Planta DOI 10.1007/s00425-017-2734-4
ORIGINAL ARTICLE
Comparative proteomic analysis of Chlamydomonas reinhardtii control and a salinity‑tolerant strain revealed a differential protein expression pattern Sayamon Sithtisarn1 · Kittisak Yokthongwattana2 · Bancha Mahong2 · Sittiruk Roytrakul3 · Atchara Paemanee3 · Narumon Phaonakrop3 · Chotika Yokthongwattana1
Received: 23 May 2017 / Accepted: 1 July 2017 © Springer-Verlag GmbH Germany 2017
Abstract Main conclusion Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as proteolytic enzymes, were remarkably up-regulated in the salinity-tolerant strain of Chlamydomonas reinhardtii. Excessive concentration of NaCl in the environment can cause adverse effects on plants and microalgae. Successful adaptation of plants to long-term salinity stress requires complex cellular adjustments at different levels from molecular, biochemical and physiological processes. In this study, we developed a salinity-tolerant strain (ST) of the model unicellular green alga, Chlamydomonas reinhardtii, capable of growing in medium containing 300 mM NaCl. Comparative proteomic analyses were performed to assess differential protein expression pattern between the ST and the control progenitor cells. Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as protein degradation, were Electronic supplementary material The online version of this article (doi:10.1007/s00425-017-2734-4) contains supplementary material, which is available to authorized users. * Chotika Yokthongwattana
[email protected] 1
Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan Rd., Bangkok 10900, Thailand
2
Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, 272 Rama 6 Rd., Bangkok 10400, Thailand
3
Genome Institute, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Rd., Pathumthani 12120, Thailand
remarkably up-regulated in the ST cells, suggesting the importance of these processes in acclimation mechanisms to salinity stress. Moreover, 2-DE-based proteomic also revealed putative salinity-specific post-translational modifications (PTMs) on several important housekeeping proteins. Discussions were made regarding the roles of these differentially expressed proteins and the putative PTMs in cellular adaptation to long-term salinity stress. Keywords Abiotic stress · Chlamydomonas · Proteomic · Salinity stress · Salt stress · Sodium chloride
Introduction Salinity stress is one of the world’s major agricultural problems affecting plant growth and development, leading to reduction in crop productivity (Ngara et al. 2012). Some economically important crop plants, such as broad bean, maize, potato, rice, soybean, are salt-sensitive or moderately sensitive (Katerji et al. 2001). High salinity level in the soil causes inhibition of photosynthetic activity, decrease in uptake of essential nutrients, increase in osmotic as well as ion toxicity, and oxidative stress (Munns 2002; Munns et al. 2006). The effects of salinity, especially NaCl, on plants can be separated into two phases (Munns and Tester 2008). The first phase of salinity stress is when plants are under osmotic stress, during which the stomata are closed. As a result, transpiration and photosynthetic rate are reduced (Brugnoli and Lauteri 1991; Munns and Tester 2008). The second phase, ionic stress, occurs later when plants are exposed to NaCl for a long period of time. Successful acclimation and adaptation of plants to salinity stress require complex synchronization of different processes such as transcription, translation, enzymatic
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reactions, and physiological and cellular adjustments. One of the key processes in the acclimation and adaptation is the modulation of gene expression. Regulation of key proteins and enzymes can be a success factor for salt tolerance in plants (Kosova et al. 2011). Alterations in transcriptome and proteome profiles in response to salinity stress have been reported in rice, potato, soybean, and others (Aghaei et al. 2008a, b; Manaa et al. 2011; Parker et al. 2006; Peng et al. 2009; Silveira and Carvalho 2016). However, those studies focused on the effects of salinity on normal cultivars of plants that are usually salt sensitive. When the treatment is prolonged, such plants may eventually die. Thus, to understand the acclimation mechanism of plants to high concentration of NaCl, study in the resistant or acclimated strains/cultivars would yield more meaningful insights into the underlying processes. Several researchers have investigated the changes in both transcript and protein levels in halophytes, for instance, Thellungiella salsuginea (VeraEstrella et al. 2014), Thellungiella halophila (Chang et al. 2015; Pang et al. 2010), Suaeda sp. (Li et al. 2011) as well as the salt-tolerant varieties of glycophytes such as rice (Domingo et al. 2016), wheat (Wang et al. 2008), and soybean (Pi et al. 2016). The results demonstrated that proteins belonging to various biological processes were differentially up- and down-regulated under different salinity treatments. Such changes comprised proteins in carbohydrate metabolism, amino acid metabolism, photosynthesis, signaling, stress, and defense. In single cell organisms, integrity of the cell is one of the key factors for survival under high saline concentration. To investigate N a+-tolerant mechanism at the cellular level, freshwater unicellular green algae that can tolerate or adapt to grow under high concentration of NaCl would offer meaningful information. In this work, a strain of the model green alga Chlamydomonas reinhardtii capable of growing in tris-acetate-phosphate (TAP) medium containing 300 mM NaCl was generated. Its physiological characteristics and proteome profiles were investigated in comparison to the progenitor cells (control) grown in normal TAP medium without NaCl supplement. Several groups of differentially expressed proteins as well as discernible changes in salt-specific post-translational modifications of proteins were detected. Discussions are made regarding possible interpretations of these observations.
Materials and methods
by daylight fluorescent lamps (Sylvania F36W/T8). The growth temperature was maintained at 25–28 °C. Cultures were manually agitated two times a day. The ST cells were cultured and maintained in TAP medium supplemented with 300 mM NaCl while keeping other culture conditions the same as the control cells. Physiology and growth characteristics Cell growth was monitored by counting under microscope using Neubauer ultraplane hemacytometer. Photosynthesis performances were assessed as the rate of O2 evolution vs. irradiance using the same protocol described by Wongratana et al. (2013) with the recorded range of light intensity between 0 and 600 µmol photons m −2 s−1. Proteomic sample preparation Total protein was extracted by lysis of cells with 5% SDS. The protein solution was centrifuged at 14,000×g for 15 min at room temperature to remove cell debris. Five volumes of 100% acetone was added to the supernatant and stored overnight at −20°C. Visible protein precipitates were collected by centrifugation at 14,000×g for 15 min at room temperature. The pellet was further resuspended in 5% SDS prior to SDS-gel electrophoresis. Protein concentrations were determined by standard Lowry method. Proteins were separated by either 2-DE or SDS-PAGE using 12% acrylamide concentration followed by staining with Coomassie Brilliant Blue R250. Proteomic analysis by GeLC–MS/MS Resolved proteins from each sample were further subjected to proteomic identification by SDS-PAGE followed by tandem mass spectrometry (GeLC–MS/MS). All the work was performed at the Proteomic Research Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology, Thailand, following the standard operating protocols as described by Chua-on et al. (2016) and Paemanee et al. (2016). Statistical analyses (P values <0.05) and subsequent database search were automatically performed by the computer software DeCyder™ MS (GE Healthcare) and Mascot Search (Matrix Science, UK).
Strains and culture conditions
Protein identification by two‑dimensional gel electrophoresis
Chlamydomonas reinhardtii strain CC-503 cw92 mt+ was cultivated mixotrophically in TAP medium under 50 µmol photons m −2 s−1 of constant illumination supplied
Protein separation and identification by 2-DE-based proteomic was performed with exactly the same protocols as described by Mahong et al. (2012).
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Results Generation of salinity‑tolerant (ST) strain of Chlamydomonas For proteomic research, working with organism that has a complete genome sequence available is ideal. Here, we used the Chlamydomonas CC-503 as it was the same strain utilized for the genome sequencing project. As Chlamydomonas is typically a salinity-sensitive alga, abrupt transition from normal growth condition to high concentration of NaCl (300 mM in this study, for example) would cause the salinity-shocked phenotype. Under the salinityshocked condition, a number of Chlamydomonas cells rapidly died within a few hours and several changes in protein expression and modification pattern could be observed (Fan and Zheng 2017; Yokthongwattana et al. 2012). Perrineau et al. (2014) reported a successful acclimation of Chlamydomonas to growth under high NaCl concentration of 200 mM. We, therefore, followed similar experiments to generate the salinity-tolerant strain (ST) by growing the Chlamydomonas CC-503 cells in the TAP medium containing the same concentration of 200 mM NaCl (Supplement Figure 1). Initially, we observed similar results to the salinity-shocked treatment. The majority of the cells died quickly within a few hours and the culture turned from green to very pale green or almost colorless. Nevertheless, after leaving the culture in the growth room for about a week, the almost colorless culture turned green again and the cells started to regrow. These cells were not as green in color as the control cells grown in the absence of NaCl. The ST cells were subcultured into a fresh TAP medium containing 200 mM NaCl to ensure a consistent phenotype. After several generations of growth in the TAP + 200 mM NaCl medium, the ST cells were challenged with higher salinity levels (250, 300 and 350 mM, respectively). From these challenges, the maximum concentration of NaCl that allowed consistent growth was 300 mM. Thus, it was the level of NaCl employed throughout the rest of the experiments. It must be noted that this ST strain was a heterogeneous culture possibly derived from multiple adapted progenitor cells. We tried to isolate single colonies from this ST culture in the hope of obtaining homogeneous cultures. However, the ST cells could not grow on solid agar medium containing 300 mM NaCl. Therefore, the subsequent experiments were conducted using this heterogeneous ST culture.
chlorophyll content within the cells. The ST cells contained only about half the amount of the chlorophyll (7.79 × 10−16 mol cell−1) when compared with the control culture of 13.34 × 10−16 mol cell−1 (Table 1). Chlorophyll a/b ratio of the control progenitor cells was ~2.6, while that of the ST cells was slightly higher, ~3 (Table 1). These values (~2.5–3) were typical for C. reinhardtii grown under low light condition (Polle et al. 2000). It was remarkable that even in the presence of 300 mM NaCl, the ST culture exhibited a typical growth pattern. When compared to the control progenitor cells grown in the absence of NaCl, the ST cells exhibited slightly slower growth rate during the initial few days after inoculation (Fig. 1a). The control culture could reach the stationary phase by day 4, while the ST population took about 6 days to reach the same stage. Interestingly, final cell densities at the stationary phase were almost the same in both ST and the control cultures. When we compared growth characteristics of the ST culture in this study to the “evolved salt (ES)” culture reported by Perrineau et al. (2014), our ST cells exhibited significantly slower growth rates. Such difference could probably be the effects of higher salt concentration used in our experiment (300 vs. 200 mM NaCl). Although the growth rates of the ST and control cultures were quite similar, their photosynthetic performances were drastically different. As assessed by the rate of O2 evolution vs. irradiance level (Fig. 1b), the control culture manifested a saturating irradiance at around 400 µmol photons m−2 s−1 with the maximum rate of photosynthesis (Pmax) around 3.5 × 10−15 mol O2 cell−1 s−1. Photosynthesis of the ST cells, on the other hand, was saturated at around 200 µmol photons m −2 s−1 and could only manage about ½ of the control’s Pmax (~1 × 10−15 mol O2 cell−1 s−1). These results were consistent with several reports in the literature that photosynthesis was prominently hampered by the high concentration of NaCl (Neale and Melis 1989; Allakhverdiev and Murata 2008; Munns et al. 2006). It is important to note that, in our experiments, both the ST and the control cultures were grown under a constant irradiance of 50 µmol photons m−2 s−1. At that level of irradiance, Table 1 Chlorophyll content of Chlamydomonas progenitor cells (control) and the salinity-tolerant strain (ST) Parameters
Control 13.34 ± 0.04 × 10−16 2.58 ± 0.14
ST 7.79 ± 0.25 × 10−16 3.03 ± 0.05
Growth and physiology of the ST vs. control cells
Chl per cell (mol/cell) Chl a/b ratio
In the mid-logarithmic phase, the ST cells appeared pale green in color, while the control cells were darker green. Such color difference was a result of dissimilar
Both cultures were grown under constant illumination of 50 µmol photons m−2 s−1 with ambient temperature controlled at 25–28 °C. The values are averages of three independent biological replicates ±SE
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Fig. 1 Growth characteristics and photosynthetic performances of ST (open circles) grown in the presence of 300 mM NaCl and control progenitor cells (solid circles) grown in standard TAP medium. a Cell densities, as counted under a microscope, after inoculation at day 0. b Light–saturation curves of photosynthesis as determined by rates of O2 evolution per cells vs. light intensities
photosynthesis rates between the two cultures were comparable (Fig. 1b), giving rise to similar growth rates shown in Fig. 1a. Proteomic analysis of ST cells The results in Fig. 1 clearly demonstrated that our ST cells could acclimate to growth in the presence of 300 mM NaCl. We hypothesized that protein expression patterns between the control progenitor and the ST culture could be significantly different. To investigate the difference in protein expression profiles between the ST and control cells, comparative proteomic approaches were deployed. To begin with, we opted for a relatively high-throughput method of using SDS-PAGE followed by in-gel tryptic digestion and LC–MS/MS, also known as GeLC–MS/MS (Chua-on et al. 2016; Paemanee et al. 2016). With this technique, a total combination of 683 unique proteins could be identified in both control and the ST samples. Using the computer analysis software, we set the cutoff value at fourfold difference (ST:control). With this stringent parameter, 93 proteins were significantly increased in abundance, while
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34 proteins exhibited significant decrease in expression level (P values <0.05). The complete lists of all the proteins with fourfold changes in their expression profiles are shown in Supplement Table 1 (up-regulation) and Supplement Table 2 (down-regulation). Figure 2 further demonstrates the relative expressions (ST:control) of individual proteins within selected seven key functional groups that had been reported in the literature with potential roles under salinity stress. The heat maps were constructed by the MeV program (Saeed et al. 2003) with green color signifying that the particular protein was expressed more in the control progenitor cells grown in the absence of NaCl. Red color, conversely, indicates that the abundance of the protein was elevated in the ST cells. The brighter the color, the greater was the difference in the protein expression levels between the two conditions. Specified numbers next to the heat map colors show relative Log2 fold changes of proteins between ST:control, where positive numbers indicate enhanced expression while negative numbers represent down-regulation. In the group of carbohydrate and energy metabolism, we found enhanced expression of several proteins consistently reported to be up-regulated under stress conditions, especially salinity stress (Fig. 2a). Examples of those elevated proteins are glyceraldehyde-3-phosphate dehydrogenase (Chang et al. 2015; Murad et al. 2014), phosphoribulokinase (El Rabey et al. 2015; Yokthongwattana et al. 2012), cytochrome c6, and cytochrome c oxidase (Ryu et al. 2003; Yan et al. 2005). Proteins in the group of membrane transport and trafficking also exhibited differential expression (Fig. 2b). Three proteins were down-regulated in the ST cells, while the expression of nine polypeptides was enhanced. Among the nine up-regulated proteins in this group, the expression level of ion transporters, especially for K + and C a2+, was increased in the ST cells. This observation was consistent with other reports that retention of K+ was one of the key factors for salinity tolerance in plants (Sun et al. 2015; Chakraborty et al. 2016), while C a2+ uptake could help counteract the toxic effect caused by high Na+ concentration outside the cell (Bacha et al. 2015; Shabala et al. 2006). Interestingly, we also found enhanced abundance of several proteins involved in trafficking and endosome system, such as subunits of SEC protein, Vamp7 of the R-SNARE system, and VPS36 of the ESCRT-II complex. This observation suggests that the endosome system as well as proteins trafficking might play important roles in salinity acclimation process of Chlamydomonas. Remarkably, we also observed the elevated expression of many proteases and proteins involved in protein degradation in the ST cells (Fig. 2c). Such up-regulation of proteolytic enzymes could suggest that protein degradation and recycling of amino acids could be an important process in plant acclimation to salinity stress. Oxidative stress
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Fig. 2 Heat map showing relative expression of proteins from seven selected functional groups between the ST vs. control progenitor cells. The map was generated using MeV software tool. Green color indicates higher expression of the particular protein in the control progenitor cells grown in the absence of NaCl. Red color signifies that the individual protein is expressed in higher amount in the ST
cells. The brighter the color, the greater are the differences. Numbers next to the heat map indicate differential L og2 fold change between the ST:control of the corresponding protein (P value <0.05). Positive numbers indicate enhanced expression of the protein in the ST cells, whereas the negative numbers represent the down-regulation of the protein
is another known side effect caused by high concentration of Na+. Up-regulation of four proteins functioning in stress and defense pathway was discerned in the ST cells of this study (Fig. 2e), including thioredoxin h (TRX), ascorbate peroxidase (APX), and superoxide dismutase (FSD). The only significant down-regulated protein we could detect was peroxiredoxin (PRX). For signaling cascades, we found changes in expression pattern of proteins involved in different signaling systems (Fig. 2d). In consequence, alteration in expression of transcriptional- and translational-related proteins were also detected (Fig. 2f, g). These results suggest that certain isoforms of proteins might function more effectively under salinity stress. According to our previously published paper on shortterm NaCl-shocked treatment of Chlamydomonas for 2 h (Yokthongwattana et al. 2012), a lot of proteins exhibited
mobility shift that could be detected as unique spots on 2-DE. Such observation suggested two possibilities: either (a) those proteins were under active degradation, leading to smaller polypeptide fragments with lower molecular weight or (b) they were post-translationally modified to have different Mr and pI from the original values. Either of those two possibilities would lead to the proteins appearing as unique spot(s) on the 2-DE images. We, thus, further investigated whether proteins differentially expressed in the ST cells would exhibit the same phenomenon. For this analysis, total proteins isolated from both ST and the control cells were resolved by 2-DE. Example gel images, from three independent biological replicates, are shown in Fig. 3. There were 370 consistent protein spots among all three biological replicates of the control cells. The 2-DE protein images of the ST cells, on the other hand, displayed
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Fig. 3 2-DE image representative of resolved proteins from a control progenitor cells grown in normal TAP medium without salt and b the ST cells cultivated in TAP medium supplemented with 300 mM NaCl. Proteins were separated on immobilized pH gradient strip of pH 4–7 in the first dimension, followed by 12.5% acrylamide SDS-
PAGE in the second dimension. Protein spots were stained with Coomassie Brilliant Blue G. Spot numbers on the gel images correspond to the arbitrary spot# assigned by the software noted in Tables 1 and 2
405 consistent spots. Cross-comparison between the two sets of samples revealed 32 and 68 protein spots that were uniquely expressed in the control and the ST cells, respectively. Those spots were excised, digested, and identified by tandem mass spectrometry. Tables 2 and 3 list all the identified exclusive protein spots that were detected in the control progenitor cells and the ST cells, respectively. Among all the exclusive proteins expressed in the control progenitor cells (Table 2), we could observe only five spots on 2-DE (#1022, 2015, 2326, 5311 and 9209) having the observed molecular weight drastically smaller than their theoretical values (the difference of more than 5 kDa). Those protein spots were identified as cobalaminindependent methionine synthase, α tubulin, β tubulin, isocitrate lyase, and α subunit of mitochondrial ATP synthase, respectively. With the same criteria, nine spots (#1327, 3427, 7523, 8115, 8514, 8523, 9212, 9224, and 9309) were detected at smaller-than-expected size in the ST cells. These spots were identified as serine protease family 10, ADP-glucose pyrophosphorylase small subunit, acetohydroxy acid isomeroreductase, hypoxanthine-guanine phosphoribosyl transferase, PEP carboxykinase, pyruvate formate-lyase, heme-containing ascorbate peroxidase, isocitrate dehydrogenase, and RbcL. Interestingly, all these nine proteins appeared to be exclusive to the ST cells, but were not detected in the short-term NaCl treatment as previously reported (Yokthongwattana et al. 2012). Such smaller
forms of the proteins might originate from active degradation of the original polypeptides. This result strengthens the notion above that active protein degradation and amino acid recycling could be more active in the ST cells. Indeed, this concept was well supported by the detection of ubiquitin conjugating enzyme E2 (spot#2017) as one of the exclusive protein spots in the ST samples. Importantly, the ubiquitin conjugating enzyme E2 did not show any retardation in its pI and Mr from the theoretical values, suggesting that it could be in its functional form. For the group of proteins that the observed molecular weight was larger than the calculated values for more than 5 kDa, 14 and 37 spots could be detected on the 2-DE images of the control and the ST cells, respectively. The larger observed Mr than the calculated value suggested that the particular protein could be a product of PTM by addition of high molecular weight adduct(s). Such putative modified proteins in the control cells belonged to a diverse group of metabolic functions. Conversely, the majority of the ST-specific modified proteins were housekeeping proteins and proteins of important functions, such as heat shock proteins and translation-related proteins. Similar shifts in protein spot localization of molecular chaperones and translational machineries were also discerned in the short-term salinity-shock treatment of the same alga (Yokthongwattana et al. 2012). Thus, it is possible that these observed putative PTMs on key cellular proteins in both the
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Planta Table 2 List of identified 2-DE-resolved exclusive protein spots from control progenitor cells of Chlamydomonas reinhardtii grown in normal TAP medium Spot #
Matched proteins
Carbohydrate and energy metabolism 416 Chloroplast ATP synthase gamma chain 1416 Glyceraldehyde-3-phosphate dehydrogenase 2613 β Subunit of mitochondrial ATP synthase 3016 NADH:ubiquinone oxidoreductase 17 kDa subunit 6622 α Subunit of mitochondrial ATP synthase 7321 Malate dehydrogenase 9209 α Subunit of mitochondrial ATP synthase 9214 Mitochondrial ATP synthase associated protein 9612 Dihydrolipoamide dehydrogenase Cell structure 2015 Alpha tubulin 2326 Beta tubulin Photosynthesis 1615 Rubisco, chain A 2131 Light harvesting protein of photosystem I LHCA1 2219 LHCBM6 chlorophyll a/b binding protein of LHCII 4324 LHCBM1 chlorophyll a/b binding protein of LHCII 5428 Photosystem II stability/assembly factor HCF136 8421 Rubisco, chain A 9415 Ferredoxin–NADP reductase Protein processing 141 Peptidyl-prolyl cis–trans isomerase, cyclophilin-type Transcription 8226 G-strand telomere binding protein 1 9207 G-strand telomere binding protein 1 9213 G-strand telomere binding protein 1 Translation 140 Ribosomal protein L12 7625 Elongation factor Tu Other metabolism 1022 Cobalamin-independent methionine synthase 5311 Isocitrate lyase 7525 Isocitrate lyase 9417 NADH-specific enoyl-ACP reductase 9614 Biotin carboxylase Unannotated proteins 135 Predicted protein 139 Predicted protein 3915 Predicted protein
Phytozome #
Observed Mr (kDa)/pI
Theoretical Mr (kDa)/pI
MOWSE scores
% Sequence coverage
Cre06.g259900 Cre01.g010900 Cre17.g698000 Cre05.g240800 Cre02.g116750 Cre03.g194850 Cre02.g116750 Cre13.g581600 Cre01.g016514
44.1/4.6 48.1/4.8 65.8/5.1 17.0/5.3 64.5/6.0 38.4/6.1 27.1/7.0 30.7/6.9 68.4/7.0
39.1/9.08 40.5/9.17 62.0/4.99 19.7/6.75 48.8/6.20 36.8/8.50 48.8/6.20 34.1/6.86 60.1/8.73
150 355 241 100 57 137 88 77 103
6 16 8 11 2 7 8 3 4
Cre03.g190950 Cre12.g542250
17.3/5.0 31.3/5.0
50.4/5.02 50.2/4.82
140 117
6 4
– Cre06.g283050 Cre06.g285250 Cre01.g066917 Cre06.g273700 – Cre11.g476750
70.7/4.8 21.3/5.0 25.0/5.0 30.5/5.4 44.5/5.7 48.6/6.5 41.1/6.8
53.1/6.04 23.5/7.98 27.6/5.96 27.0/5.96 32.7/6.95 53.1/6.04 28.90/5.90
358 63 177 103 414 258 145
12 4 8 7 22 9 10
Cre01.g002300
19.1/4.0
18.6/7.66
48
6
Cre01.g032300 Cre01.g032300 Cre01.g032300
29.6/6.5 29.6/7.0 26.6/7.0
24.2/6.78 24.2/6.78 24.2/6.78
132 393 212
7 20 9
Cre12.g528750 Cre06.g259150
19.7/4.0 60.2/6.2
17.8/9.19 45.7/5.84
222 54
25 2
Cre03.g180750 Cre06.g282800 Cre06.g282800 Cre06.g294950 Cre08.g359350
18.0/4.9 39.5/5.7 58.1/6.1 43.7/7.0 67.7/7.0
87.2/6.02 45.9/5.78 45.9/5.78 31.0/8.50 52.3/8.96
80 73 317 65 222
1 2 12 3 9
Cre01.g010400 Cre01.g010400 Cre12.g546500
20.7/4.3 20.2/4 103.7/5.6
20.7/5.10 20.7/5.10 67.6/6.41
189 129 156
18 13 4
Total proteins were resolved by 2-DE with immobilized strip of linear pH range of 4–7 for the first dimension and 12.5% SDS-PAGE on the second dimension (Fig. 3). Consistent and exclusive protein spots that were not present in the 2-DE profile of the ST cells were excised and subjected to tryptic digestion followed by identification by LC–MS/MS as described in “Materials and methods”. Spot numbers were assigned arbitrarily by the analysis software. MOWSE search scores of 47 or more are considered as significant match
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Planta Table 3 List of identified 2-DE-resolved exclusive protein spots from the ST cells of Chlamydomonas reinhardtii grown in TAP medium supplemented with 300 mM NaCl Spot # Matched proteins
Carbohydrate and energy metabolism 3415 ADP-glucose pyrophosphorylase small subunit 3424 Glyceraldehyde-3-phosphate dehydrogenase 3425 Pyruvate dehydrogenase E1 beta subunit 3427 ADP-glucose pyrophosphorylase small subunit 3830 Glycerol-3-phosphate dehydrogenase 4720 Phosphoglucomutase 7520 Pyruvate dehydrogenase, alpha subunit 7522 Phosphoribulokinase 7528 α Subunit of mitochondrial ATP synthase 8514 Phosphoenolpyruvate carboxykinase 8624 Phosphoglycerate dehydrogenase 9221 Mitochondrial ATP synthase-associated protein 9224 Isocitrate dehydrogenase 9516 ADP-glucose pyrophosphorylase large subunit 9518 Succinate-CoA ligase beta chain 9710 Phosphoenolpyruvate carboxykinase Cell division 5416 Zygote-specific Zys3 like protein Photosynthesis 7324 LHCB4 chlorophyll a/b binding protein of LHCII 7620 Rubisco, chain A 7621 Rubisco, chain A 8619 Rubisco, chain A 9309 RBCL Protein degradation 1327 Serine protease family 10 1330 Pepsin-type aspartyl protease 2017 Ubiquitin conjugating enzyme E2 3825 FtsH-like membrane metalloprotease 3827 FtsH-like membrane metalloprotease 4718 Leucyl aminopeptidase 4820 Leucyl aminopeptidase Protein processing 1621 Protein disulfide isomerase Translation 2126 2217 4822 4823
Eukaryotic initiation factor, eIF-5A Plastid 50S ribosomal protein L3-1 Chloroplast elongation factor G Chloroplast elongation factor G
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Phytozome #
Observed Mr (kDa)/pI
Theoretical Mr MOWSE (kDa)/pI scores
% Sequence coverage
Not detected in short-term salt shock treatmenta
Cre03.g188250
51.9/5.3
55.9/8.38
275
12
√
Cre01.g010900
48.3/5.3
40.5/9.17
290
17
Cre16.g677026
41.9/5.2
38.5/5.53
500
23
Cre03.g188250
49.0/5.2
55.9/8.38
131
6
√
Cre01.g053000 Cre06.g278210 Cre02.g099850 Cre12.g554800 Cre02.g116750
90.9/5.3 79.5/5.5 50.5/6.3 51.5/6.1 51.2/6.2
76.2/5.31 64.8/7.12 47.8/8.33 42.1/8.11 54.8/5.44
170 140 67 316 397
3 4 8 17 13
√ √
Cre02.g141400 Cre07.g344400 Cre13.g581600
56.9/6.5 63.8/6.6 33.4/6.7
62.4/6.23 42.7/6.28 34.1/6.86
498 752 166
17 37 8
√
Cre04.g214500 Cre13.g567950
25.4/6.9 58.4/6.8
53.9/8.98 55.7/7.41
126 124
4 5
√ √
Cre17.g703700 Cre02.g141400
54.9/6.9 78.0/6.8
44.7/8.10 62.4/6.23
313 493
14 19
Cre06.g304500
49.7/5.6
40.4/5.42
228
9
Cre17.g720250
36.0/6.3
30.0/6.22
173
13
√
– – – –
71.1/6.4 70.9/6.3 70.9/6.5 35.4/6.7
53.1/6.04 53.1/6.04 53.1/6.04 53.2/6.14
441 508 494 323
16 17 17 16
√
Cre17.g746597 Cre04.g226850 Cre12.g546650 Cre12.g485800 Cre12.g485800 Cre01.g007700 Cre01.g007700
38.6/4.7 38.9/4.8 18.5/5.1 88.8/5.3 89.3/5.3 75.5/5.4 90.9/5.4
51.7/6.02 32.6/5.44 18.9/5.08 77.7/5.70 77.7/5.70 45.0/6.55 76.2/5.31
94 56 67 429 722 150 306
2 2 8 12 20 5 6
Cre02.g088200
76.5/4.8
58.4/4.80
1130
36
Cre02.g097400 Cre48.g761197 Cre02.g076250 Cre02.g076250
22.4/5.2 28.6/5.0 97.0/5.4 97.8/5.4
18.2/4.97 27.9/10.26 79.9/5.23 79.9/5.23
130 136 767 480
19 10 19 13
√ √
√ √
Planta Table 3 (continued) Spot # Matched proteins
Phytozome #
Observed Mr (kDa)/pI
Theoretical Mr MOWSE (kDa)/pI scores
% Sequence coverage
Not detected in short-term salt shock treatmenta
5429 Mitochondrial translation factor Tu 7421 Ribosomal protein L10 8220 Plastid-specific ribosomal protein 1 Signal transduction 2330 14-3-3 protein 8221 Ran-like small GTPase Stress and defense 1623 UV excision repair protein RAD23 2720 Heat shock protein 90B 3715 Chaperonin 60A 3719 Heat shock protein 70A 3813 Heat shock protein 90A 3814 Heat shock protein 90A 3815 Heat shock protein 90A 3826 Endoplasmic reticulum associated Hsp70 4124 2-Cys peroxiredoxin 4429 NADPH-dependent thioredoxin reductase 5720 Chaperonin 60C 7326 Stromal ascorbate peroxidase 8416 Catalase 9212 L-ascorbate peroxidase, heme containing Other metabolism 3426 Gamma-glutamyl hydrolase 3515 Magnesium chelatase subunit I 7113 Thiamine-phosphate diphosphorylase 7418 Glutamine synthetase 7425 Phosphoserine aminotransferase 7523 Acetohydroxy acid isomeroreductase 7622 Acetolactate synthase, small subunit 8115 Hypoxanthine-guanine phosphoribosyl transferase 8423 Coproporphyrinogen III oxidase 8523 Pyruvate formate-lyase 8716 Acetohydroxy acid dehydratase 8831 1-Deoxy-d-xylulose-5-phosphate synthase Unannotated proteins 2132 Predicted protein 2329 Predicted protein 8322 Hypothetical protein CHLREDRAFT_188454
Cre06.g259150 Cre12.g520500 Cre05.g237450
45.1/5.8 44.0/6.3 29.5/6.6
45.8/5.90 34.7/6.07 31.9/9.18
74 260 501
3 15 29
√
Cre12.g559250 Cre03.g191050
34.2/5.0 30.1/6.6
29.7/4.90 25.7/6.24
65 252
4 21
Cre08.g366400 Cre02.g080650 Cre04.g231222 Cre08.g372100 Cre09.g386750 Cre09.g386750 Cre09.g386750 Cre02.g080700
68.0/4.6 82.9/5.1 78.0/5.2 75.9/5.4 97.9/5.2 97.7/5.2 98.2/5.2 92.8/5.3
37.3/4.48 87.5/4.80 61.9/5.49 71.5/5.25 81.0/4.99 81.0/4.99 81.0/4.99 72.7/4.99
147 191 944 381 643 635 281 73
7 3 31 14 17 14 7 1
Cre02.g114600 Cre07.g355600
23.0/5.5 46.9/5.5
21.8/5.46 37.0/5.26
142 241
21 14
Cre06.g309100 Cre02.g087700 Cre09.g417150 Cre09.g401886
79.4/5.6 37.4/6.2 51.8/6.5 32.8/6.8
57.2/5.40 35.9/8.67 56.2/6.71 40.0/8.63
482 204 133 287
18 11 4 18
Cre06.g275050 Cre06.g306300 Cre09.g396850 Cre12.g530650 Cre07.g331550 Cre10.g434750 Cre01.g055453 Cre04.g217934
41.1/5.2 53.0/5.3 24.0/6.4 48.7/6.3 44.2/6.2 51.5/6.2 68.0/6.3 19.7/6.6
41.4/5.34 45.5/6.22 15.5/5.57 41.7/7.14 44.4/8.88 60.6/8.29 52.8/8.90 25.8/6.00
210 463 130 368 128 314 107 61
10 22 9 22 6 8 6 4
Cre02.g085450 Cre01.g044800 Cre03.g206600 Cre07.g356350
42.7/6.7 53.5/6.5 79.6/6.7 91.4/6.5
41.7/8.12 93.7/6.40 64.7/7.51 79.3/7.07
177 53 228 375
15 1 9 9
√ √
Cre03.g153450 Cre08.g372000 Cre07.g333150
21.3/5.0 32.6/4.9 35.3/6.5
15.0/5.13 33.2/6.08 21.6/6.23
85 493 92
28 18 7
√
√ √
√
√ √ √
√ √ √ √
√
Total proteins were resolved by 2-DE with immobilized strip of linear pH range of 4–7 for the first dimension and 12.5% SDS-PAGE on the second dimension. Consistent and exclusive protein spots that were not present in the 2-DE profile of the control cells were excised and subjected to tryptic digestion followed by identification by LC–MS/MS as described in “Materials and methods”. Spot numbers were assigned arbitrarily by the analysis software. MOWSE search scores of 47 or more are considered as significant match a
Compared to salt-shock proteome profiles from Yokthongwattana et al. (2012)
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salinity-shocked and the ST cells might be an NaCl-specific response. Such PTMs might be initiated during short-term exposure to salinity stress and last long until the alga successfully adapts to the adverse environment. Further analyses revealed 28 other exclusive proteins in the ST cells that did not appear in any of the control or short-term salt stress proteomes (noted by checkmarks in Table 3). In addition to the nine proteins in which their observed Mr values were lower than the calculated values, the remaining 19 proteins were: spot #1330 (pepsintype aspartyl protease), #2132 (predicted protein), #2720 (HSP90B), #3415 (ADP-glucose pyrophosphorylase small subunit), #3719 (HSP70A), #3830 (glycerol-3-phosphate dehydrogenase), #4124 (2-cys peroxiredoxin), #4720 (phosphoglucomutase), #5429 (mitochondrial translation factor Tu), #7113 (thiamine-phosphate diphosphorylase), #7324 (LHCB4), #7326 (stromal ascorbate peroxidase), #7425 (phosphoserine aminotransferase), #7528 (α subunit of mitochondrial F1F0 ATP synthase), #8322 (hypothetical protein), #8416 (catalase), #8423 (coproporphyrinogen III oxidase), #9221 (mitochondrial F1F0 ATP synthase associated protein), and #9516 (ADP-glucose pyrophosphorylase large subunit). Most of these proteins appeared to perform important functions within the cells.
Discussion Adaptation of plants to growth under high concentration of NaCl requires significant changes in gene and protein expression profiles. Our proteomic data presented here portrays the differences in protein expression patterns between Chlamydomonas cells grown in normal TAP medium (control) and the ST cells grown in the presence of 300 mM NaCl. This work has provided complementary information to the already existing evidence in the literature on salinitytolerant mechanisms in plants. Evidences have shown that photosynthesis in plants and algae is hampered by increasing salinity in the environment (Allakhverdiev and Murata 2008; Munns et al. 2006; Neale and Melis 1989; Neelam and Subramanyam 2013). The ST cells in this study had lower threshold of photoinhibition as observed by lesser photosynthetic saturation irradiance level (Fig. 1b), albeit unaffected chlorophyll antenna size (similar chlorophyll a/b ratio). These observations were consistent with the previous report by Neale and Melis (1989). It was suggested that the enhanced susceptibility to photoinhibition under salinity stress was primarily due to suppression of the photosystem II repair process (Allakhverdiev et al. 2003; Neale and Melis 1989). Like other abiotic stresses, reduced photosynthetic performance under high saline environment of the ST cells, as well as other plant species, would lead to an imbalance
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of light absorption and utilization in the chloroplast. This scenario could lead to generation of several forms of ROS (Miller et al. 2010). It is generally known that effective signaling network and ROS detoxification systems are crucial factors contributing to salinity tolerance and adaptation in plants (Abogadallah 2010; Golldack et al. 2014; Miller et al. 2010). Our Chlamydomonas proteomic study here also showed that a variety of signaling proteins, especially the kinases, were accumulated more in the ST cells (see Fig. 2d). Nevertheless, whether or not the signaling network(s) transduced by these proteins could eventually lead to biosynthesis of the differentially expressed proteins found in this work requires further investigations. For ROS detoxifying enzymes, the protein levels of thioredoxins, ascorbate peroxidase, Fe–SOD, etc. were all enhanced in the ST over the control cells. Accumulation of such antioxidant proteins are often found in the prolonged salinity treatment of plants and thought to help maintain the ROS inside the cells below the toxic level (Abogadallah 2010; Li et al. 2015; Miller et al. 2010). Effective management of the ROS within the cell could be one of the important characteristics of the ST cells for successful adaptation to salinity stress. It was previously reported that salinity stress conferred suppressive effects on cellular transcriptional and translational processes (Allakhverdiev et al. 2003). As our ST cells already adapted to proliferation under high concentration of NaCl, it is plausible that the inhibitory effects of salinity on the transcription and translation had been overcome. Indeed, differential expressions of proteins in the transcription, translation, and protein processing have been observed in this study. We hypothesize that expression of different isoforms of the proteins that are more suitable to function under salinity stress could help overcome the suppressed processes. Proteins containing iron or those involved in iron uptake were drastically up-regulated in the ST cells (Supplement Table 1). This observation is in support of the existing notion that salt stress has a negative effect on iron acquisition (Yousfi et al. 2007). An abundance of plant ferritin proteins is often found to be elevated under salinity stress (Parker et al. 2006). Two ferritin genes, FER1 and FER2, exist in the Chlamydomonas genome (Long et al. 2008). We found that the amount of FER1 protein was remarkably increased in the ST cells. FER1 is the prominent protein that functions in iron homeostasis in Chlamydomonas and has been suggested to help prevent hydroxyl radical generated through the Fenton reaction (Zhang et al. 2012). Chloroplast and mitochondria are the two organelles that are considered “iron rich” as they contain a large number of Fe-containing proteins (Glaesener et al. 2013). Ferredoxin and cytochrome c-type heme lyase are up-regulated in this study. In the literature, components of cyclic photosynthetic
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electron transport, including ferredoxin, have been shown to be up-regulated under drought stress (Lehtimäki et al. 2010). Similar enhanced expression could be expected in the Chlamydomonas ST cells, as both salinity and drought stresses in plants share a lot in common in terms of responsive pathways (Golldack et al. 2014; Ye et al. 2017; Zheng et al. 2017). Expression of several membrane transporters were notably enhanced in the ST cells, insinuating functional importance of these proteins for maintaining ion homeostasis of the cells. Up-regulation of both Ca2+ and K+ channels was observed in the ST cells of this study. A high concentration of extracellular Na+ can interfere with the uptake transport of several other cations, especially K + (Bacha et al. 2015; Chakraborty et al. 2016; Nieves-Cordones et al. 2008; Shabala et al. 2003). There was a suggestion that increase in Ca2+ concentration could help alleviate the effect of Na+-induced K+ loss (Shabala et al. 2006). It is possible that enhanced expression of both C a2+ and K+ transporters found in this study could be one of the mechanisms to compensate for the impairment of the K+ channel under Na+ toxicity. In the situation of ion imbalance, keeping homeostasis of important minerals would require active transport. Here, many other types of ion transport proteins/channels were found in greater abundance in the ST cells (Fig. 2b). We also found up-regulation of several proteins involved in membrane trafficking in the ST cells, including SEC3, Vamp7 R-SNARE protein, VPS36 of the ESCRT pathway, etc. Involvement of the membrane trafficking pathways with salinity stress response is not without precedence. Under salt stress condition, Na+ was shown to promote active recycling of integral membrane proteins, especially aquaporin, between the plasma membrane, Golgi apparatus, and ER (Asaoka et al. 2013; Martinière et al. 2012; Pou et al. 2016; Ueda et al. 2016). Moreover, in yeast and higher plants, proteins in the ESCRT endosomal/vacuolar trafficking system have also been shown to confer important functions in response to increasing NaCl concentration (Logg et al. 2008; Xia et al. 2013). Thus, overexpression of these membrane trafficking proteins might be another important mechanism of the ST cells for successful adaptation to live under high salinity of 300 mM NaCl. Although not often mentioned in other publications on plant responses to salinity stress, turnover of proteins for amino acid recycling could be another key factor for acclimation to salinity stress. In this paper, we observed several protein degradation fragments on 2-DE (Fig. 3; Table 3) coincide with the elevated expression of many proteases in the ST cells found by GeLC–MS/MS (Fig. 2c). Additionally, E2 ubiquitin conjugating enzyme was also found as a unique spot on 2-DE without any mobility shift from the calculated values. In support of our result, heterologous overexpression of mung bean E2 ubiquitin ligase in
Arabidopsis was shown to confer better protection of the plant toward osmotic stress (Chung et al. 2013). Short-term exposure to high NaCl concentration leads to arrest of protein synthesis at both transcriptional and translational levels (Allakhverdiev et al. 2003; Allakhverdiev and Murata 2004). We hypothesize that when photosynthetic organisms find a way to overcome the NaCl-induced inhibition of such processes, they can successfully adapt to high salinity. Once the transcription and translation are reinstated, sources of amino acid for protein synthesis become crucial. This notion is supported by the recent metabolomic study in maize showing that relative concentrations of several amino acids were drastically reduced under salt stress (Guo et al. 2017). Thus, recycling of amino acid from less important or damaged proteins could provide a supply source of the building block. All of these observations entail the importance of protein degradation and turnover for amino acids recycling under salt stress condition. When compared with similar studies on halotolerant unicellular green alga Dunaliella salina grown under low and high concentrations of NaCl (Jia et al. 2016; Wang et al. 2016), similar patterns of differential protein expression could be found. Examples of such include elevated abundance of ATP synthase subunits, cytoskeleton proteins, molecular chaperone, etc. (Jia et al. 2016). Interestingly, Wang et al. (2016) identified a zinc-finger transcription factor, Ds-26-16, from D. salina grown under 4 M NaCl that, when expressed in E. coli, could confer resistance to salinity stress. In this study, we also observed up-regulation of several transcription factors including the zinc-finger protein (Fig. 2f), suggesting its potential role to activate salinity-responsive genes. Short-term exposure of Chlamydomonas cells to 300 mM NaCl was shown to trigger PTMs on a large number of housekeeping proteins (Yokthongwattana et al. 2012). Such PTMs led to significant deviation of the observed pI and Mr on 2-DE from the theoretical values of individual protein spot. When the Chlamydomonas, like the ST cells in this study, successfully adapted to proliferation under high concentration of NaCl, similar PTMs on the key proteins could still be detected (Table 3). This finding denotes that the PTMs imposed on those important proteins could be the specific consequence of cell exposure to excessive NaCl, as irradiance stress did not confer such mobility shift of polypeptide spots (Mahong et al. 2012). Yokthongwattana et al. (2012) gave two possible explanations regarding such putative PTMs during shortterm NaCl treatment of Chlamydomonas as follows: first, the PTMs could lead to activation of protein functions or confer enhanced stability to cope with the stress; second, the modifications would destabilize the proteins, leading to salt-sensitive nature of the alga. Since the ST cells could grow fairly well in the medium containing 300 mM NaCl,
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we could possibly rule out the latter hypothesis. Thus, we suggest that the observed putative PTMs on the key housekeeping proteins could provide better stability under salinity stress. Nevertheless, what type of modifications was made on those important proteins need further in-depth investigation. In summary, this work provides additional supplementary proteomic data on plants’ salinity tolerance at the cellular level. Proteins in the group of stress and defense, iron uptake and metabolism, proteolysis and turnover of amino acid, and membrane transport and trafficking were found to be enhanced in their expression in the ST cells. Moreover, excessive NaCl in the environment also led to salt-specific modifications that might promote extra stability or activate functions of the proteins. Author contribution statement SS performed GeLC–MS/ MS and drafted the manuscript. KY designed experiments, analyzed data, and revised the manuscript. BM performed 2-DE analysis. SR, AP, and NP provided facility and helped perform tandem mass spectrometric analyses. CY designed experiments, analyzed data, and revised the manuscript. Acknowledgements SS was supported by Graduate School, Kasetsart University. This work was conducted with financial support in part by the Kasetsart University Research and Development Institute Grant (Mor-Vor 9.55), Office of the Higher Education Commission and Thailand Research Fund (TRF) Grant No. MRG5580171 to CY. KY thanks TRF and Mahidol University for financial support.
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