Membrane association of active genes organizes the chloroplast nucleoid structure

Abstract

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.

Keywords: chromatin; organellar genome; transcription.

Fig. 1.

Protein binding to chloroplast DNA is dominated by PEP transcriptional machinery. (A) Genome-wide pattern of protein occupancy on DNA detected by IPOD. Signal from IPOD in Col-0 wild-type 14-d-old plants was calculated in 500 genomic bins and plotted throughout the entire plastid genome. Genome annotation including genomic regions, positions of annotated genes (22), and names of selected individual genes is provided on top of the plot. Average enrichment from four independent biological replicates is shown. The light blue ribbon indicates SD. (B) Previously published genome-wide pattern of PEP binding to DNA (22). Signal enrichment from ptChIP-seq using αRpoB antibody in 14-d-old Col-0 wild-type plants was calculated in 500 genomic bins and plotted throughout the entire plastid genome. Average enrichment and SD from three independent biological replicates are shown. (C) Preferential protein occupancy on PEP gene promoters. IPOD and RpoB ptChIP-seq signal (22) from 14-d-old Col-0 wild-type was calculated in 10-bp genomic bins and plotted at psbA, psbE, psbB, and rbcL loci. Average signal from four (IPOD) or three (ptChIP-seq) independent biological replicates are shown. Ribbons indicate SD. Gray vertical lines indicate positions of the annotated promoters. Genome annotation is shown on Top. (D) Protein occupancy and PEP binding are significantly correlated. IPOD signal and RpoB ptChIP-seq signal (22) were compared on annotated genes. Data points are color-coded by function and show averages from three (ptChIP-seq) or four (IPOD) biological replicates. Error bars indicate SD. The blue line represents the linear regression model.

Fig. 2.

PEP-occupied genes have reduced DNA accessibility. (A) Genome-wide pattern of DNA accessibility detected by ptATAC-seq. Signal from ptATAC-seq in 14-d-old Col-0 wild-type plants was calculated and plotted in 250 bins covering the entire plastid genome. The Y axis represents the ratio of insertions in crosslinked nucleoid to insertions in purified (naked) DNA. Genome annotation including genomic regions, positions of annotated genes (22), and names of selected individual genes is provided on top of the plot. Average signal from eleven independent biological replicates is shown. Red shading indicates significant accessibility and blue shading indicates significant protection identified using a negative binomial model FDR ≤ 0.05. Individual biological replicates are shown in SI Appendix, Fig. S2A. (B) Identification of genomic bins with significant accessibility or protection. Negative binomial model (FDR ≤ 0.05) was used to identify genomic bins with significant enrichment (accessible) or depletion (protected) of Tn5 insertions. Regions with no significant change (FDR > 0.05) were identified as undetermined. Individual data points within boxplots are averages from eleven biological replicates. (C) Protected genomic regions have high PEP binding. Previously published RpoB ptChIP-seq signal (22) plotted on genomic bins identified as accessible, protected, or undetermined (B). Individual data points within boxplots are averages from three biological replicates.

Fig. 3.

Chloroplast nucleoid is organized by association with the membranes. (A) Workflow of the SOLINA. (B) Validation of SOLINA by western blot demonstrating presence of RbcL in the soluble (stroma) fraction and LHCB1 in the insoluble (membrane) fraction. Units of MNase and fractions are labeled on the bottom of the panel. S indicates supernatant and P indicates pellet. The star indicates a nonspecific band. (C) Genome-wide pattern of membrane association identified by SOLINA. Signal from SOLINA in Col-0 wild-type 4-d-old plants was calculated in 500 genomic bins and plotted throughout the entire plastid genome. Genome annotation including genomic regions, positions of annotated genes (22), and names of selected individual genes is provided on top of the plot. Average from three independent biological replicates is shown. The light green ribbon indicates SD.

Fig. 4.

Membrane association is correlated with PEP transcription. (A) Membrane association and PEP binding are significantly correlated. SOLINA signal and RpoB ptChIP-seq signal from 4-d-old plants were compared on annotated genes. Data points are color-coded by function and show averages from three biological replicates. Error bars indicate SD. The blue line represents the linear regression model. (B) Extended dark treatment affects the pattern of PEP binding to DNA. RpoB ptChIP-seq was performed using 14-d-old Col-0 wild-type plants collected during the day or after extended dark treatment and enrichment was calculated in 500 genomic bins and plotted throughout the entire plastid genome. Genome annotation including genomic regions, positions of annotated genes (22), and names of selected individual genes is provided on top of the plot. Average enrichments from three independent biological replicates are shown. Ribbons indicate SD. (C) Extended dark treatment affects the pattern of membrane association. SOLINA was performed on 14-d-old Col-0 wild-type plants collected during the day or after extended dark treatment and signal was calculated in 500 genomic bins and plotted throughout the entire plastid genome. Average enrichments from three independent biological replicates are shown. Ribbons indicate SD. (D) Changes in membrane association and PEP binding after extended dark treatment are significantly correlated. Changes in SOLINA signal and RpoB ptChIP-seq signal were compared on annotated genes between plants collected during the day and after extended dark treatment. Data points are color-coded by function and show averages from three biological replicates. Error bars indicate SD. The blue line represents the linear regression model.

Fig. 5.

Disruption of chloroplast development may reduce the overall level of PEP transcription. (A) Reduction of PEP binding to DNA in the chl27-t mutant. RpoB ptChIP-seq enrichment in 4-d-old plants was calculated in 500 genomic bins and plotted throughout the entire plastid genome. Genome annotation including genomic regions, positions of annotated genes (22), and names of selected individual genes is provided on top of the plot. Average signal from three independent biological replicates is shown. Ribbons indicate SD. (B) The chlm mutant has no impact on the pattern of PEP binding to DNA. RpoB ptChIP-seq enrichments in 4-d-old Col-0 wild type and the chlm mutant were compared on annotated genes. Data points are color-coded by function and show averages from three biological replicates. Error bars indicate SD. The blue line represents the linear regression model. The red line represents no differences. (C) The chl27-t mutant had an overall reduction of PEP binding to DNA. RpoB ptChIP-seq enrichments in 4-d-old Col-0 wild type and the chl27-t mutant were compared on annotated genes. Data points are color-coded by function and show averages from three biological replicates. Error bars indicate SD. The blue line represents the linear regression model. The red line represents no differences. (D) The sig2 mutant had a combination of overall and locus-specific reductions of PEP binding to DNA. RpoB ptChIP-seq enrichments in 4-d-old Col-0 wild type and sig2 assayed side-by-side with chlm and chl27-t mutants were compared on annotated genes. Data points are color-coded by function and show averages from three biological replicates. Error bars indicate SD. The blue line represents the linear regression model. The red line represents no differences. (E). RpoB and RpoC1 are expressed in chlm and chl27-t mutants. Western blot was performed with whole cell extracts from 4-d-old seedlings of Col-0 wild type, chlm, and chl27-t using anti-RpoB and anti-RpoC1 antibodies. Anti-Actin antibody was used as a loading control.

Fig. 6.

PEP transcription impacts DNA membrane association. (A) Genome-wide pattern of membrane association in sigma factor mutants. Signal from SOLINA in 4-d-old Col-0 wild-type, sig2, and sig6 plants was calculated in 1 kb genomic bins distributed throughout the entire plastid genome. Genome annotation including genomic regions, positions of annotated genes (22), and names of selected individual genes is provided on top of the plot. Average signal from three independent biological replicates is shown. Ribbons indicate SD. (B) Changes in membrane association are correlated with changes in PEP binding in the sig2 mutant. Changes in SOLINA signal and previously published RpoB ptChIP-seq signal (22) between 4-d-old Col-0 wild-type and sig2 mutant were compared on annotated genes. Data points are color-coded by function and show averages from three biological replicates. Error bars indicate SD. The blue line represents the linear regression model. (C) Changes in membrane association are correlated with changes in PEP binding in the sig6 mutant. Changes in SOLINA signal and previously published RpoB ptChIP-seq signal (22) between 4-d-old Col-0 wild-type and sig6 mutant were compared on annotated genes. Data points are color-coded by function and show averages from three biological replicates. Error bars indicate SD. The blue line represents the linear regression model.

Fig. 7.

Disruption of chloroplast development impacts DNA-membrane association. (A) Genome-wide pattern of membrane association in chlorophyll biosynthesis mutants, which disrupt chloroplast development and thylakoid membrane structure. Signal from SOLINA in 4-d-old Col-0 wild-type, chlm, and chl27-t plants was calculated in 1 kb genomic bins and plotted throughout the entire plastid genome. Genome annotation including genomic regions, positions of annotated genes (22), and names of selected individual genes is provided on top of the plot. Average signal from four independent biological replicates is shown. Ribbons indicate SD. (B) The chlm mutant affects the pattern of DNA membrane association. SOLINA signal in 4-d-old Col-0 wild type and the chlm mutant were compared on annotated genes. Data points are color-coded by function and show averages from four biological replicates. Error bars indicate SD. The blue line represents the linear regression model. The red line represents no differences. (C) The chl27-t mutant affects the pattern of DNA membrane association. SOLINA signal in 4-d-old Col-0 wild type and the chl27-t mutant were compared on annotated genes. Data points are color-coded by function and show averages from four biological replicates. Error bars indicate SD. The blue line represents the linear regression model. The red line represents no differences. (D) Changes in membrane association are correlated with changes in PEP binding in the chl27-t mutant. Changes in SOLINA signal and RpoB ptChIP-seq signal between Col-0 wild-type and chl27-t mutant were compared on annotated genes. Data points are color-coded by function and show averages from three or four biological replicates. Error bars indicate SD. The blue line represents the linear regression model. (E) Membrane association of annotated genes is reduced in the chlm and chl27-t mutants. Average SOLINA signal from four biological replicates was calculated on annotated genes grouped by their functions (tRNA, rRNA, and protein-coding). Individual biological replicates are shown in SI Appendix, Fig. S7A.