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Protistology 15 (3), 153-160 (2021)
Protistology
Nitric oxide levels and CYP55 expression in Chlamydomonas reinhardtii under normoxia and hypoxia
Zhanneta Zalutskaya, Semen Dukhnov, Nataliia Leko and Elena Ermilova
Department of Microbiology, St. Petersburg State University, 199178, St. Petersburg, Russia
| Submitted August 13, 2021 | Accepted September 8, 2021 |
Summary
Nitric oxide (NO) is a vitally important molecule in many organisms, including Chlamydomonas reinhardtii. Given that NO can be toxic, its generation and degradation have to be tightly regulated. The intracellular NO can be the substrate for NO reductases producing nitrous oxide (N2O), and the cytochrome P450, CYP55, catalyzes this NO conversion in the darkness. Here we show that nitrite-dependent NO accumulation in dark cells is directly dependent on O2 levels: under hypoxia, very little NO is detected, whereas under normoxia, substantial amount of NO is measured. Following dark incubation, NO level is negatively correlated with the CYP55 expression. Moreover, we found that the transcription of CYP55 is under the complex influence of NO- and O2-dependent pathways. Finally, removal of extracellular Ca2+ by the application of EGTA leads to an increase in CYP55 transcript levels both in hypoxic and aerobic dark cells, demonstrating the importance of membrane Ca2+ influx in the regulation of this gene. Taken together, our results suggest that CYP55 expression might be the regulatory point involved in controlling of NO accumulation in the dark-incubated Chlamydomonas.
Key words: Chlamydomonas, cytochrome P450, hypoxia, nitric oxide, normoxia
Introduction
Nitric oxide (NO) is now regarded as a vitally important molecule in many organisms, including unicellular green algae (Leon and Costa-Broseta, 2020). In Chlamydomonas reinhardtii (further referred to as Chlamydomonas), NO is involved in the regulation of various cell functions, such as nitrate assimilation (Sanz-Luque et al., 2013), macronutrient stress responses (Wei et al., 2014;
Minaeva et al., 2017; De Mia et al., 2019; Filina et al., 2019), salt stress acclimation (Chen et al., 2016) and impact on putrescine and proline synthesis (Zalutskaya et al., 2020). Moreover, NO has also been implicated in responses to hypoxia (Hemschemeier et al., 2013).
The accumulated evidence suggests that Chla-mydomonas produce NO from nitrite using a dual enzymatic system of nitrate reductase (NR) and NO-forming nitrite reductase (Chamizo-
doi:10.21685/1680-0826-2021-15-3-5 © 2021 The Author(s)
Protistology © 2021 Protozoological Society Affiliated with RAS
Ampudia et al., 2016) or the mitochondrial electron transport chain (Plouviez et al., 2017; Ostroukhova and Ermilova, 2019). Although another (Arg-dependent) mechanism of NO generation has been defined, this process yet has to be characterized at the molecular level (De Mia et al., 2019).
Importantly, NO can be toxic, and therefore, its generation and degradation have to be tightly regulated. In Chlamydomonas, truncated hemoglobins might be NO scavengers that modulate NO and thereby control NO-based signaling cascades (Sanz-Luque et al., 2015; Minaeva et al., 2017; Filina et al., 2019; Grinko et al., 2021). Moreover, the intracellular NO can also be the substrate for NO reductases producing nitrous oxide (N2O) in mitochondria and chloroplasts (Plouviez et al., 2017, 2019; Burlacot et al., 2020).
Interestingly, NO reduction to N2O has been described in both prokaryotic (Kuypers et al., 2018) and non-photosynthetic eukaryotic organisms (Shoun et al., 2012). In eukaryotes (fungi), the cytochrome P450, CYP55, which is involved in the pathway NO^N2O, appears to be acquired by a horizontal gene transfer from bacteria (Shoun et al., 2012). The Chlamydomonas genome also contains a gene encoding for CYP55.
Given that in Chlamydomonas CYP55 is responsible for N2O production in the darkness (Plouviez et al., 201"/; Burlacot et al., 2020), it is likely that the cytochrome P450 plays an important role in the regulation of cellular NO levels. Nevertheless, the ratio of NO in the two types of dark cells, aerobic and hypoxic, is not known to date. In this study, we investigated nitrite-dependent NO accumulation in the dark-incubated NR-deficient cells upon normoxia and hypoxia. Our analysis demonstrates that there is a negative correlation between the NO amount and the levels of CYP55 transcripts. The expression level of CYP55 increased in the dark cells and was negatively associated with both O2 levels and membrane-regulated entry of extracellular Ca2+ ions. These data suggest that CYP55 expression might be the regulatory point involved in the control of NO levels in dark cells.
um (http://www.chlamy.org/TAP.html) under continuous illumination by white light (fluence rate of 45 ^mol/m2 s1) at 22 °C with a constant orbital agitation at 90 rpm. Depending on the nitrogen source, four variants of the TAP medium were used: with 7.5 mM NH4Cl, 4 mM KNO3, 10 mM or 20 mM KNO2.
To induce anoxic conditions, the cells grown in TAP-NH4 to log phase (2-2.5 106 cells/ml) in 1 l flasks were collected by centrifugation (4000 x g, 5 min), washed with TAP medium containing 20 mM KNO2, and then 50 ml aliquots of cells were transferred into sealed 50 ml square glass bottles. The flasks were subsequently incubated in the dark, allowing a gradual removal of dissolved oxygen by respiratory activity. Dark-aerobic conditions were established by incubating 50 ml of cell suspension in 250 ml glass beakers loosely covered with aluminum foil. Dark-aerobic and dark-anoxic experiments were performed in parallel from a single batch culture in each biological experiment. Dissolved oxygen was measured using a Clark-type electrode to ensure that anoxic conditions were achieved.
2-(N,N diethylamino)-diazenolate 2-oxide sodium salt (DEA-NONOate, Sigma-Aldrich, USA), S-nitroso-N-acetylpenicillamine (SNAP, Sigma-Aldrich, USA) and ethyleneglycol- bis (P-aminoethyl)-N,N,N',N'-tetraacetic acid (EG TA, Sigma-Aldrich, USA) were used at final concentrations of 100 ^M, 2 mM, and 2 mM, respectively.
At each harvesting time, the number of cells was measured employing a counting chamber and the viable cells were estimated microscopically with use of 0.05% (v/v) Evans blue (DIA-M, Russia) as described by Baker and Mock (1994).
Chlorophyll extraction was carried out using 80% acetone. The resulting supernatant, which contains total chlorophyll, was isolated, and absorption was measured by a spectrophotometer (SmartSpec Plus, BioRad) at 652 nm. The total chlorophyll content (^g ml-1) was quantified according to Harris (1989).
Quantitative real-time PCR
Material and methods
Growth conditions and cell treatments
The strain CC4533 (cw15 mt-) was obtained from Chlamydomonas Resource Center (USA). Cells were grown in tris-acetate-phosphate (TAP) medi-
The total RNA was isolated with Trizol according to the manufacturer's instructions (Invitrogen, USA). DNA contamination was avoided by treatment ofthe RNA samples with RNase-Free DNase I (Fermentas, Lithuania). Reverse transcription was performed with Revert Aid H Minus First Strand cDNA Synthesis Kit according to the manufacturer's instructions (Thermo Scientific,
USA). Gene expression analysis was carried out by real-time quantitative RT-PCR (RT-qPCR) on the Light Cycler Instrument (CFX96 Real-Time PCR Detection System, Bio-Rad) using SYBR Green I following a previously reported protocol (Zalutskaya et al., 2016). The primer pairs used for RT-qPCR were as follows: 5'-AG GCCAAGGCAGACAAGCTG-3' and 5'-GC CTCGGGAATGCCGATGAA-3' for CYP55 (Cre01.g007950), and 5'- CTTCTCGCCCATG ACCAC-3' and 5'-CCCACCAGGTTGTTCT TCAG-3' for RACK1 (receptor of activated protein kinase C; Cre06.g278222, formerly termed CBLP). The relative gene expression ratios were normalized with RACK1 using the ACT and AACT methods (Livak and Schmittgen, 2001). Values were obtained from at least three biological replicates; each replicate was analyzed three times. Student's t-tests were used for statistical comparisons. P-values of <0.05 were considered as significant.
Measurement of NO
Chlamydomonas cells (45 ^g chlorophyll ml-1) were incubated in TAP medium containing 20 mM KNO2 in the presence of 1 ^M (4-amino-5-methylamino-2'7'-difluorofluorescein diacetate) dye (DAF-FM DA, Sigma-Aldrich). After 15 min of treatment, the cells were washed, resuspended in the same medium, incubated for additional 30 min to allow complete de-esterification of the intracellular diacetates, and then intracellular generation of NO was evaluated using a microplate reader CLARIOstar (BMG). Excitation and emission wavelengths were set at 483+14 and 530+30 nm, respectively. Cell autofluorescence was subtracted from the total fluorescence obtained. Fluorescence levels were expressed as arbitrary units (per ^g chlorophyll 106 cells). Three technical replicates per condition were included on each plate, and each experiment was performed three times independently.
Confocal microscopy
For the NO detection by confocal microscopy, cells were treated as described above. Images were acquired with a Leica TCS-SP5 confocal microscope (Leica-Microsystems, Germany) equipped with a HC PL APO 63 * oil immersion objective. Excitation was performed with a 488-nm argon laser. The signals arising from the DAF-FM DA
were collected on the channel between 500 and 544 nm. Chlorophyll autofluorescence was monitored across a window of 600+680 nm. The experiment was performed in triplicate.
Results
NO production in the dark-incubated cells under normoxia and hypoxia
It has been shown previously that NO is important for hypoxic growth of Chlamydomonas in the dark (Hemschemeier et al., 2013). Moreover, NO was generated in the dark aerobic wild-type cells (Burlacot et al., 2019). To investigate the potential components involved in the control of NO levels in the dark-incubated cells, we used the strain CC4533. Because this strain was unable to grow on nitrate and lacked NR activity, this enzyme cannot be responsible for NO generation in these cells. However, CC4533 exhibits the growth on nitrite as nitrogen source (Fig. 1, A).
To compare the nitrite-dependent NO production in aerobic and hypoxic cells, we carried out two types of experiments. In the first type of analysis, cells grown in the light were transferred to open flasks with nitrite-containing medium in the dark. In these experiments, no significant drop in the dissolved oxygen levels was monitored, showing only slight decrease (about 5 %) after 3 h of incubation in the dark (Fig. 1, B). During the second type of experiments, illuminated aerobic cultures were transferred to sealed flasks in the dark to create hypoxic conditions. In these conditions, about 80% drop in oxygen levels was monitored after 1 h of incubation (Fig. 1, B). To detect NO production, an NO-sensitive fluorescent dye, DAF-FM diacetate, was added to the nitrite-containing medium before cells were transferred to a dark condition. Notably, NO amounts were 5-6 fold higher in cells under normoxia than under hypoxia (Fig. 1, B).
We confirmed these results using confocal microscopy. After incubation with the NO-specific dye DAF-FM DA, the dark hypoxia-treated cells demonstrated either no signal or a very weak one in the cytosol (Fig. 1, C). When Chlamydomonas was incubated in the dark aerobic conditions, the cells displayed green NO fluorescence. Thus, NO is generated under normoxia and hypoxia at very different levels.
Fig. 1. Effects of nitrite on NO production in the dark-incubated Chlamydomonas cells under normoxia and hypoxia. A — The cell growth was analyzed in 7.5 mM ammonium (1)-, 4 mM nitrate (2)-, 10 mM nitrite (3)- or 20 mM (4) nitrite-containing media; B — NO production in cells. Vegetative cells were grown in TAP medium (Con) and transferred to a 20 mM nitrite-containing medium in the dark under hypoxia or normoxia. Fluorescence intensity (indicated by grey and white squares under hypoxia and normoxia, respectively) due to intracellular NO was determined using DAF-FM DA and was expressed as arbitrary units per ^g chlorophyll 10 cells. Cell autofluorescence was subtracted from the total fluorescence obtained. Data are the means ± SE from three independent experiments. Levels of dissolved oxygen (indicated by and white circles under hypoxia and normoxia, respectively) are given as mg per l of medium; C — NO visualization by confocal microscopy. Images of cell populations grown in TAP (Con) or incubated in 20 mM nitrite-containing medium in the dark for 3 h under hypoxia or normoxia. The left-hand panels show DAF-FM fluorescence (green color) while the right-hand panels show chlorophyll autofluorescence (red color). Scale bars: 50 ^m or 10 ^m.
Effects of oxygen levels on CYP55 expression in the dark-incubated cells
Previous studies have indicated that the dark reduction of NO to N2O is mediated by cytochrome P450 (CYP55) in Chlamydomonas (Plouviez et al., 2017; Burlacot et al., 2019). We therefore wondered whether the CYP55 mRNA levels correlated with NO levels in the dark-incubated cells. We found that CYP55 mRNA transcript abundance increased 10-fold after 1 h and declined after 3 h exposure to the dark under normoxia (Fig. 2). When Chlamydomonas cells were exposed to hypoxia, we observed a more dramatic induction in the CYP55 transcript levels (29-fold increase after 1 h with the
highest induction of 62-fold after 3 h). Altogether, our results indicate a negative correlation between the CYP55 mRNA levels and NO content in Chlamydomonas cells subjected to darkness under aerobic or hypoxic conditions indicating that the transcription step is potentially the key regulatory point of the dark reduction of NO.
NO regulates CYP55 expression
In order to check whether the observed CYP55 transcript levels were related to NO, we first measured the CYP55 mRNA abundances in response to DEANONOate or SNAP as NO donors. Unexpectedly, after the treatment with 100 ^M DEA
Fig. 2. Effects of nitrite on CYP55 expression in the dark-incubated Chlamydomonas cells under normoxia and hypoxia. Cells were treated as described in Fig. 1, B. Levels of gene transcripts are calculated as times of relative abundance with respect to the housekeeping control gene (RACK1) that has a value of 1. Data are the means ± SE from three biological and three technical replicates obtained by RT-qPCR.
NONOate for 0.5 h or 1 h, transcripts of the CYP55 gene were strongly induced (Fig. 3, A). Furthermore, the addition of SNAP also strikingly enhanced the CYP55 expression (Fig. 3, A). However, in the dark-hypoxic cells, the high CYP55 mRNA transcript abundance was maintained under low levels of NO (Fig. 2, A). A possible explanation ofthis difference is that the effects of DEA NONOate and SNAP were found in the TAP grown aerobic cells in the light. We hypothesized that NO may interfere with the hypoxia-specific components involved in the regulation of this gene in the darkness. In order to test this possibility, we analyzed the effects of DEA NONOate that was added to dark cells in the TAP-NO2 (Fig. 3, B). The reduction of CYP55 expression levels in dark-hypoxic cells in the presence of exogenous NO indicates that NO-dependent signal network appears to be not the only pathway influencing the transcript levels of CYP55 mRNA under hypoxia. In contrast, the addition of DEA NONOate resulted in the increase of the dark-induced expression of CYP55 in aerobic cells (Fig. 3, B). Therefore, our results highlight that NO may negatively control the dark-induced CYP55 gene expression at low O2 levels by operating in a reverse regulatory loop. This mechanism may help to fine-tune NO levels depending on the cells' physiological status.
CYP55 gene responds to Ca2+
Since Ca2+ appears to play a role in adaptation to hypoxic conditions (Duner et al., 2018), the question arises whether this second messenger exerts any effect on the hypoxia-induced CYP55 expression. As shown in Fig. 4, the addition of the calcium chelator EGTA to washed cells prior to hypoxia treatment did affect the CYP55 transcription: the presence of 2 mM EGTA led to enhanced amplitude ofhypoxia-induced CYP55 gene expression. Moreover, this induction was diminished when an excess extracellular Ca2+ (2.1 versus 0.34 mM) was provided, despite the presence of EGTA.
Then we also tested the cells that were incubated in darkness, but in the presence of O2. Notably, EGTA had an even stronger effect on the CYP55 transcription in dark aerobic cells (Fig. 4). The results suggest that a membrane-regulated entry of extracellular Ca2+ ions is essential to regulate the dark-induced CYP55 expression and this effect is dependent on O2 levels.
Discussion
In this study, we demonstrate that dark-incubated Chlamydomonas cells are able to the nitrite-dependent NO production, which is not mediated by nitrate reductase. Moreover, we unveil that NO accumulation in dark cells is directly dependent on O2 levels: under hypoxia, very little NO is detected, whereas under normoxia, substantial amount of NO is measured (Fig. 1). This effect differs from higher plants that produce high levels of NO under hypoxic conditions (Gupta et al., 2005; Wany et al., 2017).
In Chlamydomonas, NO homeostasis is based on the balance between reduction of nitrite to NO and re-oxidation of NO to nitrate (Bellido-Ped-raza et al., 2020). It is believed that truncated hemoglobins might be NO scavengers that protect Chlamydomonas cells from an accumulation of the radical molecule (Sanz-Luque et al., 2015; Cha-mizo-Ampudia et al., 2016; Minaeva et al., 2017; Filina et al., 2019; Grinko et al., 2021). Recently it was shown that Chlamydomonas can reduce NO to N2O in the dark (Plouviez et al., 2017, 2019) and this dark reaction is mediated by CYP55 (Burlacot et al., 2020). Interestingly, we found that expression of CYP55 is under the complex influence of NO-and O2-dependent pathways (Figs 2, 3). The NO level in dark cells is negatively correlated with the CYP55 expression (Figs 1, 2). An important ques-
Fig. 3. NO-dependent expression of CYP55. A — Effects of DEA-NONOate and SNAP on the expression of CYP55gene. DEA NONOate (100 ^M) or SNAP (2 mM) were added to TAP grown cells (Con). B - Effects of DEA-NONOate on the expression of CYP55 gene under hypoxia and normoxia. Cells grown in TAP were transferred to 20 mM nitrite-containing medium and incubated for 1 h, 3 h or 5 h in the dark. DEA NONOate (100 ^M) was added 40 min before the indicated times. Expression levels of CYP55 are given as a percentage with respect to the variant without DEA NONOate at each time point, which has a value of 100%. Relative gene expression was determined as described in Fig. 2.
tion is why O2-dependent signals are required to control the expression of CYP55. One possibility is that lower NO levels have emerged to be required for the acclimation to dark hypoxia than to dark normoxia. Therefore, important regulatory layer in dark Chlamydomonas could be that the CYP55 protein amounts must be balanced in order to ensure the NO signaling mechanism to function properly.
In higher plants, NO cross talks with Ca2+ (Bes-son-Bard et al., 2008; Verma et al., 2019; Lau et al., 2021). The regulation of CYP55 transcription by Ca2+ in the cells incubated under hypoxia overlaps with that under normoxia. As we found a strict negative correlation between NO levels and the accumulation of CYP55 transcripts (Fig. 2), a retrograde signal appears to exist that is integrated at the level of CYP55. Here, one scenario would be that NO triggers Ca2+ influx through membrane Ca2+ channels, which, via a signaling cascade, finally results in a negative control of CYP55 expression. Given that we observed a more dramatic induction in the CYP55 transcript levels by EGTA in dark aerobic cells (Fig. 4), additional components might be used to regulate the transcription of this gene by Ca2+ under normoxia. Moreover, we still cannot rule out the role of NO production mechanisms. Besides, the major players involved in NO generation in dark cells at different O levels remain unknown.
This study provides a basis for further research on elucidating the components involved in the relationship between NO- and Ca2+-dependent signaling, and on the question at which level the mechanisms of NO production are coordinated with the mechanisms of NO conversion in dark cells.
Acknowledgements
This work was supported by the Russian Science Foundation (21-14-00017). We thank Dr. Anton Radaev and the Core Facility "CHROMAS" of Saint-Petersburg State University for assistance with confocal microscopy.
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Address for correspondence: Elena Ermilova. Department ofMicrobiology, St. Petersburg State University, Universitetskaya Emb. 7/9, 199034 St. Petersburg, Russia; e-mail: e.ermilova@spbu.ru
