’s instructions. The transcription levels of dxs, lcy, bkt, chy, acp, fad, sad and 18s were analysed by qRT-PCR using an ABI 7500 Real-Time PCR System with SYBR Green Master Mix (TaKaRa; Shanghai, China) according to the manufacturer’s instructions. The primers for amplification of the 18S rRNA gene, dxs, lcy, bkt, chy, acp, fad and sad are shown in Table S1. The transcription levels were normalized against the H. pluvialis 18S rRNA gene (an internal control), which was constantly expressed under all experimental conditions. PCR efficiency for each sample was determined by the LinRegPCR program, and primer efficiency (PE) obtained from the individual samples was maintained at 100 ± 5 %. For each gene, the reference sample was defined as an expression level of 1.0, and the results are expressed as a fold-increase of the mRNA level over the reference sample. Post-qRT-PCR calculations were used to analyse the relative gene expression according to the 2−∆∆CT method.34 Statistical analyses: Each experiment was performed in triplicate. All data were 10


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derived and statistically analysed using Student’s t test. The results were considered significant at P < 0.05 in two-tailed analysis.

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Results Effect of different treatment agents on the biomass and accumulation of

astaxanthin in H. pluvialis under nitrogen starvation and photoinduction conditions: Sodium nitroprusside (SNP) was used as NO donor to increase the production of NO.23 3-Isobutyl-1-methylxanthine (IBMX) is an chemical fortifier of cAMP signaling, which is responsible for inhibiting cAMP phosphodiesterase, thereby causing an accumulation of cAMP in cells.24 Carboxy-PTIO (cPTIO), which is a very popular NO scavenger, has been increasingly used in NO-related studies.23 U0126 was used as a MAPK inhibitor to decrease the level of MAPK.25 As shown in Fig. 1A, after 13 days of culture, the biomass concentration of the control, melatonin, IBMX and SNP groups generally reached 0.61, 0.59, 0.70 and 0.62 g L−1, respectively, although not differing statistically from each other. However, after adding cPTIO and U0126, the growth of algal cells was inhibited by 22.95 % and 26.23 %, respectively, compared to the control. With increasing biomass, the content of astaxanthin showed varying degrees of enhancement in all experimental groups. The astaxanthin content in H. pluvialis was 14.36 mg g-1 in the control group over a period of 0-13 d (Fig. 1B). After induction with melatonin, the astaxanthin content in the cells rapidly increased (32.37 mg g-1) by 2.25-fold compared to that in the control within 13 d of cultivation. In the IBMX and SNP treated samples, both pretreatments resulted in a significantly improved accumulation of astaxanthin in H. pluvialis compared to that in cells that did not receive a pretreatment, peaking after 13 d and reaching 23.99 and 19.69 mg g-1, 12


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respectively. By contrast, the cPTIO and U0126 pretreatments resulted in less accumulation of astaxanthin, with maximal astaxanthin contents of only 11.04 and 9.03 mg g-1, respectively. Thus, despite not exerting a positive effect on biomass, melatonin treatment combined with nitrogen starvation and photoinduction resulted in the highest astaxanthin accumulation. Changes in cell morphology during induction: Melatonin induction led to a change in the morphology of algal cells (Fig. 2). As shown in Fig. 2 A, the flagellum of a cell generally receded, leading to a change from a vegetative state (zoospore) to reddish cysts on day 5. However, after applying melatonin, the cells changed to reddish, thick-walled immotile cysts (Fig. 2 B). On day 13, the cell colour in the melatonin treatment group became more pronounced, while a red colour was observed in the control. Furthermore, the proportion of red cells was nearly 100 % in the melatonin treatment group. In addition, the diameter of the cells after melatonin treatment group was larger than that of most cells in the control group. Moreover, all of the cultures began to die and disintegrate. These results indicate that treatment of the microalgae with melatonin promotes the conversion of zoospores to cysts. Changes of the ROS level and antioxidant enzyme activities in melatonin-treated cells: Fig. 3 A shows the intracellular ROS level during nitrogen starvation and photoinduction with melatonin treatment. The ROS content was enhanced in cells exposed to nitrogen starvation and photoinduction in both the control and melatonin groups. At 5 d, the highest level of ROS in melatonin-treated cells was decreased by 9.31 % compared to the control. Subsequently, the ROS level 13



significantly decreased with the activation of antioxidant enzymes and accumulation of astananthin compared with the control. After inoculation for 1 day, the activities of POD, CAT and SOD were analysed (Fig. 3 B, C and D). After inoculation for 1 day, the POD and SOD activities were significantly higher than those in the melatonin treatment group while CAT showed no significant activity differences. The POD, CAT and SOD activities increased with melatonin treatment after 5 days, increasing by 3.19-, 1.45-, and 1.35-fold, respectively. Moreover, in both the melatonin and control groups, the POD, CAT and SOD activities showed a downward trend, except for a slight increase in POD activity at 13 d. As shown in Fig. 3 E, when microalgae were exposed to an adverse environment, the MDA content was significantly enhanced. Particularly, the control group showed a more rapid increase compared with the melatonin-treated group. After 5 d, a gradual rise in the MDA content was observed in both groups. However, after melatonin treatment, the MDA content in H. pluvialis was significantly lower than that in groups without treatment. Here, we found that melatonin treatment activated the enzymatic antioxidant and alleviated ROS accumulation. Effect of melatonin on the lipid content of H. pluvialis: The lipid content increased sharply after treated with melatonin during induction, and the highest lipid content reached 42.84 %, which was 1.22-fold greater than that of the control samples (35.19 %) (Fig. 4). This result indicates that supplementation of melatonin under nitrogen deficiency and photoinduction promotes lipid accumulation in algae. Quantification of the NO level in H. pluvialis: The results showed that 14


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melatonin and SNP pretreatment significantly increased the NO level, while the NO level in the control group showed no significant changes, while the combination of melatonin and cPTIO pretreatment dramatically reduced the intracellular NO content (Fig. 5) and also significantly reduced astaxanthin accumulation in the whole culture (Fig. 1B). These results show that melatonin mediate the nitric oxide dependency against stress conditions. The observed changes show that exogenous melatonin and SNP treatment increase NO levels. Effect of different treatments on the cAMP and MAPK content of H. pluvialis: As illustrated in Fig. 6 A, at 1- 9 days, the concentration of cAMP was not significantly increased in groups treated with melatonin, IBMX or control. After cultivation for 13 days, the intracellular cAMP levels after the addition of melatonin and IBMX resulted in a 1.24- and 1.19- fold rise over that of the control, respectively. The content of MAPK was detected by western blotting. As show in Fig. 6 B, when exposed to different treatments, MAPK content showed a significant difference at 5 d. The MAPK level was significantly enhanced in the melatonin and SNP treatment groups relative to control. By contrast, cPTIO and U0126 exposure significantly inhibited the content of MAPK. At 13 d, the MAPK content showed a significant increase in all groups compared with the levels at 5 d. However, in the melatonin and U0126 combination group, the MAPK level was lower than that of the other groups. The results suggest the possible involvement of these agents in resisting abiotic stresses in H. pluvialis. Effect of melatonin on the relative gene transcription levels in H. pluvialis 15



astaxanthin and the lipid biosynthesis pathway under stress: To determine the effects of melatonin on the relative gene transcription levels in H. pluvialis, astaxanthin and lipid biosynthesis pathway activation under nitrogen starvation and photoinduction condition, the relative expression of astaxanthin and the lipid biosynthesis pathway were quantified. The gene expression levels were normalized against the 18S rRNA gene expression level in H. pluvialis. As shown in Fig. 7, dxs, lcy, bkt and chy are the key genes in the astaxanthin biosynthesis pathway showed no significant differences in gene expression among the melatonin, SNP and melatonin plus cPTIO groups at 1-5 d, despite low expression levels compared with the cPTIO group. Fig. 7 shows that melatonin and SNP treatment elevated the transcription levels of dxs, lcy, bkt and chy at 9-13 d. The maximal transcription levels of dxs, lcy, bkt and chy after melatonin treatment were increased by 3.13-, 2.67-, 2.43- and 1.74-fold, respectively, compared to the control. The transcription levels of dxs, lcy, bkt and chy on day 13 were 2.35-, 2.09-, 2.20- and 1.98-fold higher in the SNP group compared to the control. The transcription level of the melatonin plus cPTIO group was decreased. Moreover, acp, fad and sad, the key genes in the lipid biosynthesis pathway were induced by melatonin after 5 d, as the expression level was up-regulated 1.22-, 2.44and 2.54-fold, respectively, compared to the control. The transcription levels of acp, fad and sad on day 13 were increased by 1.68-, 1.52- and 2.17-fold, respectively, in the melatonin group compared to the control. These trends were consistent with those observed for the astaxanthin and lipid accumulation levels. These results indicate that NO is involved in the melatonin regulation of astaxanthin biosynthesis-related genes 16


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and that melatonin is involved in the common regulation of lipid biosynthesis-related genes in H. pluvialis.

Discussion H. pluvialis accumulates astaxanthin through comprehensive physiological

process. In particular, H. pluvialis are described as carotenogenic microalgae because of their large accumulation of astaxanthin under stressful conditions.35 However, astaxanthin accumulation is often accompanied by a reduction in the cell number.36 Melatonin, as a signalling molecule and antioxidant, regulates the tolerance of both plants and algae.37,10 In the present study, no significant difference was observed between cells grown with melatonin compared to a control cells under adverse conditions (Fig. 1 A). The results are similar to those of a previous study. The addition of melatonin did not promote cell growth of C. reinhardtii under long day conditions.38 Notably, we observed that exogenous melatonin significantly enhanced the content of astaxanthin under nitrogen starvation and photoinduction by 2.25-fold compared to the control (Fig. 1 B). This indicates that melatonin enhanced the accumulation of astaxanthin in H. pluvialis in to stressful conditions. Additionally, compared with previously reported induction strategies, astaxanthin production with melatonin treatment combined with nitrogen starvation and photoinduction was much greater than that with H2O2, butylated hydroxyanisole (BHA), or various light-emitting diode (LED) treatments.39,

40, 41

However, due to culture media

differences, the dependence of regulators on the algal strain and species and the culture conditions, this observation requires further experimental validation. In H. 17



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pluvialis, cyst formation is the primary response to stress. On day 5, melatonin-induced green algae were rapidly converted to cysts (Fig. 2). This result may be due to the effects of melatonin on a variety of regulatory functions. ROS are ubiquitous signalling molecules that are involved in many cellular metabolic processes and are believed to play crucial roles in regulating carotenoid biosynthesis and astaxanthin accumulation in

both H.

pluvialis and C.

zofingiensis.42,43 However, ROS can potentially react with major macromolecules, such as DNA, lipids, and protein, leading to cellular damage.44 Consequently, microalgae have evolved miscellaneous systematic enzymatic and nonenzymatic detoxification mechanisms to cope with the production of these potentially toxic compounds under adverse environments.9 It is clear that a wide variety of signal transduction activities in microalgae cells are involved in processing environmental cues and directing appropriate reactions to their environment for their own survival. Thus, after exposure of algae cells to high light and nitrogen starvation, we observed that the intracellular ROS level rapidly increased in both the control and melatonin groups at 5 d (Fig. 3 A). Simultaneously, astaxanthin was synthesized by H. pluvialis at large amounts when the ROS level peaked, indicating that a rise in the ROS level triggered astaxanthin accumulation.45 Activated H. pluvialis also accumulate active antioxidative enzymes and show rapid accumulation of astaxanthin. Treatment with melatonin resulted in significantly elevated activities of POD, CAT and SOD compared with those of the control (Fig. 3 B, C and D) and led to a dramatic drop in ROS levels. However, the activities of POD, 18


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CAT and SOD decreased at 9 and 13 d. These results document that melatonin had significant effects on both antioxidant enzymes and the non-enzymatic astaxanthin content, consistent with the alleviation of abiotic stress-induced ROS accumulation in H. pluvialis. This phenomenon is similar to that observed in many green algae, which quickly respond to ROS, but with cell damage being extensive. Over time, proteins are broken and degraded, resulting in reduced enzyme activity. Elevated cellular ROS levels also often trigger autophagy, which enables the orderly degradation and recycling of cellular components.46 MDA is an indicator of membrane lipid peroxidation of cells. MDA levels in melatonin-treated cells (Fig. 3 E) confirmed the antioxidative actions in response to direct oxidative stress. These data are consistent with stress increasing cellular ROS levels in microalgae. However, there is a delicate balance between ROS generation and their scavenging. Thus, similar to plants, different cellular compartments respond to stress and have distinct profiles of ROS formation. The enzymatic detoxification mechanisms as initial stress responses maintain the stability of the intracellular environment; this includes rises in POD, CAT and SOD activities. However, a ROS burst always results in mangled and degraded proteins. As a result, cellular autophagy is often triggered. These effects lead to a reduction in the efficiency of the enzymatic defence mechanisms. Concurrently, a second set of defences, which mainly include antioxidants, is initiated. Astaxanthin acts as an antioxidant and ‘‘sunscreen’’ in this detoxification mechanism. Thus, this shift-away process not only saves the cell energy but also enables the cell to survive in an adverse environment. These data showed that 19



exogenous melatonin was an efficient antioxidant and modifier against toxic ROS and that the intracellular ROS level coordinated with the astaxanthin content. Melatonin, as an antioxidant and signalling molecule, prevents overproduction of ROS, which is a consequence of two antioxidative prosesses, to protect the cell from the oxidative damage and autophagy caused by stress. A previous study demonstrated that a single melatonin application to seeds during pre-sowing priming improved the oxidative stress tolerance of growing seedlings exposed to paraquat.47 Shi et al. also demonstrated that melatonin reduced oxidative stress in Bermuda grass by regulating antioxidative enzyme activity.48 However, the specific protective actions of melatonin which functions as a signal for signal transduction pathways and up-regulates astaxanthin accumulation in H. pluvialis under stress requires further research. Multiple signal transduction pathways in which NO functions as a key signalling molecule regulates defence responses in eukaryotic organisms. The primary targets of NO in cells might include MAPK.49 A previous study showed that melatonin treatment enhances disease resistance in Arabidopsis and H. pluvialis by up-regulating a set of defence genes that are triggered by NO.50,51 Similar results were also obtained in the present study. Melatonin treatment enhanced the NO level of H. pluvialis, in which a NO-dependent MAPK signalling cascade was activated during the defence response process induced by melatonin. The MAPK content was elevated in the melatonin group (Fig. 6 B). However, MAPK and cAMP signalling cascades were equally activated. We observed that treatment with melatonin up-regulated cAMP (Fig. 6 A). Melatonin stimulated the accumulation of cAMP in cells that co-express type II 20


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adenylyl cyclase.52 To confirm that MAPK is a target of NO action in physiological processes, chemical genetics with SNP (NO donor), cPTIO (NO scavenger) and U0126 (MAPK inhibitor) were used to alter the functions of particular signal transduction pathways. Administration of SNP significantly increased the level of NO and MAPK as well as the content of astaxanthin. By contrast, cPTIO significantly inhibited NO and MAPK in H. pluvialis, but up-regulation of the MAPK level at 13 d may have resulted from the consumption of cPTIO. Astaxanthin was at a low level in this group. After addition of a MAPK inhibitor, the accumulation of astaxanthin decreased compared to that in the cPTIO group. These observations suggest that MAPKs are targets of NO action in physiological processes and that these processes can be induced by melatonin. To confirm the function of melatonin on the cAMP signalling pathway, IBMX (3-isobutyl-1-methylxanthine), a well-known chemical modulator that increases cellular cAMP levels was used.24 The results showed than IBMX promoted the accumulation of cAMP and asatananthin in H. pluvialis. These observations in contrast to those of Choi et al.20 Furthermore, stress and chemicals or phytohormones up-regulate genes related to astaxanthin and lipid biosynthesis in microalgae.40,53 Both melatonin and NO positively modulated the expression levels of astaxanthin synthesis genes (dxs, lcy, bkt and chy). The transcriptional activation of astaxanthin synthesis genes by melatonin was also suppressed by pretreatment with cPTIO. This result suggests that the melatonin-induced NO production was essential for the activation of 21



MAPK in H. pluvialis. Lamotte et al. also confirmed that many of the identified defence-related responses in the MAPK signalling pathway, such as several different transcription factors, are up-regulated by melatonin treatment.54 Although MAPK and cAMP signaling pathways mediate an increase in environmental resistance in H. pluvialis, the signal transduction systems are complex networks. Our previous study reported another signaling pathway, i.e., salicylic acid (SA)-dependent pathway, during melatonin-regulated stress response.51 Similarly, Lee et al. reported that the application of melatonin on Arabidopsis and tobacco leaves induces not only various pathogenesis-related (PR) genes but also defense genes activated by SA and ethylene (ET), which are two key factors involved in plant defense response.55 In short, melatonin treatment enhances the signaling mechanisms that trigger a wide range of responses. Nonetheless, further studies are needed to fully understand these signaling systems and their interconnections. A recent study revealed that astaxanthin is stockpiled in triacylglycerol (TAG)-rich lipid bodies (LBs).56 Previous studies have demonstrated that melatonin is capable of increasing the lipid content in Monoraphidium.57 These results suggest that melatonin exhibited accelerated induction of lipid biosynthesis. This conclusion was drawn because melatonin promotes the synthesis of astaxanthin while inducing lipid biosynthesis. The same result was also shown in the present study. This change may be explained as follows. First, as an inducer, melatonin promotes the production of lipids to relieve the feedback inhibition of carotenogenesis by free astaxanthin. Second, as an antioxidant, melatonin prevents lipid peroxidation and maintains the 22


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normal function of biofilm in H. pluvialis. Consistently, genes related to lipid biosynthesis were up-regulated by melatonin. Based on the evidence provided herein, a novel model for melatonin and the NO-mediated rise in environmental resistance in H. pluvialis is proposed (Fig. 8). As mentioned above, environmental stress induces a burst of ROS. Melatonin is an amphipathic molecule that can easily diffuse through cell membranes into the cytoplasm and enters subcellular compartments as a broad-spectrum direct antioxidant to scavenge ROS. In addition, melatonin treatment modulates antioxidant enzymes by increasing their activity levels to protect cells from oxidative damage. Melatonin also has the ability to enhance cellular antioxidant defence mechanisms by regenerating endogenous antioxidants, such as astaxanthin and lipids, which inﬂuence cellular signalling and trigger redox-sensitive regulatory pathways. Exogenous application of melatonin significantly increased the production of NO, suggesting that melatonin first increases signalling molecule production under stress, followed by downstream signal transduction, resulting in activation of transcription factors. Finally, most of the identified genes were related to the up-regulation of biotic and/or abiotic stress responses by melatonin treatment in H. pluvialis. In summary, with these studies we unravel in part the complex roles of the melatonin regulatory mechanisms by assessing the defence response. First, melatonin treatment enhanced miscellaneous systems regarding enzymatic and non-enzymatic detoxification mechanisms, including POD, CAT, SOD and astaxanthin. Chemical genetics aided in expanding the recognition of important microalgal signalling 23



pathways in astaxanthin biosynthesis. The identified signal transduction pathways were MAPK and cAMP. However, the majority of the genes in these pathways are stress-related transcription factors that require further study.

24


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ACKNOWLEDGEMENTS

This work was funded by the National Natural Science Foundation of China (21766012), Key Science and Technology Project of Yunan Province (2018ZG003), the National Natural Science Foundation of China (21666012)

AUTHOR CONTRIBUTIONS

Yu XY conceived and directed this study, designed the experiments, analyzed the data and revised the manuscript. Ding W and Zhao YT completed experiments and statistical analysis, wrote and revised the manuscript. Zhao P, Russel J. Reiter and Xu JW statistical analysis, provided suggestions and revised the manuscript. Li T and Ma HX managed the literature edition. All authors read and approved the final manuscript.

CONFLICT OF INTEREST

The authors declared that they have no competing financial interests. Supporting Information. Culture of H. pluvialis, gene-specific primers, astaxanthin content, ROS levels and activities of POD, CAT and SOD, lipid content. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure legends Fig. 1. (A) The effect of different treatments on the biomass of H. pluvialis during induction. (B) The effect of different treatments on the astaxanthin content of H. pluvialis during induction. Vertical bars represent the means ± SD (n = 3). *indicates statistical significance at p

Melatonin: A multifunctional molecule that triggers defence responses

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