Figures
Abstract
Nitrogen assimilation is strictly regulated in cyanobacteria. In an inorganic nitrogen-deficient environment, some vegetative cells of the cyanobacterium Anabaena differentiate into heterocysts. We assessed the photosynthesis and nitrogen-fixing capacities of heterocysts and vegetative cells, respectively, at the transcriptome level. RNA extracted from nitrogen-replete vegetative cells (NVs), nitrogen-deprived vegetative cells (NDVs), and nitrogen-deprived heterocysts (NDHs) in Anabaena sp. strain PCC 7120 was evaluated by transcriptome sequencing. Paired comparisons of NVs vs. NDHs, NVs vs. NDVs, and NDVs vs. NDHs revealed 2,044 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs showed that carbon fixation in photosynthetic organisms and several nitrogen metabolism-related pathways were significantly enriched. Synthesis of Gvp (Gas vesicle synthesis protein gene) in NVs was blocked by nitrogen deprivation, which may cause Anabaena cells to sink and promote nitrogen fixation under anaerobic conditions; in contrast, heterocysts may perform photosynthesis under nitrogen deprivation conditions, whereas the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Immunofluorescence analysis of nitrogenase iron protein suggested that the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Our findings provide insight into the molecular mechanisms underlying nitrogen fixation and photosynthesis in vegetative cells and heterocysts at the transcriptome level. This study provides a foundation for further functional verification of heterocyst growth, differentiation, and water bloom control.
Citation: He H, Miao R, Huang L, Jiang H, Cheng Y (2021) Vegetative cells may perform nitrogen fixation function under nitrogen deprivation in Anabaena sp. strain PCC 7120 based on genome-wide differential expression analysis. PLoS ONE 16(3): e0248155. https://doi.org/10.1371/journal.pone.0248155
Editor: Anil Kumar Singh, ICAR-Indian Institute of Agricultural Biotechnology, INDIA
Received: August 28, 2020; Accepted: February 20, 2021; Published: March 4, 2021
Copyright: © 2021 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This study was supported by grants from the National Natural Science Foundation of China (No. 31670681; 31770723).
Competing interests: The authors declare that they have no conflict of interest.
Introduction
Cyanobacteria evolved approximately 3.5 billion years ago. As the first photosynthetic prokaryotes on earth, cyanobacteria can efficiently fix atmospheric nitrogen via a process catalyzed by nitrogenase [1]. Nitrogenase-catalyzed substrate reduction requires the association of an iron (Fe) protein and molybdenum-iron (MoFe) protein, with electron transfer occurring from the Fe protein to the MoFe protein. However, nitrogenase responsible for the reduction of N2 is extremely sensitive to O2 [2–5]. How to avoid oxygen released during photosynthesis by nitrogenase has attracted much attention. Cyanobacteria need to segregate the timing of nitrogen fixation from oxygenic photosynthesis by restricting nitrogen fixation to the dark period of diel cycles or spatially separate these processes by restricting nitrogen fixation to heterocysts [6, 7]. Under nitrogen-deprivation conditions, heterocysts differentiate from vegetative cells only in the filaments of cyanobacteria [8]. It is well-known that vegetative cells perform photosynthetic functions, whereas heterocysts perform nitrogen fixation functions in cyanobacteria. However, whether both cell types perform photosynthesis and nitrogen fixation simultaneously remains unclear.
All aerobic nitrogen fixation occurs in heterocysts in a semiregular pattern, and nitrogen fixed in heterocysts is transported to vegetative cells in the filament; vegetative cells supply carbon and reductants to heterocysts [6, 9]. The heterocyst is the site of dinitrogen fixation and provides oxygen-sensitive nitrogenase with a low-oxygen environment [10]. In general, heterocysts lack photosystem II activity and ribulose bisphosphate carboxylase, and they cannot photoreduce CO2 via the reductive pentose phosphate pathway to provide carbon skeletons for assimilation of fixed nitrogen [6, 11–13]. The heterocyst develops a special glycolipid layer that serves as a gas diffusion barrier, and the heterocyst glycolipid layer can be modified in response to the external O2 concentration [10, 14]. However, other studies suggested that heterocysts are not the only cells capable of nitrogen fixation in heterocystous cyanobacteria and that vegetative cells can fix molecular nitrogen using another nitrogenase encoded by a homologous gene cluster named as nif2 [15]. Mutants of heterocystous cyanobacteria (het−) that fail to produce heterocysts retain their nitrogen fixation ability when incubated under micro-oxic or anoxic conditions [16]. Thus, studies are needed to evaluate the nitrogen fixation ability of vegetative cells and photosynthetic ability of heterocysts in cyanobacteria.
Recent genome-wide studies have focused on cell-specific metabolism in cyanobacteria by comparing gene transcript levels under different growth conditions or stage-specific gene expression signatures in response to nitrogen step-down. RNA sequencing was used to study transcript expression levels in filaments at different periods of nitrogen step-down using a mixture of heterocysts and vegetative cells [17–19]. Using samples collected during the differentiation in three developmental states, a DNA microarray containing 6,893 gene fragments were was used to identify differentially expressed genes (DEGs) regulating the differentiation of heterocysts, akinetes, and hormogonia in the cyanobacterium Nostoc punctiforme [20]. Similarly, seven genes encoding transcriptional regulators that respond to nitrogen deprivation in Anabaena sp. strain PCC 7120 were identified by oligonucleotide microarray technology, and the results for an nrrA deletion mutant suggested that nrrA facilitates heterocyst development [21]. However, vegetative cells in the mycelium samples were not separated from heterocysts in the filamentous cyanobacteria. Therefore, the function of vegetative cells and heterocysts may have been biased in sequencing and bioinformatics analysis. Compared with vegetative cells, heterocysts cells of Anabaena have thicker walls and better resist adverse external environments, providing a theoretical basis for the separation and purification of the two types of cells. In general, two strategies are used to isolate and purify heterocysts from the mycelium: sonication treatment [22] or lysozyme treatment [23]. Vegetative cells easily crack during these treatments, resulting in the isolation of heterocysts without vegetative cells. Heterocysts can be isolated from mycelium containing both vegetative cells and heterocysts of Anabaena sp. strain 7120 with lysozyme for RNA-Seq analysis (Fig 1). We evaluated important candidate genes involved in nitrogen fixation and photosynthesis in vegetative cells and heterocysts from Anabaena sp. strain PCC 7120 at the transcriptome level and provide insight into the molecular mechanisms of nitrogen fixation and photosynthesis in both cell types.
(A1 and A2) Filaments only consisting of vegetative cells under nitrogen-replete condition. A2 is an enlarged view of black square in A1. (B1 and B2) Filaments consisting of vegetative cells and heterocysts under nitrogen deprivation condition. B2 is an enlarged view of black square in B1. (C1 and C2) The filaments were treated with lysozyme followed by centrifuge. The picture shows the mixture of heterocysts and residual vegetative cells in the sediment. The supernatant was used to extract mRNA from vegetative cells. C2 is an enlarged view of black square in C1. (D1 and D2) Pure heterocysts for RNA extraction. D2 is an enlarged view of black square in D1. Black and blue arrows show vegetative cells and heterocysts respectively.
Results
lllumina sequencing and clean read mapping
To evaluate entire gene expression profiles in nitrogen-deprived vegetative cells (NDVs), nitrogen-deprived heterocysts (NDHs), and nitrogen-replete vegetative cells (NVs), nine digital gene expression (DGE) profiling libraries of Anabaena sp. strain PCC 7120 were sequenced using the Illumina HiSeq 4000 platform, which generated approximately 26 million clean reads for each library (S1 Table), accounting for approximately 92% of the total obtained reads. In total, 243,139,646 clean reads were acquired, which were 36,714,086,546 bp in length; the average length of each clean read was 151 bp (S1 Table). For all reads in the nine libraries, statistical analysis of the distribution results of RNA-Seq mapped events showed that approximately 7%, 93%, 78%, and 1% mapped reads were queried against the intergenic regions, genes, mRNA, and rRNA, respectively, of the reference genome, suggesting that most reads were from gene sequences (S2 Table). For all nine libraries, clean read assembly generated 5,842 genes with a mean length of 988 bp.
Global gene expression in NDVs, NDHs, and NVs
We found that 5,808, 5,812, and 4,792 genes were expressed in NVs, NDVs, and NDHs, respectively. The number of expressed genes in NDHs was smaller than that in NVs and NDVs. These results suggest that more genes are necessary for the growth of vegetative cells than for the growth of heterocysts. Furthermore, the number of expressed genes in NVs and NDVs was similar. Fragments per kilobase of transcript per million mapped reads (FPKM) values were used to calculate gene expression levels; the results suggested that the average FPKM values of NDHs were lower than those of NVs and NDVs (Fig 2A). Consistent with these findings, the densities of NDH genes with medium and low FPKM values were lower and higher, respectively, than those of NV and NDV samples, suggesting relatively low expression of genes in NDHs (Fig 2B).
(A) The FPKM boxplot distribution is shown with a box plot. (B) FPKM density distribution for all transcripts. Note: FPKM, fragments per kilobase of transcript per million mapped reads. ‘NDV’ and ‘NDH’ refers to vegetative cells and heterocysts cultured respectively under nitrogen deprivation condition, and ‘NV’ refers to vegetative cells cultured under nitrogen-replete condition.
Identification of DEGs in three paired comparisons
In total, 2,044 DEGs were identified in NV vs. NDH, NV vs. NDV, and NDV vs. NDH comparisons (Fig 3A). Among these, 251, 420, and 149 DEGs were uniquely expressed in NDV vs. NDH, NV vs. NDH, and NV vs. NDV samples, respectively, and 116 DEGs were commonly expressed in these comparisons (Fig 3B).
(A) DEGs amount in each paired comparison; (B) Venn diagram of all identified DEGs. In three paired comparisons, X vs. Y refers to Y/X; ‘NDV’ and ‘NDH’ refers to vegetative cells and heterocysts cultured respectively under nitrogen deprivation condition, and ‘NV’ refers to vegetative cells cultured under nitrogen-replete condition.
Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis of DEGs
KEGG significant enrichment analysis of DEGs can reveal metabolic pathway and signaling pathway information. KEGG functional enrichment analysis of all DEGs was performed to determine the pathways involved in regulating the responses of vegetative cells and heterocysts to nitrogen deprivation conditions. Fourteen significantly enriched pathways were identified, and 7, 3, and 4 KEGG pathways were enriched in NV vs. NDH, NV vs. NDV, and NDV vs. NDH comparisons, respectively (Table 1). In the NDV vs. NDH comparison, five pathways were related to metabolism, including those related to carbohydrates, nucleotides, and biosynthesis of other secondary metabolites. In the NV vs. NDV comparison, two pathways were involved in energy and amino acid metabolism and one pathway was involved in replication and repair. In the NDV vs. NDH comparison, three pathways were related to the metabolism of cofactors and vitamins, terpenoids and polyketides, and nucleotides. These results suggest that most DEGs were involved in metabolism, carbon fixation, and the synthesis of important nitrogenous biomacromolecules including, but not limited to, arginine, DNA, and purine, providing important insight into screening of candidate genes involved in regulating vegetative cells and heterocysts in response to nitrogen deprivation conditions.
To determine the main biological functions of the identified DEGs, GO functional enrichment analysis of the DEGs was performed. All 2,044 DEGs were assigned to three GO categories, including biological processes, cellular components, and molecular functions (Fig 4). Most of these DEGs were involved in the following functions: cofactor transferase activity, cofactor binding, substrate-specific transporter activity, catalytic activity, acting on a protein, and transition metal ion binding. Moreover, most DEGs contributed to cellular and metabolic processes, including those pertaining to organic cyclic compounds, heterocyclic compounds, small molecules, and phosphorus. Most corresponding proteins were located in the cell membrane including the intrinsic and integral parts of the membrane.
(A) NDV vs. NDH; (B) NV vs. NDV; (C) NV vs. NDH. “Rich factor” means that the ratio of the DEGs number and the number of genes have been annotated in this term. The greater of the Rich factor, the greater the degree of enrichment. The different color represents different false discovery rate (FDR) values. ‘NDV’ and ‘NDH’ refers to vegetative cells and heterocysts cultured respectively under nitrogen deprivation condition, and ‘NV’ refers to vegetative cells cultured under nitrogen-replete condition. In three paired comparisons, X vs. Y refers to Y/X.
Cluster analysis of DEGs
To explore the relationship between the expression pattern of these DEGs and their biological functions, we performed cluster analysis of all identified DEGs. Hierarchical clustering analysis of the 2,044 DEGs in NV vs. NDH, NV vs. NDV, and NDV vs. NDH paired comparisons indicated that they could be classified into nine clusters, and DEGs belonging to a given cluster showed a similar expression pattern (Figs 4B and 5A). Among all DEGs, a few highly expressed DEGs were found in clusters 1, 4, 5, and 6. Many DEGs in cluster 1 were highly expressed in NDVs and NDHs, whereas their expression in NVs was relatively lower, and most highly expressed DEGs (|log2fold-change| > 3) were involved in nitrogen metabolism. The expression levels of highly expressed DEGs (|log2fold-change| > 3) in clusters 4, 5, and 6 differed in the NDH and NDV samples; some were involved in gas vesicle protein biosynthesis. Among the highly expressed DEGs belonging to clusters 6, 7, and 9, genes encoding phosphate ABC transporter permease, transposase, and cation efflux system proteins were strongly downregulated in the NDH sample.
(A) Heat map of the hierarchical cluster analysis of gene expression in NDH, NDV and NV. Each row represents a single gene. Each column represents a single sample. Green squares indicate transcript levels below the mean; black squares, transcript levels equal to the mean; red squares, transcript levels greater than the mean; gray squares, technically inadequate or missing data. Colored bars leftmost adjacent to groups in which the cluster of genes is expressed. (B) Trend analysis of different clusters. The gray line in the figure shows the expression pattern of different DEGs in each cluster. The blue line represents the average expression level of all DEGs in the cluster in different samples. ‘NDV’ and ‘NDH’ refers to vegetative cells and heterocysts cultured respectively under nitrogen deprivation condition, and ‘NV’ refers to vegetative cells cultured under nitrogen-replete condition.
DEG validation by qRT-PCR and RT-PCR
To validate the DEGs identified in the NV vs. NDH, NV vs. NDV, and NDV vs. NDH paired comparisons by RNA-Seq technology, four DEGs that may be involved in nitrogen fixation and photosystems were selected for validation by qRT-PCR analysis, and two DEGs that may be involved in nitrogen fixation were selected for validation by RT-PCR analysis. In general, the log2 (fold-change) values obtained using the Illumina sequencing platform were consistent with those obtained by qRT-PCR analysis (Fig 6). Results of RT-PCR tests were shown in S1 Fig. These results indicate that our mRNA sequencing results were reliable.
The relative expression levels of four chosen DEGs were obtained by RNA-seq and qRT-PCR. Bars represent mean ± standard deviation (n = 3). Different low case letter above each column indicated significant difference at P = 0.05. ‘NDV’ and ‘NDH’ refers to vegetative cells and heterocysts cultured respectively under nitrogen deprivation condition, and ‘NV’ refers to vegetative cells cultured under nitrogen-replete condition. In three paired comparisons, X vs. Y refers to Y/X.
Immunofluorescence analysis of nitrogenase iron protein (NifH)
4’,6-Diamidino-2-phenylindole (DAPI) and fluorescein 5-isothiocyanate (FITC) were used to visualize NifH expression in the filaments of Anabaena sp. strain PCC 7120. DAPI is a fluorescent dye that can tightly combine with DNA. Our second antibody of NifH was labeled with FITC. DAPI and FITC emitted blue and green fluorescence after excitation respectively. Under nitrogen-replete condition, filaments consisting of only vegetative cells (Fig 7A1–7A4), and weak green fluorescence of vegetative cells were detected (Fig 7A3). Under nitrogen deprivation conditions, in filaments consisting of vegetative cells and heterocysts, there was one heterocyst in every approximately 10 cells (Fig 7B1–7B4). Compared with vegetative cells cultured under nitrogen-replete conditions, the green fluorescence intensity of vegetative cells cultured under nitrogen deprivation conditions was clearly stronger (Fig 7A3 and 7B3), indicating that nitrogen deprivation induced the expression of NifH. Under nitrogen deprivation conditions, the green fluorescence of vegetative cells was not lower than that of heterocysts. Thus, there was almost no difference in the NifH expression level between vegetative cells and heterocysts under nitrogen deprivation conditions.
(A1-A4) Filaments only consisting of vegetative cells under nitrogen-replete condition. A1, vegetative cells under white light; A2 and A3, vegetative cells was excited at 365 nm (DAPI excitation wavelength) and 488 nm (FITC excitation wavelength) respectively; A4, merged imagine of A2 and A3. (B1-B4) Filaments consisting of vegetative cells and heterocysts under nitrogen deprivation condition. B1, vegetative cells and heterocysts under white light; B2 and B3, vegetative cells and heterocysts were excited at 365 nm and 488 nm respectively; B4, merged imagine of B2 and B3. Double fluorescent labeling were used in the experiment. DAPI was used to visualize the DNA, and FITC was used to visualize NifH. DAPI, 4’, 6-diamidino-2-phenylindole; FITC, fluorescein 5-isothiocyanate. In B1-B4, arrows show locations of heterocysts; the large square frame is an enlarged view of the small square frame.
Discussion
Nitrogen deprivation induces heterocyst differentiation and inhibits gas vesicle development in vegetative cells
Gas vesicles are gas-filled prokaryotic organelles that function as flotation devices [24–26]. A range of bacteria and archaea produces intracellular gas-filled proteinaceous structures to maintain a suitable depth in an aqueous environment [24]. Under conditions of nitrogen-replete conditions, cyanobacterial filaments consisted of only vegetative cells (Fig 1A); under nitrogen deprivation conditions, these filaments consisted of both vegetative cells and heterocysts (Fig 1B). These results suggest that nitrogen deprivation induced heterocyst differentiation, which is consistent with previously reported findings [27]. In total, we identified eight DEGs encoding the gas vesicle protein (Table 2). In the comparison of NVs vs. NDVs, six DEGs were highly downregulated, suggesting that nitrogen deprivation inhibits the expression of gas vesicle genes in vegetative cells; these results are consistent with most of the results obtained previously on gas vesicle protein regulation [28]. Furthermore, in the NDV vs. NDH comparison, three DEGs encoding gas vesicle genes were upregulated, suggesting that the expression of gas vesicle genes is higher in heterocysts than in vegetative cells under nitrogen deprivation conditions (Table 2). Thus, the density of vegetative cells increases via inhibition of gas vesicle protein expression under nitrogen deprivation conditions. Our results are consistent with the phenomenon of water bloom under eutrophication conditions [29, 30] and improve the understanding of the formation mechanism of water blooms at the transcription level.
Heterocysts may perform photosynthetic functions under nitrogen deprivation conditions
In heterocyst-forming cyanobacteria, thylakoid membranes in vegetative cells contain photosystem I (PS-I) and PS-II, which use water as the electron donor and produce oxygen. Whether heterocysts have both PS-I and PS-II remains controversial [31]. Thylakoid membranes in vegetative cells have been suggested to contain PS-I and PS-II, whereas those in heterocysts predominantly contain PS-I [32]. The water-oxidizing activity of PS-II, as well as fluorescence emission at 685 nm associated with PS-II, has been reported to be absent in intact heterocysts from several filamentous strains [33, 34]. The reaction center proteins of PS-II were suggested to be degraded during heterocyst differentiation, although PS-II proteins can still be found in mature heterocysts [35–37]. In our study, KEGG enrichment analysis showed that carbon fixation in the photosynthetic pathway (ko00710) was significantly enriched in the NV vs. NDV comparison, suggesting that nitrogen deprivation alters the photosynthetic capability of Anabaena sp. strain PCC 7120 (Table 1). We also detected the expression of 13 and 19 genes encoding PS-I and PS-II proteins, respectively (S3 Table), including two DEGs encoding PS-I and five DEGs encoding PS-II. Comparison of NV vs. NDV suggested that nitrogen deprivation inhibited the expression of both genes encoding PS-I and PS-II, as shown by downregulation of psaB/K in PS-I and psbN in PS-II. Under nitrogen deprivation conditions, the expression of PS-I and PS-II genes was more active in heterocysts than in vegetative cells, shown by upregulation of isiA (PS-II), psbAII (PS-II), psbAIII (PS-II), psbAIV (PS-II), and psbN (PS-II). Thus, both PS-I- and PS-II-related genes were expressed in heterocysts, which is not consistent with the results of a previous study reporting that heterocysts lack PS-II [31–35].
Nitrogen fixation capability of vegetative cells is promoted by nitrogen deprivation
Heterocysts differentiate from vegetative cells of some filamentous cyanobacteria to fix nitrogen for the entire filament under oxic growth conditions [38]. Anabaena sp. strain PCC 7120 contains two Mo-dependent nitrogenases [38, 39]. Among these, nif1 is expressed exclusively in heterocysts and functions under oxic growth conditions [40], whereas nif2 is expressed only under anoxic conditions in vegetative cells shortly after nitrogen step-down and long before heterocysts are formed [38]. Both nitrogenases fix nitrogen and supply it to the filament for growth [40]. We identified 23 nitrogen fixing genes, 21 of which were differentially expressed, including 10 highly-modulated DEGs (S4 Table). GO enrichment analysis of the DEGs showed that most of these genes encoded proteins with cofactor transferase activity, cofactor binding, and catalytic activity (Fig 4); these results are consistent with the properties of the nitrogenase-processing Mo cofactor. KEGG enrichment analysis indicated that multiple nitrogen-related KEGG pathways were significantly enriched, including amino sugar and nucleotide sugar metabolism (ko00520), purine metabolism (ko00230), DNA replication (ko03030), and arginine biosynthesis (ko00220) (Table 1). Cluster analysis revealed that the DEGs were grouped into cluster 1, indicating higher gene expression in NDV and NDH samples than in the NV sample. Comparison of NVs vs. NDVs showed that FKXN, NifD, NifE, NifH1, NifK, NifN, and NifX were highly expressed in the NDV sample (S4 Table), suggesting that this set of DEGs mainly contributed to promoting nitrogen fixation under nitrogen deprivation conditions. Immunofluorescence analysis of NifH indicated that its expression in vegetative cells was low when the nitrogen supply is sufficient; moreover, nitrogen deprivation induced and promoted the expression of NifH in both vegetative cells and heterocysts (Fig 7). These results confirm the nitrogen fixation function of this set of DEGs in NDVs.
Conclusion
Overall, our data confirm that directional RNA deep sequencing is a more thorough method of analyzing transcriptional regulation in heterocysts and vegetative cells by cell separation procedures, providing a systematic view of transcriptome level differences between heterocysts and vegetative cells under conditions of nitrogen step-down. Our results show that heterocysts and vegetative cells function by coordinating activities related to photosynthesis and nitrogen fixation. This direct comparison of transcript levels in NVs, NDVs, and NDHs revealed many characterized genes that are differentially regulated in the three cell types which should be further studied in heterocyst-forming cyanobacteria under nitrogen deprivation conditions.
Materials and methods
Experimental strain and treatments
Anabaena sp. strain PCC 7120 is a model strain useful for performing molecular studies of cell differentiation and heterocyst-forming cyanobacteria; this strain was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology with accession number of FACHB-418. The initial strain was cultured in Blue-Green medium (BG11) for 3 days, and then centrifuged and used in subsequent experiments. Nitrogen-replete medium (BG11) [41] and nitrogen deprivation medium (BG110) were used to culture PCC 7120 at an initial inoculation concentration OD730 of 0.1. Before inoculation, the strain was centrifuged and washed three times with BG11 or BG110 for purification and enrichment. The culture period was 72 h. To maintain the relative stability of nutrient elements in the culture medium, the strains were centrifuged and replaced with fresh culture medium every 24 h. After 72 h of culture, the strain OD730 in BG11 medium and BG110 medium were 0.997 and 0.621, respectively. The strain was cultured in 250-mL flasks sealed with plastic ventilating film, and the culture volume was 100 mL. PCC 7120 cultivation was carried out in a double layer shaping incubator under the following conditions: light of 300 μmol·m−2·s−1, 30°C, and shaking at a speed of 140 rpm. All centrifugation steps were performed at 500 ×g for 5 min. After 72 h of cultivation, the formation of vegetative cells and heterocysts in the filaments was confirmed by laser scanning confocal microscopy (TCS SP2, Leica Microsystems, Wetzlar, Germany) (Fig 1A1, 1A2, 1B1 and 1B2).
RNA extraction from vegetative cells and heterocysts
In total, three RNA samples (NDV, NDH, and NV) were used for extraction, each with three biological replicates. “NDV” refers to vegetative cells of PCC 7120 cultured in BG110 (N-deficient medium), “NDH” refers to heterocysts of the PCC 7120 strain cultured in BG110, and “NV” refers to vegetative cells of the PCC 7120 strain cultured in BG11 (nitrogen-replete medium).
RNA was extracted from NDVs and NVs as described by Golden et al [42] with some modifications: 500 mL NDVs and NVs was sedimented at 500 ×g for 5 min at 4°C. To ensure that only vegetative cells were broken down, the concentration of lysozyme was low and enzymolysis time was short. The precipitate was resuspended in 1 mL lysozyme solution (1 mg/mL lysozyme in TE buffer) at 30°C for 5 min, shaking each tube for 15 s at 1-min intervals. Next, 5 mL of guanidine isothiocyanate cell lysis solution (mixed solution in the EASYspin Plus Bacteria RNA kit, Aidlab, Beijing, China) was added to terminate the reaction as quickly as possible. This solution was centrifuged to remove the precipitate, and the supernatants were used for RNA isolation according to the instructions of the EASYspin Plus Bacteria RNA kit (Aidlab). The sediment obtained after this treatment contained vegetative cells and heterocysts (Fig 1C1 and 1C2).
NDHs were purified and enriched using as described by Golden et al [42] with some modifications: 500 mL NDHs was cultured in BG110 for 72 h and sedimented at 500 ×g for 5 min at 4°C. To eliminate contamination by vegetative cells, NDHs were treated with a high concentration of lysozyme, Triton X-100, and long-term cell lysis to ensure that all vegetative cells were completely lysed. The precipitate was re-suspended in 50 mL lysozyme solution for 1 h at 25°C (5 mg/mL lysozyme and 0.1% Triton X-100 in TE buffer). Most vegetative cells were lysed during this period, whereas the heterocysts remained intact, as determined by laser scanning confocal microscopy (Leica TCS SP2) (Fig 1D1 and 1D2). The microscopy results showed that heterocysts were mixed with impurities produced by lysed vegetative cells because of the strong adhesion of heterocysts (Fig 1D1 and 1D2). This phenomena were also observed during heterocyst isolation by Park et al [22], who suggested that the impurities were ruptured remains of vegetative cell or heterocyst protoplasts, and heterocysts mixed with cell contents were used to extract RNA from the heterocysts. To eliminate possible RNA residue from the ruptured remains of vegetative cells, the heterocyst-containing sediment was re-suspended in 500 μL TE buffer containing 50 μg/mL RNAase at 37°C for 1 h, followed by three washes with TE buffer. Finally, the purified heterocysts were mixed with mill silica sand (0.6–1.5 mm), ground into powder in liquid nitrogen, and diluted in deionized water for RNA extraction according to the instructions of the kit mentioned previously.
RNA extraction, library construction, and sequencing
To investigate the changes in gene expression in NDVs, NDHs, and NVs, nine DGE profiling libraries were constructed using RNA samples of NDV1, NDV2, NDV3, NDH1, NDH2, NDH3, NV1, NV2, and NV3. RNA pretreatment was performed before sequencing, including rRNA depletion, RNA fragmentation, cDNA synthesis, end repair, A-tailing, adapter ligation, and PCR (S1 File). rRNA depletion was performed using the Ribo-Zero Magnetic Kit for bacteria (Epicentre Biotechnologies, Madison, WI, USA). The samples were then cleaned using RNAclean XP beads (Beckman Coulter, Brea, CA, USA). The RNA was fragmented into 130–170 nt by adding fragmentation buffer (Ambion, Austin, TX, USA) to the samples and incubating the samples at 70°C. The samples were purified again with RNAclean XP beads. Purified RNA was used for cDNA synthesis. First-strand cDNA was synthesized using First Strand Master Mix and Super Script II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The mixture was incubated at 42°C for 50 min followed by inactivation at 70°C for 15 min. Using second-strand Master mix, second-strand cDNA was synthesized. Before PCR, end repair and poly-A tail addition was performed using End Pair Repair Mix and A-tailing mix simultaneously. RNA index adapters were added to the adenylated 3′ end of the DNA using T4 DNA ligase. This DNA sample was used for PCR amplification, which was performed using PCR Master Mix and a PCR primer cocktail, for several rounds. The library was validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to determine the average molecular length. The profiling libraries were sequenced on a HiSeq 4000 system (Illumina, San Diego, CA, USA) at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). The raw transcriptome data were deposited into the NCBI sequence read archive under accession number SAMN12877542-12877550.
Data analysis and mapping of DGE tags
After filtering reads with an adaptor at the 3′ end and quality values less than Q20 using Cutadapt [43], clean reads were assembled. Functional annotation was performed using five databases including Non-Redundant Protein Sequence Database (NR), GO, KEGG, and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggnog) databases [44]. Three pairs of DEG profiles for different sample libraries (NV vs. NDH, NV vs. NDV, and NDV vs. NDH; in each paired comparison, the former was used as a control and the latter as the experimental group) were compared to identify DEGs in vegetative cells and heterocysts of Anabaena sp. strain PCC 7120.
After assembly, the clean reads were mapped to the reference Nostoc sp. PCC 7120 genome (https://www.ncbi.nlm.nih.gov/genome/genomes/13531. BioSample = SAMD00061094, BioProject = PRJNA244; BioSample = SAMN10102199, BioProject = PRJNA492407) and gene expression levels were calculated for each sample. DEGs were identified using the DESeq R package (version 1.18.0) using thresholds of log2|fold-change| >1 and P-value < 0.05. KEGG pathway enrichment analysis of the DEGs was performed using BLAST by searching the KEGG database (http://www.kegg.jp/kegg/). P ≤ 0.05 was set as the threshold for significant enrichment of KEGG pathways. GO functional enrichment of DEGs was performed using the topGO R package [45], and terms with a P-value less than 0.05 were regarded as significant. Hierarchical clustering analysis of the DEGs was performed using Multi Experiment Viewer (http://mev.tm4.org/#/welcome).
qRT-PCR analysis
DEGs obtained by Illumina sequencing were verified by qRT-PCR. The RNA samples used for qRT-PCR were identical to those used for the DEG experiments. cDNA was synthesized using the TUREscript One Step RT-PCR Kit (Aidlab) according to the manufacturer’s instructions. Primers were designed using Premier 6.0 software (Premier Biosoft, Palo Alto, CA, USA) and synthesized commercially (Sangon Biotech Shanghai Co., Ltd., Shanghai, China) (S5 Table). The 20-μL reaction mixture contained 1 μL cDNA template, 1 μL each primer (10 μM), and 10 μL 2× SYBR Green qPCR (Aidlab); qRT-PCR was performed using the 2× SYBR Green qPCR kit (Aidlab) according to the manufacturer’s protocol, and relative expression levels of the genes were calculated by the 2−ΔΔCT method [46]. In this calculation, the relative mRNA levels of target genes were normalized by rnpB [47] using the ΔCT method [ΔCT = av CT(target gene)–av CT (rnpB)]. To determine the fold-change, gene expression in NVs was used as a control, and the results were calculated using the ΔΔCT (comparative threshold cycle) method: ΔΔCT = (av CT(target gene)–av CT(rnpB))sample–(av CT(target gene)–av CT(rnpB))control. In three paired comparisons, X vs. Y refers to Y/X. Y was used as the target gene and X was used as a control.
Immunofluorescence analysis of nitrogenase iron protein
Immunofluorescence analysis of nitrogenase iron protein was performed as previously described with minor modifications [48]. After 72 h culture under nitrogen-replete and deprivation conditions, strain PCC 7120 was fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 25°C. The samples were rinsed three times with PBS containing 0.1% Triton X-100 (PBST), and then incubated for 5 min in PBST at 25°C. The samples were incubated for 1 h with 6 μg/mL affinity-purified anti-NifH antibody (Agrisera, Vaesterbotten, Sweden) diluted in PBST containing 1 mg/mL bovine serum albumin and three washes with PBST. The samples were incubated in the dark with rabbit anti-chicken IgY conjugated to FITC (Sigma, St. Louis, MO, USA) for 45 min and then washed twice with PBST. The samples were mounted in Vectashield medium containing DAPI (Vector Laboratories, Burlingame, CA, USA H-1500) and viewed under a laser scanning confocal microscope (Leica TCS SP2).
Supporting information
S1 Fig. NifH1 and nifH2 gene expression analysis by RT-PCR.
https://doi.org/10.1371/journal.pone.0248155.s002
(DOC)
S1 Table. RNA-seq reads of Anabaena sp. strain PCC 7120 mapped to reference genome.
https://doi.org/10.1371/journal.pone.0248155.s003
(DOCX)
S2 Table. Distribution statistics results of RNA-Seq mapped events for all reads.
https://doi.org/10.1371/journal.pone.0248155.s004
(DOCX)
S3 Table. The DEGs encoding PS I, PS II in three comparison pairs.
https://doi.org/10.1371/journal.pone.0248155.s005
(DOCX)
S4 Table. The DEGs encoding nitrogenase genes in three comparison pairs.
https://doi.org/10.1371/journal.pone.0248155.s006
(DOCX)
References
- 1. Soo RM, Hemp J, Parks DH, Fischer WW, Hugenholtz P. 2017. On the origins of oxygenic photosynthesis and aerobic respiration in Cyanobacteria. Science 355:1436–1440. pmid:28360330
- 2. Peterson RB, Wolk CP. 1978. High recovery of nitrogenase activity and of 55Fe-labeled nitrogenase in heterocysts isolated from Anabaena variabilis. Proc Natl Acad Sci USA 75:6271–6275. pmid:16592599
- 3. Martinez L, Silver MW, King JM, Alldredge AL. 1983. Nitrogen fixation by floating diatom mats: a source of new nitrogen to oligotrophic ocean waters. Science 221:152–154. pmid:17769213
- 4.
Rees DC, Kim J, Georgiadis M, Chan MK, Komiya H, Woo D, et al. 1993. Structures and functions of the nitrogenase proteins, p 83–88. In Palacios R, Mora J, Newton WE (ed), New horizons in nitrogen fixation. Springer, Dordrecht.
- 5. Howard JB, Rees DC. 2006. How many metals does it take to fix N2? A mechanistic overview of biological nitrogen fixation. Proc Natl Acad Sci USA 103:17088–17093. pmid:17088547
- 6. Berman-Frank I, Lundgren P, Chen YB, Kupper H, Kolber Z, Bergman B, et al. 2001. Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium. Science 294:1534–1537. pmid:11711677
- 7. Fredriksson C, Malin G, Siddiqui PJA, Bergman B. 2008. Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002. New Phytol 140:531–538.
- 8. Muro-Pastor AM, Hess WR. 2012. Heterocyst differentiation: from single mutants to global approaches. Trends Microbiol 20:548–557. pmid:22898147
- 9. Weare NM, Benemann JR. 1973. Nitrogen fixation by Anabaena cylindrica-I. Localization of nitrogen fixation in the heterocysts. Arch Mikrobiol 90:323–332.
- 10. Stal LJ. 2017. The effect of oxygen concentration and temperature on nitrogenase activity in the heterocystous cyanobacterium Fischerella sp. Sci Rep 7:5402. pmid:28710405
- 11. Zheng Z, Omairi-Nasser A, Li X, Dong C, Lin Y, Haselkorn R, et al. 2017. An amidase is required for proper intercellular communication in the filamentous cyanobacterium Anabaena sp PCC 7120. Proc Natl Acad Sci USA 114:e1405–e1412. pmid:28159891
- 12. Wolk CP. 1996. Heterocyst formation. Annu Rev Genet 30:59–78. pmid:8982449
- 13.
Wolk CP. 2000. Heterocyst formation in Anabaena, p 83–104. In YV B (ed), Prokaryotic development. American Society of Microbiology, Washington DC.
- 14. Molot LA. 2017. The effectiveness of cyanobacteria nitrogen fixation: Review of bench top and pilot scale nitrogen removal studies and implications for nitrogen removal programs. Environ Rev 25:292–295.
- 15. Thiel T, Lyons EM, Erker JC, Ernst A. 1995. A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium. Proc Natl Acad Sci USA 92:9358–9362. pmid:7568132
- 16. Spence DW, Stewart WDP. 1987. Heterocystless mutants of Anabaena PCC 7120 with nitrogenase activity. FEMS Microbiol Lett 40:119–122.
- 17. Flaherty BL, Van Nieuwerburgh F, Head SR, Golden JW. 2011. Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp strain PCC 7120 to combined-nitrogen deprivation. BMC Genomics 12:332. pmid:21711558
- 18. Flores E, Picossi S, Valladares A, Herrero A. 2019. Transcriptional regulation of development in heterocyst-forming cyanobacteria. Biochim Biophys Acta Gene Regul Mech 1862:673–684. pmid:29719238
- 19. Mitschke J, Vioque A, Haas F, Hess WR, Muro-Pastor AM. 2011. Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp PCC 7120. Proc Natl Acad Sci USA 108:20130–20135. pmid:22135468
- 20. Campbell EL, Summers ML, Christman H, Martin ME, Meeks JC. 2007. Global gene expression patterns of Nostoc punctiforme in steady-state dinitrogen-grown heterocyst-containing cultures and at single time points during the differentiation of akinetes and hormogonia. J Bacteriol 189:5247–5256. pmid:17483217
- 21. Ehira S, Ohmori M. 2010. NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp strain PCC 7120. Mol Microbiol 59:1692–1703.
- 22. Park JJ, Lechno-Yossef S, Wolk CP, Vieille C. 2013. Cell-specific gene expression in Anabaena variabilis grown phototrophically, mixotrophically, and heterotrophically. BMC Genomics 14:759. pmid:24191963
- 23. Privalle LS, Burris RH. 1983. Permeabilization of isolated heterocysts of Anabaena sp strain 7120 with detergent. J Bacteriol 155:940–942. pmid:6409890
- 24. Pfeifer F. 2012. Distribution, formation and regulation of gas vesicles. Nat Rev Microbiol 10:705–715. pmid:22941504
- 25. Walsby AE. 1994. Gas vesicles. Microbiol Rev 58:94–144. pmid:8177173
- 26. Klemer AR, Feuillade J, Feuillade M. 1982. Cyanobacterial blooms: carbon and nitrogen limitation have opposite effects on the buoyancy of Oscillatoria. Science 215:1629–1631. pmid:17788490
- 27. Zhang H, Wang S, Wang Y, Xu X. 2018. Functional overlap of hetP and hetZ in regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120. J Bacteriol 200:e00707–17. pmid:29440250
- 28. Konopka A, Kromkamp J, Mur LR. 1987. Regulation of gas vesicle content and buoyancy in light- or phosphate-limited cultures of Aphanizomenon flos-aquae (cyanophyta). J Phycol 23:70–78.
- 29. Ekvall MK, Martin JDLC, Faassen EJ, Gustafsson S, Lürling M, Hansson LA. 2013. Synergistic and species-specific effects of climate change and water colour on cyanobacterial toxicity and bloom formation. Freshwater Biol 58:2414–2422.
- 30. Levine SN, Lewis WM. 1987. A numerical model of nitrogen flxation and its application to Lake Valencia, Venezuela. Freshwater Biol 17:265–274.
- 31. Ferimazova N., Felcmanová K., Šetlíková E., Küpper H., Maldener I., Hauska G., et al. 2013. Regulation of photosynthesis during heterocyst differentiation in Anabaena sp. strain PCC 7120 investigated in vivo at single-cell level by chlorophyll fluorescence kinetic microscopy. Photosynth Res 116:79–91. pmid:23918299
- 32. Cardona T, Battchikova N, Zhang P, Stensjo K, Aro EM, Lindblad P, et al. 2009. Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme. Biochim Biophys Acta 1787:252–263. pmid:19366613
- 33. Tel-Or E, Stewart WDP. 1977. Photosynthetic components and activities of nitrogen-fixing isolated heterocysts of Anabaena cylindrica. Proc R Soc Lond B Biol Sci 198:61–86.
- 34. Thomas J. 1972. Relationship between age of culture and occurrence of the pigments of photosystem II of photosynthesis in heterocysts of a blue-green alga. J Bacteriol 110:92–95. pmid:4622907
- 35. Black K, Osborne B. 2004. An assessment of photosynthetic downregulation in cyanobacteria from the Gunnera-Nostoc symbiosis. New Phytol 162:125–132.
- 36. Kumazaki S, Akari M, Hasegawa M. 2013. Transformation of thylakoid membranes during differentiation from vegetative cell into heterocyst visualized by microscopic spectral imaging. Plant Physiol 161:1321–1333. pmid:23274239
- 37. Wang Y, Gao Y, Li C, Gao H, Zhang CC, Xu X. 2018. Three substrains of the cyanobacterium Anabaena sp. strain PCC 7120 display divergence in genomic sequences and hetC function. J Bacteriol 200:e00076–18. pmid:29686139
- 38. Thiel T, Pratte B. 2001. Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413. J Bacteriol 183:280–286. pmid:11114927
- 39. Pratte BS, Eplin K, Thiel T. 2006. Cross-functionality of nitrogenase components NifH1 and VnfH in Anabaena variabilis. J Bacteriol 188:5806–5811. pmid:16885448
- 40. Schrautemeier B, Neveling U, Schmitz S. 1995. Distinct and differently regulated Mo-dependent nitrogen-fixing systems evolved for heterocysts and vegetative cells of Anabaena variabilis ATCC 29413: characterization of the fdxH1/2 gene regions as part of the nif1/2 gene clusters. Mol Microbiol 18:357–369. pmid:8709854
- 41. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61.
- 42. Golden JW, Whorff LL, Wiest DR. 1991. Independent regulation of nifHDK operon transcription and DNA rearrangement during heterocyst differentiation in the cyanobacterium Anabaena sp strain PCC 7120. J Bacteriol 173:7098–7105. pmid:1938911
- 43. Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12.
- 44. Muller J, Szklarczyk D, Julien P, Letunic I, Roth A, Kuhn M, et al. 2010. eggNOG v2.0: extending the evolutionary genealogy of genes with enhanced non-supervised orthologous groups, species and functional annotations. Nucleic Acids Res 38:D190–D195. pmid:19900971
- 45.
Alexa A, Rahnenfuhrer J. 2010. topGO: enrichment analysis for gene ontology. R package version 2.8.
- 46. Cheng Y, Liu J, Zhang H, Ju W, Zhao Y, Geng W, et al. 2015. Transcriptome analysis and gene expression profiling of abortive and developing ovules during fruit development in hazelnut. Plos One 10:e0122072. pmid:25836368
- 47. Chua JPS, Wallace EJS, Yardley JA, Duncan EJ, Dearden PK, Summerfield TC. 2012. Gene expression indicates a zone of heterocyst differentiation within the thallus of the cyanolichen Pseudocyphellaria crocata. New Phytol 196:862–872. pmid:22931432
- 48. Geisler E, Bogler A, Rahav E, Bar-Zeev E. 2019. Direct detection of heterotrophic diazotrophs associated with planktonic aggregates. Sci Rep 9:9288. pmid:31243322