Planta DOI 10.1007/s00425-016-2528-0

ORIGINAL ARTICLE

The role of nitric oxide signalling in response to salt stress in Chlamydomonas reinhardtii Xiaodong Chen1 • Dagang Tian2 • Xiangxiang Kong3 • Qian Chen3 Abd_Allah E.F.4 • Xiangyang Hu5 • Aiqun Jia1

•

Received: 20 October 2015 / Accepted: 11 April 2016 Ó Springer-Verlag Berlin Heidelberg 2016

Abstract Main conclusion Nitric oxide signal and GSNOR activity play an essential role for Chlamydomonas reinhardtii response to salt stress. The unicellular alga Chlamydomonas reinhardtii is one of the most important model organisms phylogenetically situated between higher plants and animals. In the present study, we used comparative proteomics and physiological approaches to study the mechanisms underlying the response to salt stress in C. reinhardtii. We identified 74 proteins that accumulated differentially after salt stress, including oxidative enzymes and enzymes associated with

Electronic supplementary material The online version of this article (doi:10.1007/s00425-016-2528-0) contains supplementary material, which is available to authorized users. & Xiangyang Hu [email protected]; [email protected] & Aiqun Jia [email protected] 1

School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China

2

Biotechnology Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, Fujian, China

3

The Germplasm Bank of Wild Species, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, China

4

Plant Production Department, College of Food and Agricultural Sciences, King Saud University, P.O. Box. 2460, Riyadh 11451, Saudi Arabia

5

Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai, China

nitric oxide (NO) metabolism, cell damage, and cell autophagy processes. A set of antioxidant enzymes, as well as S-nitrosoglutathione reductase (GSNOR) activity, were induced to balance the cellular redox status during shortterm salt stress. Enzymes involved in DNA repair and cell autophagy also contribute to adaptation to short-term salt stress. However, under long-term salt stress, antioxidant enzymes and GSNOR were gradually inactivated through protein S-nitrosylation, leading to oxidative damage and a reduction in cell viability. Modulating the protein S-nitrosylation levels by suppressing GSNOR activity or adding thioredoxin affected the plant’s adaptation to salt stress, through altering the redox status and DNA damage and autophagy levels. Based on these data, we propose that unicellular algae use multiple strategies to adapt to salt stress, and that, during this process, GSNOR activity and protein S-nitrosylation levels play important roles. Keywords Autophagy  Cellular redox status  Chlamydomonas  Proteomics  Salt stress  S-nitrosoglutathione reductase Abbreviations APX Ascorbate peroxidase ATG Autophagy related protein DHAR Dehydroascorbate reductase GSNOR S-Nitrosoglutathione reductase GST Glutathione S-transferase MDHAR Monodehydroascorbate reductase NO Nitric oxide NR Nitrate reductase cPTIO 2-(4-Carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide RNS Reactive nitrogen species ROS Reactive oxygen species

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SNO TRXh5

S-Nitrosothiol Thioredoxin-h5

Introduction Plants have evolved complex mechanisms to respond to environmental stress. During this response, a series of small molecules act as essential messengers to mediate the plant defense response (Xiong and Zhu 2001; Lu and Huang 2008; Akpinar et al. 2012). Nitric oxide (NO) is a hydrophobic diffusible gaseous molecule that participates in various aspects of plant growth and development, including germination, flowering, root initiation, stomatal closure, and programmed cell death (He et al. 2004; Zemojtel et al. 2006; Sirova et al. 2011; Yu et al. 2014). Recently, NO has emerged as an essential signal that modulates the plant’s response to environmental stress, including salinity, drought, mechanical injury, heavy metals, and pathogen attack (Hancock et al. 2001). NO synthase (NOS) and nitrate reductase (NR) are two potential enzymatic sources of NO in plants (Desikan et al. 2002; He et al. 2004). Although NOS has not yet been identified in plants, studies using specific inhibitors of the animal NOS suggest that the L-arginine pathway functions in NO production (He et al. 2004). NR generates NO from nitrite in an NAD(P)H-dependent manner. In Arabidopsis, NR is encoded by two genes, NIA1 (NITRATE REDUCTASE 1) and NIA2. NIA2 is responsible for 90 % of the total NR activity, while NIA1 is responsible for the remaining NR activity (Desikan et al. 2002; Kolbert and Erdei 2008; Hao et al. 2010; Vitor et al. 2013). Furthermore, NO can be generated in the apoplast of plant cells via enzyme-dependent or non-enzymatic pathways (Wilson et al. 2008; Baudouin and Hancock 2013; Domingos et al. 2015). NO also plays essential roles in unicellular algae, such as Chlamydomonas reinhardtii. For example, treatment with mastoparan, a peptide toxin that activates GTP-binding regulator protein, induces cell death in C. reinhardtii, accompanied by ethylene and NO generation (Yordanova et al. 2010). NO acts as a regulator of chloroplast biogenetics and thylakoid protein stability upon nitrogen starvation (Wei et al. 2014), and controls nitrate and ammonium assimilation in C. reinhardtii (Sanz-Luque et al. 2013). NR is reported to be involved in NO production in green algae (Sakihama et al. 2002). However, the physiological and biochemical mechanisms underlying NO production in algae remain largely unknown.

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Autophagy is activated in response to multiple abiotic stresses, pathogen infection, and senescence in plants (Hayward et al. 2009; Han et al. 2011; Liu and Bassham 2012). The basic autophagy process is conserved in eukaryotes ranging from yeast to animals and plants (Kim et al. 2012). Several types of autophagy have been described in many species, including microautophagy, macroautophagy and chaperone-mediated autophagy, and organelle-specific autophagy. Autophagy is mediated by a set of proteins encoded by ATG (autophagy-related) genes. In yeast, the autophagy pathway includes two ubiquitinlike conjugation systems that involve ubiquitin-like proteins Atg8 and Atg12. Whereas yeast has single ATG8, ATG4, and ATG12 genes, Arabidopsis contains nine members of the AtATG8 family (AtATG8a-AtATG8i), two members of the AtATG4 family (AtATG4a and AtATG4b), and two members of the AtATG12 family (AtATG12a and AtATG12b). Homologs of ATG genes have been reported in plant and algal genomes, indicating that autophagy is also conserved in photosynthetic organisms, including C. reinhardtii (Perez–Perez and Crespo 2010). Nitrogen and carbon depletion trigger autophagy in C. reinhardtii (DiazTroya et al. 2008; Perez–Perez et al. 2010), as do environmental stresses, such as H2O2 and methyl viologen (MV), which cause reactive oxygen species (ROS) levels to increase. These findings indicate that ROS may trigger autophagy in Chlamydomonas (Perez-Martin et al. 2014). Exogenous environmental agents, such as ultraviolet (UV) light, ionizing radiation, genotoxic chemicals, and endogenous byproducts of metabolism, including ROS, alter or damage the DNA structure (Mannuss et al. 2012). DNA damage, which induces specific cell responses such as cell death and senescence, is also an adaptation strategy for plants in response to environmental stress (Hemnani and Parihar 1998). Recent evidence demonstrates that DNA damage is associated with cell autophagy (Huang et al. 2013; Filippi-Chiela et al. 2015); however, the mechanism by which environmental stress triggers DNA damage and autophagy in plants and in unicellular algae, remains unclear. In the present study, we conducted comparative proteomic and physiological analyses to investigate the response of unicellular C. reinhardtii to salt stress. We found that 74 proteins were differentially expressed in C. reinhardtii after salt stress. Among these, proteins associated with the NO metabolism pathway, including NR and GSNOR, and with cell autophagy and DNA damage were differentially regulated after salt stress. Further physiological and biochemistry analysis reveal that NO signal act as the important regulator to adjust C. reinhardtii tolerance to salt stress. This finding provides a basis for engineering strategies that aim to improve the tolerance of unicellular algae to environmental stress.

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Materials and methods Materials and salt treatment The Chlamydomonas reinhardtii wild-type strain was provided by Dr. Junchao Huang (Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming). Cells were cultured under continuous light at 23 °C in Trisacetate-phosphate (TAP) medium as described (Ramundo et al. 2014). For the salt treatment, cells in the stationary phase (about 106 cells per milliliter) were treated with 100 mM sodium chloride. At different time points, cells were removed for further analysis. Viability assay Evans blue dye E2129 (Sigma, St. Louis, MO, USA) was used as a marker of cell death, as reported previously (Baker and Mock 1994). Briefly, 450 ml of C. reinhardtii cells (about 106 cells per milliliter) was incubated with 0.1 % (w/v) Evans Blue for 5 min, washed once with 500 ml of TAP (Tris-acetate-phosphate) medium, and resuspended in an equal volume of TAP medium. Viability was determined by counting the colored cells in a hemacytometer, and ten random counts were performed. Control tests of viability were performed with suspension cultures fixed in 70 % ethanol or in FAA (5 % formaldehyde, 5 % glacial acetic acid, 70 % ethanol, by vol.). Cell viability was also evaluated using fluorescein diacetate (FDA) staining (Jones and Senft 1985). Briefly, the algae were incubated in 0.01 % (w/v) FDA solution for 30 min, and then viewed under a confocal laser scanning microscope with a 109 objective. Using an argon ion laser, samples were excited at a wavelength of 488 nm, and emission were detected between 505–530 nm and 650–730 nm. Viable algae exhibited strong green fluorescence. Average viability was determined for 10 different fields of view. Comet assay Nuclei were isolated from C. reinhardtii cells after various treatments, and alkaline/neutral assays were performed using the CometAssay Kit (Trevigen, Gaithersburg, MD, USA). A total of 50 or 100 comets per experimental point were analyzed using CometScore (Trevigen, Gaithersburg, MD, USA). Analysis of chlorophyll fluorescence intensity and malondialdehyde (MDA) content Chlorophyll fluorescence was determined using a dual PAM 100 (Walz, Effeltrich, Germany) with the emitter and detector units attached to the cuvette holder (Yang et al.

2013). Cell samples (3 ml) were stirred with a magnetic stirring bar at the bottom of a standard cuvette with 1 cm2 square and 6 cm height. Each sample was transferred directly from the turbidostat to the cuvette and dark adapted for 15 min, whereupon Fv/Fm was determined with a saturating pulse of 10,000 mmol quanta m-2 s-2. Malondialdehyde (MDA) were measured as previous method (Yang et al. 2013). NO and H2O2 content detection NO content was measured using a Nitric Oxide Fluorometric Assay Kit (Biovision Inc.). Briefly, cells (about 106 cells per milliliter) were suspended in TAP medium containing the NO indicator 4,5-diaminofluorescein diacetate (DAF-2DA, 10 lM), incubated at 25 °C for 1 h in dim light (40 lmol m-2 s-1), and then washed twice (1000g for 5 min) with growth medium. The fluorometric detection of NO was carried out using a fluorescence spectrophotometer (BioRad, Hercules, CA, USA). The excitation and emission wavelengths for the NO indicator were 495 and 515 nm, respectively. NO measurements were carried out in darkness at 25 °C. The reaction mixture contained 20 mM potassium phosphate (pH 7.0), 5 mM NaHCO3, and C. reinhardtii cells. The reaction was initiated by adding 10 mM NaNO2. The H2O2 content was measured using an AmplexÒ Red Hydrogen Peroxide/ Peroxidase Assay Kit (Molecular Probes, Invitrogen). Briefly, 50 ll of cells (about 106 cells per milliliter) was incubated with an equal volume of AmplexÒ Red reagent/ HRP working solution for 30 min in darkness. The H2O2 levels were determined using a Synergy2 Multi-mode Reader (Biotek, Winooski, VT, USA) equipped for excitation in the range of 530–560 nm and fluorescence emission detection at *590 nm. The H2O2 concentration was calculated using a H2O2 standard curve. S-Nitrosothiol (SNO) content and GSNOR activity measurements Total SNO content was determined using a previously reported method with minor modifications (Bai et al. 2011). Briefly, SNO measurement was based on the reductive decomposition of nitroso species by an iodine/ triiodide mixture, and the released NO was measured using a gas-phase chemiluminescence method and a NO analyzer (Model 410, 2B Technologies, Boulder, CO, USA). SNOs are sensitive to mercury-induced decomposition, in contrast to nitroso species such as nitrosamine (RNNOs) and nitrosyl hemes. Samples (10 ml) after exposure to different treatments were centrifuged at 30009g for 10 min at 25 °C, and the pellet was homogenized in 1 ml of extraction buffer (50 mM Hepes–KOH, pH 7.5, 1 mM DTT,

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1 mM EDTA, 7 mM cysteine, 100 lM diethylenetriaminepentaacetic acid, DTPA) (1:5; w/v), and centrifuged at 3000g for 10 min at 25 °C. The supernatants were then incubated with an equal volume of 10 mM N-ethylmaleimide (NEM) for 15 min at 4 °C. For each sample, two aliquots were prepared; aliquot (1) was treated with 10 mM sulphanilamide for 15 min at 4 °C to eliminate nitrite and aliquot (2) was treated with 10 mM sulphanilamide and 7.3 mM HgCl2 for 15 min at 4 °C to eliminate nitrite and SNOs, respectively. These samples were analyzed using the NO analyzer. The data from aliquots (1) and (2) represent the total SNO concentration. The entire procedure was performed under a red safety light to protect the SNOs from light-dependent decomposition. GSNOR activity was determined spectrophotometrically at 25 °C by monitoring the oxidation of NADH at 340 nm (Bai et al. 2011). The treated samples (5 ml) were centrifuged at 3000g for 15 min at 4 °C, the pellet was then quickly homogenized in liquid nitrogen and extracted with assay mixture (20 mM Tris–HCl (pH 8.0), 0.2 mM NADH, and 0.5 mM EDTA) at 4 °C and then centrifuged at 3000g for 10 min at 25 °C. The supernatants were subjected to further assays, and the reaction was initiated by adding GSNO (Calbiochem, San Diego, CA, USA) to the supernatants at a final concentration of 400 mM. The activity was expressed as nanomoles NADH consumed per minute per milligram of protein (e340 6.22 mM-1 cm-1). Antioxidant enzymes activities assay To determine the antioxidant enzyme activities, 10 ml of cultured algae was centrifuged at 3000g for 15 min at 4 °C. The pellet was then quickly homogenized in liquid nitrogen and extracted with 5 ml of extraction buffer [50 mM sodium phosphate buffer (pH 7.0), 0.2 mM EDTA, and 2 % polyvinylpolypyrrolidone (PVPP)] for 10 min. The homogenates were filtered through two layers of cheesecloth and centrifuged at 4 °C at 15,0009g for 15 min. Supernatants were desalted on a Sephadex G-50 column and used to measure the enzymatic antioxidant activities. The total soluble protein concentration of supernatants was determined by the Bradford method (Bradford 1976). The activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), ascorbate peroxidase (APX), and glutathione reductase (GR) were determined as described (Yang et al. 2013). Nitrate reductase measurements Nitrate reductase activity was measured following an established method (Zhao et al. 2009). The cultured algae (10 ml, about 106 cells per milliliter) were centrifuged at

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3000g for 15 min at 4 °C and the pellet was then quickly homogenized in liquid nitrogen and extracted with 5 ml of cold extraction buffer [250 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 lM Na2MoO4, 3 mM dithiothreitol (DTT), 1 % BSA, 12 mM b-mercaptoethanol, and 250 lM phenylmethylsulfonyl fluoride]. One volume of pre-warmed (25 °C) assay buffer [40 mM KNO3, 8 mM Na2HPO4, 20 mM NaH2PO4 (pH 7.5), and 0.2 mM NADH] was added and the mixture was incubated at 25 °C. After 0, 5, 10, and 15 min, 100 ll aliquots were removed from the assay mixture and the reaction was stopped by adding 25 ll of 0.6 M zinc acetate. The samples were maintained at room temperature for 20 min, and then a solution (100 ll) of 1 % sulphanilamide dissolved in 3 N HCl supplemented with 100 ll 0.02 % N-(1-naphthyl)ethylenediamine was added. The mixture was incubated for 20 min at room temperature, centrifuged at 18,000g, and the nitrite content in the supernatant was measured by spectroscopy at 540 nm. Protein extraction and 2D electrophoresis Protein extraction and 2D separation were performed according to a reported method with minor modifications (Yang et al. 2013). Cultured algae (50 ml) subjected to various periods of 100 mM salt treatment were centrifuged at 3000g for 15 min at 4 °C. The algae without salt treatment were used as the control. The pellet was ground in liquid nitrogen and the total soluble protein was extracted at 4 °C in 5 ml of 50 mM Tris–HCl buffer (pH 7.5) containing 20 mM KCl, 13 mM DTT, 2 % (v/v) NP40, 150 mM PMSF, and 1 % (w/v) PVP. The homogenates were centrifuged (12,000g, 15 min, 4 °C) and the supernatants were added to five volumes of acetone containing 10 % (w/v) TCA and 1 % (w/v) DTT. The samples were maintained at -20 °C for 4 h and then centrifuged (25,000g, 30 min, 4 °C). The resulting pellets were washed with acetone containing 1 % (w/v) DTT at -20 °C for 1 h and then centrifuged at 25,000g for 30 min at 4 °C, and the wash step was repeated. The final pellets were vacuum-dried and then dissolved in 8 M urea, 20 mM DTT, 4 % (w/v) CHAPS, and 2 % (w/v) ampholyte (pH 3–10). The samples were vortexed thoroughly for 1 h at room temperature and then centrifuged (25,000g, 20 min, 20 °C). The supernatants were collected for 2D electrophoresis (2DE). Each experiment was repeated three times. Extracted proteins were first separated by isoelectric focusing (IEF) using gel strips to form an immobilized nonlinear pH gradient from 3 to 10 (Immobiline DryStrip, pH 3–10, 17 cm; Bio-Rad) and then by SDS-PAGE using 12.5 % polyacrylamide gels. The strips were rehydrated for

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16 h in 450 ll of dehydration buffer containing 800 lg of total protein. The strips were focused at 20 °C for a total of 64 kV/h using the PROTEAN IEF system (Bio-Rad). After IEF, the strips were equilibrated for 15 min in equilibration buffer [6 M urea, 0.375 M Tris, pH 8.8, 2 % (w/v) SDS, 20 % (v/v) glycerol, and 2 % (w/v) DTT]. For 2D SDSPAGE, the strips were placed on top of 12.5 % (w/v) SDSPAGE gels. Gel electrophoresis was performed at 25 mA for 5 h. The gels were stained using the colloidal CBB staining method. After staining, the gels were scanned using a GS-800 Calibrated Densitometer (Bio-Rad) and PDQUEST software (Bio-Rad), on the basis of their relative volume. Parameters were optimized as follows: saliency, 2.0; partial threshold, 4; and minimum area, 50. Spots were quantified by determining the ratio of the volume of a single spot to the entire set of spots on the gels. The relative volume of each spot was assumed to represent its expression level. Cut-offs of 1.5- and 0.6-fold were set to indicate upregulation and downregulation of proteins, respectively, and P \ 0.05 (t test) was used to indicate significance. To compensate for subtle differences in sample loading or gel staining/destaining during individual experiments, the volume of each spot was normalized. Experiments were performed in biological triplicate. In-gel digestion Protein spots showing significant changes in abundance during the treatments were excised manually from colloidal CBB-stained 2DE gels. Protein digestion with trypsin was performed as follows. Individual spots of interest were excised from the 2DE gels using sterile tips and placed in 1.5 ml sterile tubes. Each polyacrylamide spot was destained with 50 mM NH4HCO3 for 1 h at 40 °C, reduced with 10 mM DTT in 100 mM NH4HCO3 for 1 h at 60 °C, and then incubated with 40 mM iodoacetamide in 100 mM NH4HCO3 for 30 min. The gel pieces were minced and allowed to dry, and then rehydrated in 12.5 ng/ll trypsin (sequencing grade; Sigma) in 25 mM NH4HCO3 at 37 °C overnight. The trypsin peptides were extracted from the gel grains with 0.1 % trifluoroacetic acid in 50 % acetonitrile three times. Supernatants were concentrated in a SpeedVac (Savant Instruments Inc., Farmingdale, NY, USA) concentrator to approximately 10 ll and desalted using ZipTips (C18 resin, P10, Millipore Corporation, Bedford, MA, USA). Peptides were eluted from the column with 50 % acetonitrile/0.1 % trifluoroacetic acid. The protein spots that changed more than 1.5-fold or below 0.6, and passed Student’s t test (P \ 0.05) were selected and identified by mass spectrometry (MS) analysis.

MALDI-TOF/TOF analysis and database searching The lyophilized peptide samples were dissolved in 0.1 % trifluoroacetic acid (TFA). Mass spectrometry (MS) analysis was conducted using a MALDI-TOF/TOF Mass Spectrometer 4800-plus Proteomics Analyzer (Applied Biosystems, Framingham, MA, US). MS acquisition and processing parameters were reflector positive mode and 800–3500 Da acquisition mass range. The laser frequency was 50 Hz, and 700 laser points were collected for each sample signal. For each sample, 4–6 ion peaks with signalto-noise ratios of greater than 100 were selected as precursors for secondary MS analysis. The TOF/TOF signal for each precursor was accumulated with 2000 laser points. The primary and secondary MS data were transferred into Excel files as inputs to search against an NCBI non-redundant database (NCBInr 20141005), and the search was restricted to Chlamydomonas reinhardtii using the MASCOT search engine (http://www.matrixscience.com). The search parameters were set as follows: no restriction of protein molecular weight; one missed trypsin cleavage allowed; cysteine treated by iodoacetamide; and oxidation of methionine. The peptide tolerance was 100 ppm and the MS/MS tolerance was 0.75 kD. Protein identifications were validated manually, with at least two peptides matching. Keratin contamination was removed and the MOWSE score threshold was greater than 60 (P \ 0.05). According to the MASCOT probability analysis, only significant hits were accepted for the identification of the protein sample. Immunoblot analysis Immunoblotting was performed as previously described (Yang et al. 2013). Total protein extracts were separated on a 15 % SDS-PAGE gel. Equal amounts of sample were loaded into the wells. For immunoblot analysis, the protein samples were electroblotted onto polyvinylidene difluoride (PVDF) membranes using a Trans-Blot well (Bio-Rad). After transfer, the membranes were probed with the appropriate primary antibodies and HRP-conjugated goat secondary antibody (Promega, Madison, WI, USA), and the signals were detected using an ECL Kit (GE Company, Evansville, IN, USA). The primary antibodies against C. reinhardtii Actin, ATG4, ATG8, and NR were obtained from AgriSera (Va¨nna¨s, Sweden), and diluted as following: 1:3000 for anti-Actin, 1:1000 for ATG4 and ATG8, and 1:2000 for NR. The antibodies against APX and GSNOR were prepared by immunizing a rabbit with synthesized peptides from the N-terminus of C. reinhardtii APX pro-

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tein (MQSARVSRTARHT) and (MSTIGKPIECKAA), respectively.

GSNOR

protein

Results Salt treatment suppressed the cell viability of C. reinhardtii To characterize the effect of salt (NaCl) on C. reinhardtii, we first examined how different salt concentrations influenced C. reinhardtii growth. A 3-day exposure to a salt concentration of below 100 mM slightly reduced the biomass of the algal culture, while a salt concentration of above 200 mM strongly reduced the biomass (Suppl. Fig. S1), resulting in a degree of cell death of above 50 %, as determined by Evans blue staining (Suppl. Fig. S2). Thus, we selected a salt concentration of 100 mM for further experiments. A 100 mM salt treatment gradually suppressed cell growth. Specifically, cells not subjected to salt treatment were dark green, whereas those subjected to 5-day salt treatment remained pale green (Fig. 1a). Fluorescein diacetate (FDA) staining is a marker of plant cell viability, with strong fluorescence indicating live cells (Jones and Senft 1985). We found that salt treatment markedly reduced the FDA fluorescence after 3 or 5 days of salt treatment, indicating that salt stress reduces cell

viability (Fig. 1b). Environmental stress, such as strong light, suppresses the photosynthetic ability of C. reinhardtii (Erickson et al. 2015). Salt treatment markedly reduced the ratio of Fv/Fm, reflecting a reduction in photosynthetic capability (Fig. 2a). Electrolyte leakage and malondialdehyde (MDA) are markers of oxidative damage to cell membrane lipids. Salt treatment also increased the degree of electrolyte leakage and MDA (Fig. 2b), suggesting that salt stress compromised cell membrane stability. Thus, based on these physiological indices, we conclude that salt stress (100 mM) suppresses cell viability and growth of C. reinhardtii. Protein profile changes after exposure to salt stress To investigate the mechanism underlying the response to salt stress in C. reinhardtii, we used a comparative proteomics approach to monitor changes in protein dynamics. A total of 143 proteins spots were reproducibly detected that showed significant changes in response to salt stress (P \ 0.05) compared to the control (Fig. 3a, b). Overall, 74 out of 143 differential protein spots were successfully identified using MALDI-TOF/TOF analysis. The identified proteins were divided into seven groups based on their putative biological functions (Table 1, Suppl. Table S1). Most groups of proteins were associated with material and energy metabolism or involved in antioxidant enzyme

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Fig. 1 The effects of salt on C. reinhardtii cell growth. a Time course analysis of the effect of 100 mM salt (NaCl) on C. reinhardtii cell growth. Cells were inoculated into TAP medium at a ratio of 1:20 (v/v), resulting in a cell density of about 3–5 9 105 cells ml-1. After the indicated periods of salt treatment, photographs were taken to assess the phenotype. Left (-) cells not subjected to salt treatment

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(control); right (?) cells subjected to salt treatment (100 mM). b Cell viability as determined by fluorescein diacetate (FDA) staining. After the indicated periods of salt treatment, the viability of cells stained with FDA was assessed based on FDA fluorescence. WL white light. Bar 100 lm

Planta Fig. 2 The effect of salt treatment on cell viability and biomass of C. reinhardtii. Data represent the means of five replicate experiments (±SD). a Time course analysis of the effect of salt treatment on photosynthetic ability (Fv/Fm) and biomass in C. reinhardtii. b Time course analysis of the effects of salt treatment on electrolyte ion leakage and malondialdehyde (MDA) in C. reinhardtii

b -1 MDA content ( mol ml ) Electrolyte leakgage ( s g FW)

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Fig. 3 Dynamic protein changes in C. reinhardtii upon exposure to salt stress. Salt stress treatment was performed as described in the legend to Fig. 1. After 1, 3, and 5 days of treatment, 1 mg of total protein was extracted from the treated samples and loaded in a gel. The experiment was repeated three times with similar results.

a Representative BR-20-stained 2D gels of total protein from the control sample. b Enlarged windows (a–e) of spot changes (arrows) marked in panel a in representative gels loaded with total proteins from the sample after different periods of salt treatment

systems, cellular structure, or the defense response, or were transcription factors, chloroplast-related proteins, or unknown proteins (Fig. 4a; Table 1 and Suppl. Table S1). The differential up-regulation or down-regulation of the 74

identified proteins after various periods of salt tress is presented in Fig. 4b. Proteins with similar expression profiles during salt stress treatment were grouped together based on hierarchical cluster analysis (Fig. 4c).

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NCBI accession no.a

Material and energy related 10 gi|159487741 17 gi|159488061 19 gi|159462978 21 gi|158275026 38 gi|159477849 42 gi|159473683 46 gi|158282886 48 gi|158277735 49 gi|159489926 53 gi|158279204 60 gi|159466052 75 gi|159474436 76 gi|159471580 80 gi|158272091 81 gi|159487437 98 gi|735959 104 gi|294846036 Defense response 7 gi|159486599 10 gi|159472671 29 gi|159478256 30 gi|159468684 31 gi|159487489 36 gi|386363671 108 gi|159482406 110 gi|159481010 112 gi|159485966 121 gi|159464168 124 gi|159472883 Photosynthesis related 1 gi|53794017 9 gi|159487669 50 gi|159472705 85 gi|159475641 86 gi|159491492

Spot no.

72.7/7.46 72.1/8.00 80.3/6.98 85.2/5.74 50.4/6.00 59.8/7.25 47.1/6.88 46.1/7.66 51.9/8.23 38.2/4.73 46.6/5.99 43.8/7.06 34.3/6.84 33.3/8.33 19.7/3.53 27.4/7.81 15.4/4.57 72.7/5.03 69.8/5.34 60.1/4.63 61.0/4.81 51.6/4.50 61.7/5.40 16.2/5.31 16.3/5.83 15.4/5.72 12.6/5.13 11.7/5.87 72.1/3.36 84.0/5.23 41.8/3.68 26.8/5.15 27.1/5.29

Heat shock protein 70A Heat shock protein 70C Endoplasmic reticulum oxidoreductin Chaperonin 60B2 Glutathione peroxidase a APX Glutathione-S-transferase Glutathione peroxidase b Heat shock 22 kDa protein, chloroplastic Thioredoxin M-type Thioredoxin h1

Plastid-specific ribosomal protein-7 precursor Chloroplast elongation factor G Ribosomal protein S1 homologue Minor chlorophyll a–b binding protein of photosystem II Light-harvesting complex II chlorophyll a–b binding protein M3

Exp. Mw/pIb

Transketolase Acetyl CoA synthetase Pyruvate-formate lyase Nitrate reductase Ubiquinol: cytochrome c oxidoreductase 5 Phosphoenolpyruvate carboxykinase S-Adenosylmethionine synthetase Adenylosuccinate synthase 3-Phosphoshikimate-1-carboxyvinyltransferase Magnesium chelatase subunit I Low-CO2 inducible protein Isocitrate lyase GSNOR enzyme Fructose-1,6-bisphosphate aldolase Coproporphyrinogen III oxidase Aconitate hydratase LEU1 Sm

Protein name

60.0/4.12 79.9/5.23 43.9/4.18 30.7/5.38 27.4/5.68

71.5/5.25 65.4/5.6 60.4/4.97 61.9/5.46 58.23/4.80 60.8/5.9 23.95/5.34 18.01/6.32 16.7/6.08 11.5/5.09 11.8/5.83

78.4/6.92 74.1/7.02 91.4/6.49 99.7/5.9 55.2/5.89 62.3/6.23 43.1/6.03 50.2/7.72 53.8/8.02 45.6/6.01 48.0/6.57 45.9/5.9 40.26/6.76 41.3/8.92 41.7/8.12 28.9/5.90 23.8/6.18

Theo. Mw/pIc

245/13.93 283/15.83 200/11.7 374/20.42 248/24.12

186/13.52 263/14.83 231/13.24 312/22.7 286/15.98 248/19.09 216/27.52 341/48.77 320/42.68 352/39.29 278/41.59

183/10.86 209/12.14 185/10.95 178/5.60 225/12.93 239/13.64 195/17.94 290/16.2 220/12.7 235/18.47 182/20.78 254/14.39 290/16.18 263/14.85 371/20.27 305/21.96 252/24.3

Scored/ coverage (%)e

0.26 0.99 1.96 1.22 0.99

1.67 1.56 1.88 1.45 1.53 1.88 1.48 1.56 1.84 1.64 2.11

0.74 2.36 0.34 1.63 0.31 0.67 0.44 0.30 2.25 0.73 1.58 1.23 2.17 0.75 0.36 1.20 0.43

1 day/ CK

Ratiof

0.032 0.001 0.033 0.024 0.039

0.043 0.046 0.018 0.037 0.018 0.015 0.009 0.044 0.002 0.034 0.023

0.006 0.019 0.015 0.030 0.032 0.010 0.011 0.032 0.044 0.033 0.046 0.044 0.047 0.027 0.021 0.007 0.039

P valueg

0.37 1.45 1.85 1.55 1.25

2.230 2.340 1.670 2.340 1.870 1.980 2.120 1.980 1.540 1.310 2.350

0.96 1.58 0.27 1.86 0.43 0.43 0.34 0.45 1.45 0.63 1.99 2.35 1.78 0.67 0.47 1.53 0.32

3 days/ CK

0.018 0.035 0.038 0.034 0.050

0.044 0.033 0.043 0.011 0.022 0.050 0.014 0.027 0.042 0.003 0.029

0.036 0.014 0.005 0.023 0.005 0.024 0.009 0.040 0.044 0.002 0.037 0.020 0.046 0.041 0.008 0.034 0.044

P valueg

0.65 1.56 1.39 1.47 1.46

1.890 1.780 1.130 2.100 1.260 1.17 1.150 1.350 1.270 1.130 1.580

0.40 0.46 0.19 1.08 0.65 0.27 0.21 0.67 0.99 0.45 2.01 0.74 1.19 0.34 0.75 1.61 0.22

5 days/ CK

Table 1 Identification of C. reinhardtii proteins that are differentially expressed by more than 1.5-fold or less than 0.6-fold after salt treatment using MALDI-MS/MS analysis

0.039 0.043 0.042 0.017 0.027

0.040 0.037 0.016 0.009 0.021 0.035 0.039 0.007 0.012 0.040 0.032

0.039 0.015 0.033 0.030 0.021 0.014 0.026 0.049 0.019 0.017 0.039 0.014 0.003 0.001 0.044 0.036 0.024

P valueg

Planta

NCBI accession no.a

23

gi|158277507

Cell structure related 32 gi|158270806 44 gi|157836652 52 gi|158273131 111 gi|159486191 117 gi|520519 Protein synthesis and refolding 28 gi|4104541 52 gi|159467709 55 gi|159466510 Protein kinase 72 gi|158277205 77 gi|159482940 107 gi|159487006 Signal transduction related 27 gi|158283649 88 gi|158271029 102 gi|159482892 101 gi|159467397 DNA damage and repair 18 gi|158277392

87 gi|159475641 89 gi|159473144 100 gi|159477687 115 gi|167428 123 gi|159468772 Autophagy 34 gi|159462550 39 gi|41179009 57 gi|159483493 105 gi|159465465 113 gi|159482262

Spot no.

Table 1 continued

35.7/6.21 39.0/7.26 15.0/4.88 51.8/3.86 23.9/4.67 17.8/4.72 19.8/6.95 80.3/8.17

Phosphoribulokinase Phosphoglycerate kinase Galactose kinase

Calreticulin 2, calcium-binding protein 14-3-3 Protein Caltractin Ran-like small GTPase

DNA damage checkpoint protein

91.9/5.38

63.6/4.44 35.5/4.49 43.8/5.18

Protein disulfide isomerase Peptidyl-prolyl cis–trans isomerase Eukaryotic initiation factor 4A-like protein

DNA repair protein

51.0/4.68 53.2/6.74 40.6/4.96 20.1/6.60 18.1/7.62

57.1/5.63 58.1/6.55 33.5/5.17 14.2/4.48 15.5/6.91

Autophagy protein VPS30 ATP-dependent Clp protease proteolytic subunit Autophagy protein 3 Vacuolar trafficking protein Autophagy protein 8

Alpha tubulin 2 Chain F Actin Flagellar associated protein Gbp1p

24.6/5.06 19.4/4.56 22.5/6.95 15.6/6.91 11.1/5.89

Exp. Mw/pIb

Minor chlorophyll a–b binding protein of photosystem II Oxygen-evolving enhancer protein 1 of photosystem II Prohibitin Oxygen-evolving enhancer protein 2 Light-harvesting protein of photosystem I

Protein name

102.98/ 9.81 152.38/ 5.44

47.4/4.54 29.7/4.9 19.4/4.7 25.7/6.24

42.2/8.11 49.2/8.92 55.9/6.17

58.4/4.8 44.8/5.37 47.3/5.50

50.2/5.01 53.1/6.04 42.1/5.3 24.9/6.32 24.2/6.78

54.30/5.37 59.5/6.27 33.66/5.04 12.9/4.46 215.26/ 6.75

30.7/5.38 30.7/8.26 31.2/6.37 25.9/9.14 22.9/9.04

Theo. Mw/pIc

188/6.11

153/9.351

276/25.48 230/28.19 252/34.32 373/40.36

231/24.27 334/23.38 320/17.69

286/15.98 302/21.79 304/26.88

380/25.72 261/14.74 321/17.77 322/17.81 265/14.93

218/17.58 218/12.6 273/15.36 395/41.46 344/23.88

284/15.92 365/19.93 219/27.66 254/29.39 245/38.97

Scored/ coverage (%)e

1.39

1.37

0.14 0.96 1.24 0.67

0.29 0.38 1.24

1.07 1.15 0.24

0.34 0.95 1.20 1.11 0.28

1.27 1.30 1.16 0.93 1.28

0.64 0.31 0.42 1.39 1.66

1 day/ CK

Ratiof

0.002

0.018

0.028 0.035 0.007 0.027

0.019 0.027 0.013

0.005 0.014 0.034

0.021 0.001 0.028 0.046 0.037

0.01 0.033 0.045 0.038 0.027

0.037 0.027 0.049 0.012 0.025

P valueg

1.62

1.74

0.61 1.34 1.78 0.45

0.47 0.56 1.36

1.68 1.56 0.44

0.32 1.41 1.47 1.79 0.32

1.89 1.75 1.59 0.61 1.98

0.53 0.34 0.56 2.2 1.58

3 days/ CK

0.02

0.039

0.031 0.04 0.047 0.018

0.04 0.042 0.001

0.027 0.05 0.007

0.008 0.027 0.022 0.046 0.008

0.007 0.036 0.02 0.046 0.015

0.002 0.029 0.028 0.017 0.036

P valueg

1.55

1.52

0.87 1.64 2.54 0.31

0.4 0.36 1.53

1.37 1.75 0.73

0.37 1.65 1.61 1.39 0.41

0.86 0.75 0.89 1.49 0.84

0.28 0.35 0.89 2.4 1.23

5 days/ CK

0.008

0.048

0.012 0.004 0.036 0.023

0.022 0.025 0.024

0.044 0.045 0.044

0.032 0.04 0.034 0.028 0.011

0.005 0.042 0.039 0.026 0.022

0.026 0.045 0.037 0.034 0.010

P valueg

Planta

123

123

gi|56199778

NCBI accession no.a

Set3p

Protein name

Sequences coverage

Mascot search score against the database of NCBInr

Theoretical Mw/pI

Experimental Mw/pI

Database accession numbers according to NCBInr

398.34/ 5.66 49.96/4.88 15.4/4.90 36.90/5.52 31.8/5.26 44.1/7.82 42.10/6.70 12.00/6.53 72.1/5.31 26.3/6.26 42.7/6.28 35.5/6.26 21.6/4.85 30.4/7.56

73.5/4.57 99.9/6.72 46.9/7.26 34.1/5.72 25.4/4.26 17.5/5.98

Theo. Mw/pIc

100.3/ 6.98 51.0/4.68 33.0/3.86 42.9/5.36 35.5/5.26 3.1/5.49 46.8/6.55 13.1/6.09

Exp. Mw/pIb

243/13.84 201/31.73 231/23.27 228/28.08 288/31.08 240/28.72

374/25.42 332/47.36 337/28.57 384/25.9 332/23.29 330/23.21 240/28.7

192/11.28

Scored/ coverage (%)e

0.67 0.27 0.4 0.26 0.27 0.62

0.92 0.65 0.93 1.55 1.58 1.22 1.52

1.18

1 day/ CK

Ratiof

0.042 0.005 0.050 0.013 0.050 0.021

0.026 0.009 0.014 0.012 0.027 0.021 0.042

0.002

P valueg

0.35 0.48 0.79 0.35 0.33 0.48

1.12 0.44 1.38 1.72 1.97 1.55 1.87

1.61

3 days/ CK

0.005 0.011 0.044 0.029 0.046 0.032

0.024 0.039 0.001 0.012 0.024 0.027 0.041

0.048

P valueg

0.31 0.35 0.49 0.65 0.53 0.32

1.63 0.38 1.63 1.96 2.01 1.42 1.42

1.78

5 days/ CK

0.049 0.007 0.045 0.040 0.005 0.010

0.007 0.013 0.006 0.018 0.016 0.049 0.013

0.002

P valueg

Protein spots showed a significant change in abundance(fold change) by a factor [1.5-fold or \0.6-fold compared to the control analyzed by LSD test. A P value of \0.05 was considered statistically significant

g

Different protein spots intensity ratios of 100 mM salt treatment at different time point; 1 day/CK: the different protein spot intensity ratio of salt treatment for 1 day to the control; Al-3 days/ Control: the different protein spot intensity ratio of Al treatment for 3 days to the control; Al-1 day ? SNAP/Control: the different protein spot intensity ratio of Al treatment plus 30 lM SNAP for 1 day to the control; Al-3 days ? SNAP/Control: the different protein spot intensity ratio of Al treatment plus 30 lM SNAP for 3 days to the control

f

e

d

c

b

a

32 gi|158282216 DNA damage inducible protein 51 gi|159472699 RAN binding protein, RANBP1, partial 54 gi|45685351 Putative DNA repair protein RAD51 56 gi|30038276 REX1-B 66 gi|37545634 RNA-binding protein RB38 68 gi|20750301 Serine acetyl transferase 120 gi|159483809 Putative defender against death (DAD) protein Hypothetical or unknown protein 4 gi|257307175 Unknown protein 20 gi|159478443 Hypothetical protein CHLREDRAFT_95661 47 gi|159468534 Predicted protein 65 gi|159475703 Hypothetical protein CHLREDRAFT_80327 82 gi|159466034 Hypothetical translation initiation factor 111 gi|300659064 Unknown protein

24

Spot no.

Table 1 continued

Planta

Planta

a

c

Material and energy related

1 d/CK 3 d/CK 5 d/CK 1 d/CK 3 d/CK 5 d/CK

Defense response 6%

4%

Photosynthesis related

24%

7%

NO signal related

7%

DNA damage and repair

9%

Hypothetical or unknown protein 16% 13% 14%

1 d/CK 3 d/CK 5 d/CK

Autophagy Cell structure related Signal transduction related

Antioxidant enzymes

Protein kinase 1 d/CK 3 d/CK 5 d/CK

b 1 d/CK

2

3 d/CK

9

11

1 d/CK

4

6

7 0

3 d/CK

Cell autophagy

0

1 d/CK 3 d/CK 5 d/CK

10 9

6

1

4 5

1d/CK 3d/CK

Cell death related

5d/CK

5 d/CK

5 d/CK

Up-regulation

Down-regulation

Log2(Ratio)

Fig. 4 Expression profile of C. reinhardtii protein species that are affected by exposure to salt stress for various periods of time. Functional classification (a), Venn diagram analysis (b), and hierarchical clustering (c) of protein species expression profiles based on samples obtained after the indicated periods of salt stress. The hierarchical clustering analysis of proteins involved in the NO signal,

antioxidant enzymes activity, cell autophagy, and cell death are also presented. Hierarchical cluster analysis was conducted using Cluster 3.0 and Treeview software. The different colors correspond to the logtransformed values of the protein species change-fold ratio shown in the bar at the bottom

Interestingly, proteins involved in NO metabolism and the antioxidant system, such as NR, GSNOR, and glutathione S-transferases (GST) were clustered together and exhibited similar accumulation profiles under salt stress (Fig. 4c), suggesting that NO signaling is involved in the salt stress response in C. reinhardtii. The proteins associated with cell autophagy were also clustered together and showed similar pattern changes following exposure to salt stress, suggesting their potential role and possible cross talk between autophagy and the NO signal during salt stress. To avoid the complexity of sample analysis, we only used the sample at 0 day as control, and did not use the sample of 1, 3, or 5 days, without salt treatment, as controls against the samples of salt stress at corresponding days. Thus, we could not exclude the possibility that some different proteins in the samples of 1, 3 or 5 days of treatment result from the cell growth status but are not directly associated with salt stress. Yet we further used the samples of 1, 3 or 5 days without salt stress as the control in the next

physiological and biochemical experiments, to confirm those protein that were really associated with salt stress. Salt stress-induced ROS accumulation and antioxidant enzyme activity Because our proteomics results showed that salt stress induced the accumulation of a set of antioxidant enzymes and because ROS, including H2O2, are important signals that mediate the plant’s response to abiotic stress (Desikan et al. 2001; Hancock et al. 2002), we investigated whether salt stress-induced ROS accumulation and whether the induced antioxidant enzymes are involved in ROS scavenging. We first quantified H2O2 levels in C. reinhardtii exposed to salt stress. In the absence of salt stress, C. reinhardtii cells accumulated low levels of H2O2, while salt treatment strongly induced H2O2 production (Fig. 5a). After 5 days of salt treatment, cells stained in situ with 3,30 -diaminobenzidine (DAB) were dark, indicating high

123

2.5

Control Salt

c

2.0 1.5 1.0 .5 0.0 0

.5

1

2

3

5

Treatment time (days)

b

CK

1d

APX activity MDHAR activity -1 -1 (nmol min mg protein) (nmol min-1mg-1 protein)

3.0

400

d c

300

160

d

140

e b

b 200

c b

100 80

a

a

120

60 40

100

20 0

c

600

c

b 400

0

d

250

d

b

b

200

a

a a

150 100

200

50 0

0 0

1

2

3

5

0

1

2

3

DHAR activity GR activity -1 -1 -1 -1 (nmol min mg protein) (nmol min mg protein)

a H2O2 content ( M)

Planta

5

Treatment time (days)

3d

5d

Fig. 5 Changes in H2O2 content and antioxidant enzyme activity in C. reinhardtii in response to various periods of salt stress. a Changes in H2O2 content in C. reinhardtii in response to the indicated periods of salt stress treatment. b In situ detection of H2O2 changes in C. reinhardtii in response to the indicated periods of salt stress treatment. Bar 10 lm. c The antioxidant activities of MDHAR, DHAR, APX,

and GR in C. reinhardtii in response to the indicated periods of salt stress treatment. The salt stress treatments were performed as described in the legend to Fig. 1. Data represent the means of five replicate experiments (±SD). The means denoted by different letters indicate significant differences according to Tukey’s test (P \ 0.05)

levels of H2O2 accumulation (Fig. 5b). The cells also clustered together after salt stress, indicating that stress affected cell growth (Fig. 5b). The antioxidant enzyme system maintains ROS at acceptable levels during environmental stress (Foyer and Noctor 2011). Our proteomics results showed that salt stress increased the accumulation of a series of antioxidant-related proteins, including glutathione peroxidase (spot 31), glutathione-S-transferase (spot 108), thioredoxin M-type (spot 121), and thioredoxin h1 (spot 124), suggesting that these proteins are involved in the plant’s response to salt stress. We thus monitored the antioxidant enzyme activities of MDHAR, DHAR, APX, and glutathione reductase (GR) (Fig. 5c), and found that salt stress gradually induced the activity of these enzymes after 1 or 3 days of salt treatment, and that activity declined after 5 days of salt treatment, in a pattern similar to the expression profiles of these proteins. These data indicate a possible role for these antioxidant enzymes in suppressing the over-accumulation of ROS during salt stress.

Salt stress induced the NO signal and GSNOR activity

123

NO is an important regulator of multiple physiological processes in plants, including plant growth and development and the plant’s response to environmental stress (Hancock et al. 2001; He et al. 2004; Wilson et al. 2008; Baudouin and Hancock 2013; Domingos et al. 2015). Because salt treatment induced the accumulation of NR (spot 21, gi|158275026), which is the main enzyme catalyzing NO generation in C. reinhardtii (Sakihama et al. 2002), we sought to establish the function of the NO signal during salt stress. We firstly measured NO production in C. reinhardtii cells exposed to salt stress. As show in Fig. 6a, salt treatment markedly induced NO production in C. reinhardtii following salt treatment. Salt-induced NO production could completely be abolished by additional NO scavenger cPTIO treatment (Fig. 6a). These data are in agreement with previous studies showing that the heavy

Planta

a

1.8 Control Salt Salt+cPTIO

1.4 1.2 1.0 .8

d

.6

.2 0

1

3

4

5

Control Salt

10

6 4 2

9

0 0

8

-1

5

GSNOR activity (nmol g FW)

6

4 3 0

1

2

3

5

Treatment time (days) 12

b

b

10

1

2

3

5

8 Control Salt

7 6 5 4 3

8

0 6

.5

Treatment time (days)

e

7

c -1 -1

8

11

-

-1 -1

2

Treatment time (days)

b

-

Control Salt

10

.4

0.0

NR activity (mmol NO2 min g pr)

12

SNOs (µM)

NO content (µM)

1.6

NR activity (mmol NO2 min g pr)

Fig. 6 Salt stress induced the accumulation of NO and SNOs, and the activities of NR and GSNOR in C. reinhardtii. Salt stress induced the accumulation of NO (a) and SNOs (d) in C. reinhardtii. For the NO scavenger cPTIO treatment, cPTIO at 30 lM was added with salt together. Salt stress activated NR activity (b) and GSNOR activity (e) in C. reinhardtii. c Effect of different inhibitors on the activity of NO in C. reinhardtii after exposure to salt stress. Salt stress treatment was performed as described in the legend to Fig. 1. For the inhibitor treatments, the NR inhibitors KCN (1 mM), tungstate (30 mM), and L-NAME (10 mM) were added to the TAP culture medium along with the salt, and NR activity was analyzed after 3 days of treatment. Data represent the means of five replicate experiments (±SD). The means denoted by different letters indicate significant differences according to Tukey’s test (P \ 0.05)

.5

1

2

3

5

Treatment time (days)

a

4

d c

2 0 CK

Salt

KCN

metal copper induces NO production in C. reinhardtii (Zhang et al. 2008). Because NO has essential functions in modulating many physiological responses in higher plants, it has been proposed that the unicellular alga C. reinhardtii uses the same NO signal to adapt to environmental stress (Yordanova et al. 2010). It was previously reported that NR, but not NOS-like enzyme, is the main enzyme responsible for NO accumulation in C. reinhardtii, and that the C. reinhardtii mutant cc-2929, which lacks NR activity, is unable to generate NO (Sakihama et al. 2002). Our proteomics data show that NR protein content increased after 1 or 3 days of salt stress (Fig. 4c; Table 1), hinting at a possible function for NR during salt stress. We then monitored NR activity in C. reinhardtii, and found that salt treatment induced NR activity. NR activity increased after 1 or 3 days of salt treatment, and then gradually declined after 5 days of salt treatment (Fig. 6b). KCN and tungstate are widely used as the NR inhibitor. Suppressing NR

Trungstate L-NAME

enzyme activity using KCN or tungstate also abolished NO generation in cells exposed to salt stress (Fig. 6c). However, the NOS-like enzyme inhibitor, L-NAME, did not affect NO generation in C. reinhardtii cells after salt stress. These data support a role for the NR-dependent NO signal in the C. reinhardtii response to salt stress. The ROS accumulated in cells exposed to salt stress interact with NO to form RNS, which react with the thiol group in the tyrosine residues to form S-nitrosothiols (SNOs), in a process called S-nitrosylation modification (Morisse et al. 2014). Protein S-nitrosylation alters the activity, localization, or conformation of target proteins (Spadaro et al. 2010; Spoel and Loake 2011). Cellular SNO levels are controlled by the conserved cytosolic enzyme GSNO reductase (GSNOR) (Frungillo et al. 2014). Our proteomics data demonstrated that salt stress induces the accumulation of GSNOR (spot 76, gi|566159968), suggesting a possible role for GSNOR in regulating the

123

Planta

3

b 5

days anti-NR

anti-GSNOR

anti-ATG8

anti-ATG3

SNO-GSNOR1 anti-GSNOR

SNO-NR

anti-NR

anti-APX

SNO-APX

anti-actin

anti-APX

Fig. 7 Effect of different inhibitors on the accumulation of saltresponsive proteins in C. reinhardtii. a Effects of salt stress on the expression of salt-responsive proteins as determined by immunoblot analysis. Total proteins were extracted from C. reinhardtii subjected to salt stress for the indicated periods, and the level of protein expression was analyzed using the corresponding antibody. Anti-actin is included as the loading control. b Modification of protein S-nitrosylation status by salt stress. The C. reinhardtii samples were

123

Salt+DA

1

CK

0

Salt+TXR

Treatment time

a

CK

Autophagy is the sophisticated strategy activated in plants in response to multiple abiotic stresses, pathogen infection,

Salt

Salt treatment-induced cell autophagy and DNA damage

and senescence (Hayward et al. 2009; Han et al. 2011; Liu and Bassham 2012). Upon induction of autophagy, portions of the cytoplasm are engulfed by double membrane structures termed autophagosomes and are delivered to the vacuole for degradation (Han et al. 2011). In C. reinhardtii, cell autophagy is also induced by nutrient limitation, oxidative stress, or the accumulation of misfolded protein in the endoplasmic reticulum (Perez–Perez and Crespo 2010). Homologs of yeast and plant ATG8 genes have been identified as molecular markers of autophagy in C. reinhardtii (Perez-Martin et al. 2014). A previous study demonstrated that autophagy protein VPS30, the ATP-dependent Clp protease proteolytic subunit, vacuolar trafficking protein, Atg3, and ATG8 are involved in cell autophagy in C. reinhardtii (Perez–Perez and Crespo 2010). Here our proteomics data revealed that a series of proteins associated with cell autophagy, including protein VPS30 (spot 43, gi|159462550), ATP-dependent Clp protease proteolytic subunit (spot 39, gi|41179009), vacuolar trafficking protein (spot 105, gi|159465465), ATG3 (spot57, gi|159483493), and ATG8 (spot113, gi|159482262), were induced after 1 or 3 days of salt treatment. Our immunoblot analysis using anti-ATG3 and anti-ATG8 antibodies confirmed that salt induced the accumulation of ATG3 and ATG8 (Fig. 7). The immunoblot analysis also revealed that ATG3 and ATG8 were

CK

cytosolic redox status and SNO levels. To evaluate this possibility, we firstly measured the level of SNOs in C. reinhardtii cells exposed to salt stress. Similar to the pattern observed for NO generation, salt treatment gradually induced the accumulation of SNOs in C. reinhardtii (Fig. 6d). Salt stress also induced GSNOR activity, which peaked after 3 days of treatment (Fig. 6e). This result agrees with the proteomics data presented above. However, GSNOR activity declined after 5 and 7 days of salt treatment, although the activity remained higher than that of control leaves not subjected to salt treatment (Fig. 3d), suggesting the existence of a feedback mechanism that suppresses the increase in GSNOR activity after 5 days of salt treatment. In agreement with our proteomics and physiological data, our immunoblot analysis showed that GSNOR protein accumulation could be partially induced after 1 day of salt treatment, and then reduced after 3 days of salt treatment (Fig. 7a), suggesting the negative feedback regulation of GSNOR in C. reinhardtii during salt stress.

treated with 100 mM NaCl or with 100 mM NaCl plus different inhibitors, respectively, for 5 days. The degree of protein S-nitrosylation was measured by immunoblot analysis using the biotin switch technique. Total NR, APX, and GSNOR proteins were detected with the corresponding antibody before the biotin switch assay. Ponceau staining was used to confirm the equal loading of different samples. The signal ratio shows the ratio of S-nitrosylated protein to total protein in C. reinhardtii cells exposed to salt stress

Planta Fig. 8 Comet analysis showing that salt stress induced DNA damage in C. reinhardtii. The cells were treated with 100 mM NaCl with or without thioredoxin (TRX, 50 lM) or dodecanoic acid (DA, 30 mM), respectively. After the indicated periods, the nuclei were isolated and DNA damage was assessed by comet analysis. Bar 50 lm

Salt

Salt+TXR

Salt+DA

Control

1d

3d

induced after 1 or 3 days of salt treatment (Fig. 7a). These data support the notion that cell autophagy participates in the response to salt stress in C. reinhardtii. It is possible that salt-induced oxidative stress induced cell autophagy in C. reinhardtii. Consistent with our data, Perez-Martin et al. (2014) reported that oxidative stress induced by the ER stressors tunicamycin or dithiothreitol triggered cell autophagy in C. reinhardtii, and that this effect is more pronounced in the C. reinhardtii sor1 mutant, which shows increased expression of oxidative stress-related genes. The DNA damage response (DDR) protects organisms against the detrimental effects of stress, by coordinating the regulation of cell cycle checkpoints and transcription of DDR genes, and ultimately inducing programmed cell death. Environmental agents, such as ultraviolet (UV) radiation, ionizing radiation, and numerous genotoxic chemicals, induce DNA damage (Hemnani and Parihar 1998; Huang et al. 2013). Our proteomics data showed that many proteins related to the DDR process, including the DNA damage checkpoint protein (spot 18, gi|158277392), DNA repair protein (spot23, gi|158277507), Set3p (spot24, gi|56199778), DNA damage inducible protein (spot 32, gi|158282216), RAN binding protein RANBP1 (spot51, gi|159472699), RAD 51 (spot54, gi|45685351), REX1-B (spot56, gi|30038276), and putative defender against death (DAD) protein (spot120, gi|159483809), were differentially induced after exposure to salt stress, suggesting that DNA damage occurs in C. reinhardtii cells exposed to salt stress. To test this possibility, we quantified the degree of DNA

damage in C. reinhardtii cells exposed to salt stress using the comet assay. We found that nuclei isolated from C. reinhardtii cells subjected to salt stress exhibited significantly more DNA damage than did those from control cells not exposed to salt stress (Fig. 8). These data suggest that DNA damage plays a role in the response to salt stress in C. reinhardtii. Recent evidence indicates that DNA damage induces autophagy (Bozhkov and Jansson 2007; Lin et al. 2015; Zhang et al. 2015). In agreement with this, our observations suggest that autophagy plays an integral part in the DNA damage response in C. reinhardtii cells subjected to salt stress. However, it is clear that oxidative stress triggers both DNA damage and cell autophagy in response to environmental stress (Rodriguez-Rocha et al. 2011; Chen et al. 2014; Filomeni et al. 2015). In our study, salt-induced oxidative stress is possibly the main cause of cell autophagy and DNA damage in C. reinhardtii. NO signal modifies the protein S-nitrosylation status and enzyme activity under salt stress NO-derived SNOs suppress GSNOR enzyme activity by protein S-nitrosylation (Feechan et al. 2005; Malik et al. 2011; Morisse et al. 2014). Here, we also measured the protein S-nitrosylation status of C. reinhardtii cells subjected to salt stress. As shown in Fig. 7b, we found that salt treatment markedly induced the protein S-nitrosylation of GSNOR. Furthermore, we found that salt treatment also induced protein S-nitrosylation of the antioxidant GST

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Fig. 9 The effects of different inhibitors on ROS/RNS and their related enzymes in C. reinhardtii cells exposed to salt stress. a The effect of different inhibitors on salt-induced H2O2 and SNO accumulation in C. reinhardtii cells exposed to salt stress. The cells were treated with 100 mM NaCl with or without thioredoxin (TRX, 50 lM) or dodecanoic acid (DA, 30 mM), respectively. The content of H2O2 and SNOs was measured after 3 days of treatment. Effect of different inhibitors on APX and GNSOR activity (b) and on biomass (c) of C. reinhardtii cells after a 3-day treatment with 100 mM NaCl

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treatment markedly reduced the protein S-nitrosylation status of GSNOR, GST, and ATG8 in C. reinhardtii cells after salt treatment (Fig. 7b). These data suggest that GSNOR enzyme activity controls the protein S-nitrosylation level in C. reinhardtii after exposure to salt stress. Our proteomics data showed that, in contrast to 1 or 3 days of salt treatment, long periods of salt treatment (i.e., 5 days) reduced the accumulation of most proteins (Table 1). A 5-day salt treatment increased the content of SNOs, indicating that salt-induced SNOs increase the level of protein S-nitrosylation, thereby inactivating GST and GSNO. To explore the potential role of protein S-nitrosylation in altering the response to salt stress, we treated C. reinhardtii with the GSNOR inhibitor DA to increase the level of protein S-nitrosylation, or with thioredoxin to reduce the level of protein S-nitrosylation, during salt stress, and then quantified cell viability. We found that dodecanoic acid (DA) treatment increased, while thioredoxin treatment reduced, the accumulation of H2O2 and SNOs during salt stress (Fig. 9a). DA treatment further suppressed the activity of APX and GSNOR, but thioredoxin treatment partially increased the activities of APX and GSNOR enzymes (Fig. 9b). These data coincide with

protein and ATG8, suggesting the potential role of S-nitrosylation in regulating GST enzyme activity and ATG8 function in C. reinhardtii cells exposed to salt stress. In agreement with this possibility, a 5-day salt treatment markedly suppressed GSNOR activity (Fig. 6e). These data suggest the negative regulatory role of protein S-nitrosylation in the response to salt stress in C. reinhardtii. Because GSNOR scavenges SNOs and reduces the degree of protein S-nitrosylation upon exposure to environmental stress (Chaki et al. 2009), we tested the possible role of GSNOR in modulating the degree of protein S-nitrosylation in C. reinhardtii cells after salt stress treatment. C. reinhardtii cells treated with the GSNOR inhibitor dodecanoic acid (DA) during exposure to salt stress exhibited a higher level of protein S-nitrosylation of GSNOR, APX, and ATG8 than did cells treated only with salt (Fig. 7b). A previous study showed that the oxidoreductase thioredoxin-h5 (TRXh5) reverses SNO modifications by acting as a selective protein-SNO reductase, and that TRXh5 selectively removes the excessive protein-SNO during the plant immune response (Kneeshaw et al. 2014). Thus, we also monitored the effect of thioredoxin on saltinduced protein S-nitrosylation status. Thioredoxin

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our above finding that the protein S-nitrosylation levels of APX and GSNOR were increased by DA treatment, but reduced by thioredoxin treatment (Wang et al. 2015). Similarly, the comet assay showed that nuclei isolated from C. reinhardtii cells treated with salt plus DA exhibited more damage than those isolated from cells exposed only to salt treatment or from control cells not exposed to salt treatment, while nuclei isolated from cells treated with salt plus thioredoxin exhibited less damage (Fig. 8). These data are consistent with our above findings that DA treatment increased the level of protein S-nitrosylation, possibly inactivating ATG8 and thereby resulting in more DNA damage.

Discussion In this study, we used a comparative proteomics and physiological approach to investigate the mechanism underlying the response to salt stress in the unicellular alga C. reinhardtii. We successfully identified 74 proteins showing significant differential accumulation after salt stress, including a set of proteins associated with NO metabolism, oxidation, cell damage, and cell autophagy (Fig. 3; Table 1). Our physiological experiments also found the over production of ROS and the increased activities and antioxidant enzymes (Fig. 5). This data coincide with previous study that salt stress induced the over accumulation of ROS, and the increase of antioxidant enzymes in unicellular alga, not only in high plants, could scavenge the over generation of ROS to avoid the ROS damage (Desikan et al. 2001; Hancock et al. 2002). NO is reported as the putative messenger to mediate multiple physiological response in high plant (Hancock et al. 2001; He et al. 2004; Wilson et al. 2008; Baudouin and Hancock 2013; Domingos et al. 2015). Here, we found salt stress induced the quick production of NO in C. reinhardtii (Fig. 6a). Such an effect could be suppressed by NR inhibitor, but not the NOS-like enzyme inhibitors (Fig. 6b, c). These data suggest that NR, but not the NOS-like enzyme, is mainly responsible for NO production in C. reinhardtii after salt stress. Consistence with our result, Sakihama et al. (2002) showed that NR played the main role in NO production in C. reinhardtii. Based on these evidences we propose that, like in higher plants, NO plays an indispensable role in enhancing the adaptation of the unicellular alga C. reinhardtii to salt stress, possibly through increasing antioxidant enzymes activities and reducing ROS damage. Though NO plays a vital role to control multiple physiological responses (Hancock et al. 2001; He et al. 2004; Wilson et al. 2008; Baudouin and Hancock 2013; Domingos et al. 2015), NO at a high level also forms RNS to

damage the protein activity through S-nitrosylation, covalent attachment of NO to cysteine residues to form SNOs (Feechan et al. 2005; Malik et al. 2011; Morisse et al. 2014). In plants, GSNOR can modulate the SNOs level to avoid the RNS damage (Chaki et al. 2009). It has been reported that GSNOR itself regulated the S-nitrosylation level to control NO production in Arabidopsis during nitrogen assimilation (Frungillo et al. 2014). Our proteomics data showed that salt stress induced the accumulation of SNOs (Fig. 6d). Salt stress also induced the protein accumulation of GSNRO and its activity in C. reinhardtii (Fig. 6e; Table 1). Suppressing GSNRO activity by its special inhibitors obviously increased SNOs and ROS levels (Fig. 9a), and reduced antioxidant enzyme activates (Fig. 9b). We found that GSNOR inhibitor treatment enhanced the S-nitrosylation levels of GSNOR, NR and APX, and reduced their activities of GSNOR and APX (Figs. 7, 9), suggesting the potential negative role of protein S-nitrosylation level modification in C. reinhardtii after salt stress. Previous studies showed that ROS could induce DNA damage and cell autophagy (Rodriguez-Rocha et al. 2011; Chen et al. 2014; Filomeni et al. 2015). In our experiments, we also found that saline stress induced the DNA damage and cell autophagy in C. reinhardtii (Figs. 7, 8). In addition, the increase of the RNS level by reducing GSNOR activity could increase DNA damage and cell autophagy after salt stress, suggesting the role of GSNOR in salt-induced DNA damage and cell autophagy. It has been reported that TRXh5 selectively removes the excessive protein-SNO during the plant immune response (Kneeshaw et al. 2014). We found that TRXh5 treatment obviously reduced salt-induced protein S-nitrosylation level for GSNOR, NR and APX (Fig. 7b), TRXh5 treatment also assuaged salt-induced DNA damage and the degree of cell autophagy (Fig. 8), reduced salt-induced SNOs and H2O2 productions (Fig. 9a) and increased antioxidant enzymes activities (Fig. 9b). Thus, we propose that GSNOR-mediated protein S-nitrosylation level strictly controls the activities of downstream proteins associated with cellular redox status, cell damage, and autophagy processes. Based on our physiological and proteomics analyses, we found that the rapid accumulation of NO acts as a messenger to trigger the defense response during the early event of C. reinhardtii response to salt stress. A set of antioxidant enzymes and GSNOR were induced following exposure to salt stress, presumably to scavenge the resulting ROS and RNS and thereby limit cellular damage. However, more ROS and RNS accumulated, and the scavenging limit of antioxidant enzymes and GSNOR was exceeded after long-term (i.e., beyond 3 days) salt treatment. As a result, RNS-induced protein S-nitrosylation, which inactivated the antioxidant enzymes and GSNOR,

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and finally increased ROS and RNS levels, which reduced cell viability. Modulating GSNOR activity and protein S-nitrosylation level could adjust the adaptation of C. reinhardtii to salt stress. Our result propose the essential role of NO homeostasis in modulate C. reinhardtii response to salt stress, and provide new insights into enhancing algae tolerance to environment stress through genetically modifying GSNOR activity and protein S-nitrosylation levels. Author contribution statement X.C. and X.H. designed the research. X.C., D.T., X.K. and Q.C. performed the research. X.C., A.J. and A.E. analyzed the data. X.H and A.J wrote the article. Acknowledgments The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this Research Group NO (RG-1435-014).

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