Dual positive and negative control of Chlamydomonas PII signal transduction protein expression by nitrate/nitrite and NO via the components of nitric oxide cycle
- 241 Downloads
The PII proteins constitute a large superfamily, present in all domains of life. Until now, PII proteins research in Chloroplastida (green algae and land plants) has mainly focused on post-translation regulation of these signal transductors. Emerging evidence suggests that PII level is tightly controlled with regard to the nitrogen source and the physiological state of cells.
Here we identify that a balance of positive (nitrate and nitrite) and negative (nitric oxide) signals regulates Chlamydomonas GLB1. We found that PII expression is downregulated by ammonium through a nitric oxide (NO)-dependent mechanism. We show that nitrate reductase (NR) and its partner, truncated hemoglobin 1 (THB1), participate in a signaling pathway for dual control of GLB1 expression. Moreover, NO dependent guanilate cyclase appeared to be involved in the negative control of GLB1 transcription.
This study has revealed the existence of the complex GLB1 control at transcription level, which is dependent on nitrogen source. Importantly, we found that GLB1 gene expression pattern is very similar to that observed for nitrate assimilation genes, suggesting interconnecting/coordinating PII-dependent and nitrate assimilation pathways.
KeywordsChlamydomonas reinhardtii Nitrate Nitrite NO signaling PII signal transduction protein Truncated hemoglobin
- DAF-FM DA
2-(N,N-diethylamino)-diazenolate 2-oxide sodium salt
Nitric oxide-forming nitrite reductase
Truncated hemoglobin 1
Inorganic nitrogen (N) acts as one of the most important mineral nutrients for all autotrophic organisms including plants. In natural ecosystems, the availability of nitrogen is often a limiting factor for plant growth. Plants have evolved highly efficient and selective systems for nitrogen acquisition to ensure an appropriate utilization of the scarce resources. In all domains of life [1, 2, 3] with representatives in most bacteria and in many archaea [4, 5] as well as in oxygenic eukaryotic phototrophs , regulation of N metabolism at various levels are coordinated by members of PII signal transduction proteins [7, 8]. PII proteins act as reporters of the metabolic state of the cell by interdependent binding of ATP/ADP and 2-oxoglutarate (2-OG) [9, 10, 11]. The conserved mode of PII function is based on the control of PII – target protein interactions via the effector molecules binding . Furthermore, in plants, the cellular glutamine levels are additionally sensed via PII signaling [8, 12, 13].
A second, phylogenetically diverse regulatory mechanism is covalent modification of apical residues of the T loop in PII proteins that allows the integration of additional signals. In proteobacteria, actinobacteria and cyanobacteria PII proteins can be covalently modified by uridylylation, adenylylation and phosphorylation at the T loop residues, respectively [14, 15, 16].
However, in many other organisms, this second regulatory layer of covalent modification of the T loop is apparently missing, as in Archaea , Bacillus , and plant PII proteins [6, 18]. The lack of this regulatory level can be partially compensated by control of PII-encoding genes at transcription level . In Chloroplastida (green algae and land plants) PII-encoding GLB1 genes are nuclear-encoded and, in Rhodophyta they are coded by the plastid genome . It is believed that regulation of PII in plants may be transcriptional [20, 21, 22]. However, unlike bacteria, the transcriptional control of plant PII expression remains poorly understood.
Chlamydomonas reinhardtii (Chlamydomonas in the following) is a model alga that shares with higher plants the capability of controlling by PII the activity of N-acetyl-L-glutamate kinase (NAGK) that leads to arginine formation . Although Chlamydomonas efficiently uses nitrate and nitrite, ammonium is preferred N source and many genes involved in nitrate/nitrite assimilation are repressed in the presence of ammonium [23, 24]. It is also important to note that amino acids are extracellularly deaminated by Chlamydomonas and only ammonium enters the cells . Given that ammonium depletion induces Chlamydomonas GLB1 upregulation , we hypothesized that ammonium may play a role in such negative regulation and this gene may respond to a balance of negative and positive signals.
In Chlamydomonas, ammonium and nitric oxide (NO) inhibit the expression of high-affinity nitrate/nitrite transporters and nitrate reductase (NR) . During the cycle NO3− → NO2− → NO→ NO3− the negative signal of NO can be converted back to the positive signal of nitrate. Recent publications have uncovered the function of NR in this cycle . NR acts as an essential partner protein of the nitric oxide-forming nitrite reductase (NOFNiR) that catalyzes the formation of NO from nitrite . Furthermore, NR is a protein partner of truncated hemoglobin 1 (THB1) for the conversion of NO into nitrate . In spite of the key role of nitrate as a major nutrient and signal molecule, its possible regulatory effects on GLB1 transcription have not been analyzed. In order to understand the processes PII is involved in, it is important to know how GLB1 gene expression is regulated and when the amount of this protein is increased.
This apparent gap in the information about plant PII control motivated us to investigate the role of the components of cycle NO3− → NO2− → NO →NO3− in regulating GLB1 transcription in Chlamydomonas cells. In this work, we unveil that GLB1 expression responds to an extracellular NO3−/NH4+ balance. Moreover, we show that nitrate and nitrite induce GLB1, and NO represses this gene. Collectively, these results suggest that the NR and its partners, NOFNiR and THB1, participate in a signaling pathway for dual control of GLB1 expression.
Algal strains, growth conditions and cell treatment
The following Chlamydomonas reinhardtii strains were used: wild-type cw15–325 (mt+, cw15, arg7), which was kindly provided by Dr. M. Schroda (University of Kaiserslautern, Germany) and transformants with reduced THB1 obtained from cw15–325 amiTHB1–11 (mt+, cw15), amiTHB1–14 (mt+, cw15) and amiTHB1–23 (mt+, cw15) . The 305 mutant (mt−nit1) affected in NAD(P) H-NR activity and without diaphorase-NR activity was originally obtained from the wild type 6145c (mt−) . The 305 and 6145 strains were kindly provided by Dr. E. Fernández (University of Cόrdoba, Spain).
Cells were grown mixotrophically in tris-acetate-phosphate (TAP) medium (https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/) containing 7.5 mM NH4Cl instead of NH4NO3 under continuous illumination with white light (fluence rate of 45 μmol m− 2 s− 1) at 22 °C with constant orbital agitation at 90 rpm. The TAP medium was supplemented with 100 mg L− 1 of arginine when required. Cells were collected at the midexponential phase of growth by centrifugation (4000 g, 5 min), washed twice with 10 mM potassium phosphate, pH 7, before being transferred to the induction media containing the different sources of nitrogen and chemicals. At each harvesting times the number of viable cells were counted microscopically with use of 0.05% (v/v) Evans blue (DIA-M, Russia) as described . Non-viable (stained) and viable (unstained) cells were counted. Four-hundred cells from each sample were scored for three biological replicates.
The compounds DEA-NONOate [2-(N, Ndiethylamino)-diazenolate 2-oxide sodium salt] and ODQ [1H-(1,2,4])oxadiazolo(4,3-a) quinoxalin-1-one] are from Sigma-Aldrich.
Gene expression analysis
The total RNA was isolated with Trizol according to the manufacturer’s instructions (Invitrogen, USA). To remove genomic DNA, the RNA samples were treated with RNase-Free DNase I (Fermentas). Subsequently, RNA concentration and purity (260/280 nm ratio) was determined using spectrophotometer (SmartSpec Plus, Bio-Rad).
Revert Aid HMinus First Strand cDNA Synthesis Kit (Thermo Scientific) was used for reverse transcription reaction. The primer pairs for RTqPCR are given in Additional file 1: Table S1. RT qPCR was performed with a CFX96 Real-Time PCR Detection System (Bio Rad) using SYBR Green I according to . Gene expression ratios were calculated with the ΔΔCt method . The RACK1 (receptor of activated protein kinase C; Cre13.g599400) gene was chosen as the control housekeeping gene. All reactions were performed in triplicate with at least three biological replicates. Significant differences between experiments were evaluated statistically by standard deviation and Student’s t-test methods.
Protein gel blot analysis
The protein content was determined with amido black staining and protein gel blot analysis was performed as described [33, 35]. After separation by SDS-PAGE on a 12% polyacrylamide gel (w/v), the proteins were transferred to nitrocellulose membranes (Carl Roth, Karlsruhe) with use of semidry blotting (Trans-blot SD BioRad). The dilutions of the primary antibodies used were as follows: 1:5,000 anti-CrPII and 1:2000 anti-HSP70B. As a secondary antibody, the horseradish peroxidase-conjugated anti-rabbit serum (Sigma) was used at a dilution of 1:10,000. The peroxidase activity was detected via an enhanced chemiluminescence assay (Roche). For quantification, films were scanned using Bio-Rad ChemiDocTMMP Imaging System, and signals were quantified using the Image LabTM software (version 5.1).
After eliminating the cells by centrifugation at 3000 g, nitrate concentrations in the medium were determined by dual-wavelength ultraviolet spectrophotometry as A220 - 2A275 using standard curve . For the measurements, media with 4 mM nitrate were diluted 50-fold. 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
Cells were treated with DEA-NONOate or nitrite, then they were incubated with in the presence of 1 μM (4-amino-5-methylamino-2′7’-difluorofluorescein diacetate) dye (DAF-FM DA, Sigma-Aldrich), at concentration of 45 μg/ml chlorophyll. After 15 min the cells were washed, resuspended in indicated medium and used for the fluorometric detection of NO. The supernatant was collected in a test tube and then used to detect NO in the medium. The measurement of NO was carried out with a microplate reader CLARIOstar (BMG) as described . The excitation and emission wavelengths for the NO indicator were 483 ± 14 and 530 ± 30 nm, respectively. Fluorescence intensity was calculated as arbitrary units per chlorophyll or protein as described previously .
NO detection by confocal microscopy
Cells were treated as described above. Images were acquired with a Leica TCS-SP5 confocal microscope (Leica-Microsystems, Germany) as described . All experiments were performed in triplicate.
GLB1 is induced by nitrate
Interestingly, GLB1 demonstrated a rather similar expression pattern to NIT1 (Fig. 1a, insertion). However, in contrast to GLB1, the higher levels of NIT1 up-regulation were reached when cells were exposed to 4 mM nitrate.
Next, we asked the question whether the nitrate-induced upregulation of GLB1 expression is accompanied by an increase in the PII protein. Compared to cells grown in ammonium, levels of PII were indeed higher in cells incubated in nitrate (Fig. 1c). The data suggest that the PII is induced by nitrate.
GLB1 transcription responds to the NH4 +/NO3 − balance
GLB1 is repressed by NO
Nitrite induces GLB1 gene and NR promotes NO-dependent GLB1 repression
Next, we asked the question whether the increased GLB1 mRNA levels correlate with a change in PII protein content in nitrite-induced cells. Although the kinetics of changes in GLB1 mRNA levels were not similar to the kinetics of changes in PII protein levels in both strains, the difference in PII protein abundance between parental strain 6145c and mutant 305 (Fig. 4b) was evident. The data suggest that GLB1 upregulation is dependent of nitrite. This result is in agreement with the fact that the NR mutant without diaphorase-NR activity did not show significant NO signal (Fig. 4c and d). In contrast to the mutant, very strong NO fluorescence appeared in the 6145c strain, supporting a correlation between NO generation and GLB1 mRNA abundance. Thus, these experiments allow nitrite to be added as a player in the control of GLB1 expression together with nitrate and NO. Taken together, the results strengthen the idea that control of GLB1 expression is regulated by a complex mechanism in which NO produced via NR/NOFNiR plays a crucial role.
THB1 controls GLB1 expression via the detoxification of NO
NO-dependent GLB1 repression is released by guanylate cyclase (GC) inhibitor
Plant chloroplasts contain cyanobacterial-like PII homologues . However, unlike PII proteins from cyanobacteria, plant PII proteins seem not to be covalently modified [20, 21]. Furthermore, in cyanobacteria, PII signaling is involved in the regulation of nitrate assimilation and gene expression through co-activator of the global nitrogen control factor NtcA . Importantly, no homologues of PipX and NtcA are conserved in plants . In representative plants, PII proteins are regulated at the transcriptional level [20, 21, 22]. In this study, we demonstrate that expression of Chlamydomonas PII is under the complex control of positive signals (i.e., nitrate and nitrite) and negative signals (NO), and GLB1 gene expression pattern is very similar to that observed for nitrate assimilation genes.
Ammonium is the preferred nitrogen source for Chlamydomonas. In ammonium-containing medium Chlamydomonas PII is expressed at low levels . Interestingly, the second major nitrogen source, nitrate, induces GLB1 transcription (Fig. 1). The cells might use this transcription regulation to limit PII levels under optimal nutritional conditions. It would thus be interesting to test whether GLB1 is strictly sensitive to ammonium or responds to a balance of ammonium and nitrate. We found that changes in the nitrate concentration modulate the response of GLB1 gene to ammonium (Fig. 2). Together these data stress the point that, a balance of positive and negative signals regulates GLB1.
A part of the response to ammonium/nitrate balance is a change in the intracellular concentration of NO . We have shown herein that NO represses expression of GLB1 (Fig. 3). Interestingly, GLB1 exhibits a similar transcription pattern to NIT1 (Fig. 3a, insertion) and other genes from the nitrate assimilation cluster [24, 38, 42, 43], suggesting that the expression of PII is tightly controlled with regard to the nitrogen source and the physiological state of cells. Physiological studies of Arabidopsis suggested a role of PII in nitrite uptake . In addition, GLB1 expression is up-regulated in the presence of nitrite (Fig. 4a). Together, these observations allow us to speculate that PII protein may also play some uncharacterized roles in control of nitrogen assimilation in Chlamydomonas cells.
In the cytoplasm, nitrite is converted into NO by NR that is partnered with NOFNiR . Thus, NR modulates both the levels of NO and the amounts of nitrite available for metabolism. Importantly, the diaphorase-NR activity is required for supplying NAD(P)H electrons to nitrite . We propose that GLB1 transcription is dependent on the dual system of NR and NOFNiR through fine tuning of NO levels. The fact that the diaphorase-NR activity is required to repress PII levels in the presence of nitrite (Fig. 4a; b) supports this idea. In agreement with these data, spectrofluorometric assays (Fig. 4c) and confocal microscopy (Fig. 4d) with DAF-FM DA allowed us to detect higher fluorescence levels in parental strain 6145c than nit1 mutant. More generally we could propose that nitrite-dependent NO production plays role in the control of PII expression dynamics, ensuring possible interconnecting/coordinating PII-dependent and nitrate assimilation pathways.
As NO is toxic, plants have protective mechanisms to defend themselves. Like higher plants, Chlamydomonas cells use hemoglobins to convert NO into nitrate [28, 45, 46]. It has been previously shown that a truncated hemoglobin 1, THB1, has NO-dioxygenase activity . In amiTHB1 strains, the nitrite-responsive accumulation of GLB1 transcripts is impaired (Fig. 5a). As expected, DEA-NONOate in nitrite-containing medium resulted to higher fluorescence levels in THB1-knockdown transformants than in parental strain (Fig. 5b). Taken together, these results strengthen the notion that NO acts as a signaling molecule for the transcriptional regulation of GLB1 gene, and THB1 is involved in this NO-dependent pathway.
Our main conclusion is that PII level is tightly controlled with regard to the nitrogen source and the physiological state of cells. We provide evidence on that NO via the components of nitric oxide cycle is involved in the negative control of GLB1. On the other hand, nitrate and nitrite induce this gene transcription. Therefore, important regulatory layer in the PII-dependent signal transduction system in Chlamydomonas could be that the concentration of the PII protein must be balanced in order for the signaling mechanism to function properly – the system is fine-tuned.
We thank Dr. Anton Radaev and the Core Facility “CHROMAS” of Saint-Petersburg State University for assistance with confocal microscopy. We also thank Dr. Michael Schroda (TU Kaiserslautern, Germany) for kind providing of HSP70B antibody and the strain cw15-325. The supply of strains 6145c and 305 by Dr. Emilio Fernández (University of Cόrdoba, Spain) is gratefully acknowledged.
This work was supported by Saint-Petersburg State University (research Grant No. 184.108.40.2067) to EE.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
ZZ and LK performed all of the biological analyses. ZZ, LK and EE analyzed the data. EE designed the study and wrote the manuscript. All authors approved the final version of the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 18.Smith CS, Morrice NA, Moorhead GB. Lack of evidence for phosphorylation of Biochim. Biophys Acta. 1699;2004:145–54.Google Scholar
- 23.Fernández E, Galván A, Quesada A. Nitrogen assimilation and its regulation. In: Rochaix JD, Goldschmidt-Clermont M, editors. Molecular Biology of Chlamydomonas: Chloroplast and Mitochondria. Dordrecht: Kluwer Academic Publishers; 1998. p. 637–59.Google Scholar
- 25.Muños-Blanco J, Hidalgo-Martinez J, Cárdenas J. Extracellular deamination of L-amino acids by Chlamydomonas reinhardtii cells. Planta. 1990;182:194–8.Google Scholar
- 32.Harris EH. The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. San Diego: Academic Press; 1989.Google Scholar
- 36.Dong G, Zhang W, Yang R, Yang Y, Yu Y, Zhang X. Determination of nitrate nitrogen in soil based on K ratio spectrophotometry. Int Conf Instr Meas. 2014. https://doi.org/10.1109/IMCCC.2014.117.
- 43.Loppes R, Ohresser M, Radoux M, Matagne RF. Transcriptional regulation of the Nit1 gene encoding nitrate reductase in Chlamydomonas reinhardtii: effect of various enviromental factors on the expression of a reporter gene under the control of the Nit1 promoter. Plant Mol Biol. 1999;41:701–11.CrossRefGoogle Scholar
- 46.Johnson EA, Rice SL, Preimesberger MR, Nye DB, Gilevicius L, Wenke BB, Brown JM, Witman JB, Lecomte JTJ. Characterization of THB1, a Chlamydomonas reinhardtii truncated hemoglobin: linkage to nitrogen metabolism and identification of lysine as the distal heme ligand. Biochemistry. 2014;53:4573–89.CrossRefGoogle Scholar
- 49.Gonzalez-Ballester D, Sanz-Luque E, Galvan A, Fernandez E, de Montaigu A. Arginine is a component of the ammonium-CYG56 signalling cascade that represses genes of the nitrogen assimilation pathway in Chlamydomonas reinhardtii. PLoS One. 2018. https://doi.org/10.1371/journal.pone.0196167.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.