Fig 1.
Enrichment and purification heterocysts in Anabaena sp. strain PCC 7120.
(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.
Fig 2.
General distribution of gene expression in Anabaena sp. strain PCC 7120.
(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.
Fig 3.
Differentially expressed genes (DEGs) in three paired comparison of Anabaena sp. strain PCC 7120.
(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.
Table 1.
Significantly enriched KEGG pathway in three comparison pairs.
Fig 4.
The 20 most enriched GO terms of different expression genes in Anabaena sp. strain PCC 7120.
(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.
Fig 5.
Differential expressed genes cluster analysis of vegetative cells and heterocysts from Anabaena sp. strain PCC 7120.
(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.
Fig 6.
Validation of DEGs by qRT-PCR analysis in Anabaena sp. strain PCC 7120.
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.
Fig 7.
Immunofluorescence analysis of nitrogenase iron protein (NifH) in filaments of Anabaena sp. strain PCC 7120 under nitrogen deprivation and nitrogen-replete condition.
(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.
Table 2.
DEGs encoding gas vesicle proteins in three comparison pairs.