The YlmG protein has a conserved function related to the distribution of nucleoids in chloroplasts and cyanobacteria

Abstract

Background: Reminiscent of their free-living cyanobacterial ancestor, chloroplasts proliferate by division coupled with the partition of nucleoids (DNA-protein complexes). Division of the chloroplast envelope membrane is performed by constriction of the ring structures at the division site. During division, nucleoids also change their shape and are distributed essentially equally to the daughter chloroplasts. Although several components of the envelope division machinery have been identified and characterized, little is known about the molecular components/mechanisms underlying the change of the nucleoid structure.

Results: In order to identify new factors that are involved in the chloroplast division, we isolated Arabidopsis thaliana chloroplast division mutants from a pool of random cDNA-overexpressed lines. We found that the overexpression of a previously uncharacterized gene (AtYLMG1-1) of cyanobacterial origin results in the formation of an irregular network of chloroplast nucleoids, along with a defect in chloroplast division. In contrast, knockdown of AtYLMG1-1 resulted in a concentration of the nucleoids into a few large structures, but did not affect chloroplast division. Immunofluorescence microscopy showed that AtYLMG1-1 localizes in small puncta on thylakoid membranes, to which a subset of nucleoids colocalize. In addition, in the cyanobacterium Synechococcus elongates, overexpression and deletion of ylmG also displayed defects in nucleoid structure and cell division.

Conclusions: These results suggest that the proper distribution of nucleoids requires the YlmG protein, and the mechanism is conserved between cyanobacteria and chloroplasts. Given that ylmG exists in a cell division gene cluster downstream of ftsZ in gram-positive bacteria and that ylmG overexpression impaired the chloroplast division, the nucleoid partitioning by YlmG might be related to chloroplast and cyanobacterial division processes.

Figure 1

Phenotypes of the AtYLMG1-1 overexpressers. (A) Three-week-old seedlings of the FOX line (FN026 and FN028), and plants with a 35S promoter-At3g07430 transgene (35S-AtYLMG1-1). Chloroplasts in single leaf mesophyll cells of FN026, FN028, and the 35S-AtYLMG1-1 transgenic plant. Bars = 10 mm (left) and 10 μm (right). (B) The average of the chloroplast diameter is shown in each graph along with the standard deviation. n = 50. (C) Levels of the AtYLMG1-1 transcript in the wild type, FOX lines and the 35S-AtYLMG1-1 transgenic plants. Transcript levels were analyzed by RT-PCR in the wild type (lane 1 and 5), FN026 (lane 2 and 6), FN028 (lane 3 and 7), and the 35S-AtYLMG1-1 transgenic plants (lane 4 and 8). A micro litter (lane 1-4) or 0.1 μl (lane 5-8) of reverse-transcription product was used as the PCR template. GAPDH was used as the quantitative control. Triangle indicates the RT-PCR products of AtYLMG1-1, and asterisk indicates that of GAPDH. (D) Levels of the AtYLMG1-1 protein in the wild type, FOX line, and the 35S-AtYLMG1-1 transgenic plants. Total proteins extracted from 3-week-old seedling of the wild type (WT), FOX line (FN028), and the 35S-AtYLMG1-1 transgenic plants (35S-AtYLMG1-1) were analyzed with the anti-AtYLMG1-1 antibodies raised against a peptide fragment of AtYLMG1-1. Fifty micrograms of proteins were loaded in each lane. The Rubisco small subunit (Rubisco SSU) was detected by Coomassie brilliant blue (CBB) staining as the quantitative control. (E) Localization of FtsZ in the wild type and the AtYLMG1-1 overexpresser. Localization of FtsZ2-1 in mesophyll cells was examined under immunofluorescence microscopy. The green fluorescence shows the localization of FtsZ2-1 and the autofluorescence of chlorophyll is depicted in red. Bars = 5 μm.

Figure 2

Phylogenetic relationships in the YlmG family of proteins. (A) Amino acid sequence alignment of the YlmG family. The amino acid sequences were collected from the National Center for Biotechnology Information database. The alignment includes the YlmG family of proteins of A. thaliana (ATH), the red alga Cyanidioschyzon merolae (CME), the cyanobacteria S. elongatus PCC7942 (S7942), and S. pneumoniae (SPN). The locus IDs or GI numbers of the sequences are indicated with the name of the species. (B) Phylogenetic tree of the YLMG family. The tree shown is the maximum-likelihood tree constructed by the PHYML program [48]. The numbers at the selected nodes are posterior probabilities by the Bayesian inference (left) and local bootstrap values provided by the maximum-likelihood analysis (right). The tree includes proteins of photosynthetic eukaryotes; A. thaliana (ATH), Oryza sativa (OSA), Chlamydomonas reinharditii (CRE), Ostreococcus tauri (OTA), C. merolae (CME), Thalassiosira pseudonana (TPS), and Phaeodactylum tricornutum (PTR), apiconplexa; Plasmodium vivax (PVI) and Theileria annulata (TAN), cyanobacteria; Synechocystis sp. PCC 6803 (S6803), S. elongatus PCC7942 (S7942), Gloeobacter violaceus PCC 7421 (G7421), and Prochlorococcus marinus str. MIT 9312 (P9312), other bacteria; Escherichia coli (ECO), Bacillus subtilis (BSU), Streptococcus pneumoniae (SPN), Chlamydophila caviae (CCA), Rhizobium etli (RET), Rhodospirillum rubrum (RRU), Caulobacter sp. K31 (C-K31), Chloroflexus aggregans (CAG), Chromohalobacter salexigens (CSA), and Pseudomonas syringae (PSY). The locus IDs or GI numbers of the sequences are shown with the name of the species. White boxes indicate non-photosynthetic organisms. * indicates proteins whose gene disruptants showed no effects on the activity of the photosystems, while ** indicates proteins whose gene disruptants reduced the photosystem activity [29,30,32]. Posterior probabilities and bootstrap values for all branches are shown in Additional file 1.

Figure 3

Phenotype of the AtYLMG1-1 knockdown plant. (A) Three-week-old seedlings of the wild type (WT) and the AtYLMG1-1 knockdown line (AS#1 and AS#2). Bars = 10 mm. (B) Levels of AtYLMG1-1 protein in the wild type and the two AtYLMG1-1 knockdown lines. Total proteins extracted from the wild type (WT) and the two AtYLMG1-1 knockdown lines (AS#1 and AS#2) were analyzed with the anti-AtYLMG1-1 antibodies. Fifty micrograms of proteins were loaded in each lane. The Rubisco small subunit (Rubisco SSU) was detected by CBB staining as the quantitative control. (C) Levels of AtYLMG1-1 and other YLMG gene transcripts in the wild type and two independent AtYLMG1-1 knockdown lines (AS#1 and AS#2). The levels of AtYLMG1-1, AtYLMG1-2, AtYLMG2, and AtYLMG3 transcripts were analyzed by RT-PCR in the wild type, AS#1, and AS#2. UBQ1 was used as the quantitative control. (D) Chloroplasts in leaf mesophyll cells of the wild type and the AtYLMG1-1 knockdown line. Chloroplasts in expanding leaf cells and the basal part of expanding leaf cells of the wild type (WT) and the AtYLMG1-1 knockdown line (AS#1) are shown. Bars = 10 μm. (E) Localization of FtsZ in the wild type and the AtYLMG1-1 knockdown line (AS#1). Localization of FtsZ2-1 in mesophyll cells was examined by immunofluorescence microscopy. The green fluorescence shows the localization of FtsZ2-1 and the autofluorescence of chlorophyll is depicted in red. Bars = 5 μm.

Figure 4

Effects of the overexpression and knockdown of AtYLMG1-1 on the morphology of the chloroplast nucleoids. (A) Morphology of the chloroplast nucleoids in the overexpresser and the knockdown lines. Expanding leaf or the basal part of expanding leaf cells of the wild type (WT), the AtYLMG1-1 knockdown line (AS), and the AtYLMG1-1 overexpresser (OX) were stained with DAPI. The white portion indicates DAPI fluorescence showing the localization of DNA. Nuclei (N) are also observed in some panels. Magnified images are also shown in the lower panels. Bars = 5 μm. All images were obtained with the same exposure time. (B) Morphology of the nucleoids in dividing chloroplasts. Young emerging leaves of the wild type were stained with SYBR GREEN I. The white portion indicates the SYBR GREEN I fluorescence showing the localization of DNA. Arrowheads indicate dividing chloroplasts. Other dividing chloroplasts are also shown in the right panels. Bars = 10 μm. (C) Comparison of the quantity of chloroplast DNA by DNA-blot analysis. Total genome DNA of the wild type (WT), the AtYLMG1-1 overexpresser (OX), and the AtYLMG1-1 knockdown line (AS) was extracted and then was digested with HindIII. Three micrograms of digested DNA were loaded in each lane. Chloroplast DNA (cp) was detected with a psbA probe and nuclear DNA (nu) was detected with a PsbO probe. Nuclear DNA was detected as the quantitative control. (D) Morphology of the chloroplast nucleoids in ftsZ2-1, arc5, and arc6 mutants. Mature leaves of the ftsZ2-1, arc5, and arc6 mutants were stained with DAPI. The white portion indicates DAPI fluorescence showing the localization of DNA. Bars = 5 μm. All images were obtained with the same exposure time.

Figure 5

Localization of the AtYLMG1-1 protein. (A) Immunoblot analysis showing the chloroplast localization of AtYLMG1-1. Total proteins extracted from whole plants and isolated chloroplasts (cp) from the wild type were analyzed with the anti-AtYLMG1-1 antibodies. Fifty micrograms of protein were loaded in each lane. The Rubisco small subunit (Rubisco SSU) was detected by CBB staining as the quantitative control. (B) Localization of AtYLMG1-1 in the chloroplast. Chloroplasts were lysed in hypotonic solution and separated into pellet and supernatant fractions by centrifugation. The total chloroplast protein (total cp), pellet (pellet), and supernatant (sup) fractions were analyzed. TOC34 was detected as a marker of the membrane protein and the Rubisco small subunit was detected as a marker of the stromal protein. (C) Localization of AtYLMG1-1 in the chloroplast membranes. Isolated chloroplasts from the wild type were lysed and separated into thylakoid and envelope membranes. Proteins of the total chloroplast (total cp), the envelope fraction (env), and the thylakoid fraction (thy) were examined with the anti-AtYLMG1-1 antibodies. Lhcb1 was detected as a marker of the thylakoid protein and TOC34 was detected as a marker of the envelope protein. (D) Localization of AtYLMG1-1 examined by immunofluorescence microscopy. Isolated chloroplasts from the wild type were immunostained with the anti-AtYLMG1-1 antibodies. The green fluorescence indicates the localization of AtYLMG1-1 and the red shows the chlorophyll fluorescence. Bar = 5 μm. (E) Relationship between AtYLMG1-1 puncta and chloroplast nucleoids. Isolated chloroplasts were immunostained with the anti-AtYLMG1-1 antibodies and counterstained with DAPI. The red indicates the localization of AtYLMG1-1 and the blue is DAPI fluorescence showing the localization of DNA. A merged image is also shown. Arrowheads indicate the overlap between the AtYLMG1-1 puncta and nucleoids. Bar = 5 μm.

Figure 6

Effects of disruption or overexpression of SylmG1 in Synechococcus elongatus. (A) Gene disruption of SylmG1. Genotypic character of the wild type (WT) and kanamycin-resistant mutants (lines #1-5) which were subjected to PCR analysis using primer 1 (5'-TGACGGACTTCTTCGACCAGATG-3') and primer 2 (5'-ATTGAACCGCGTTGGGACAAGG-3'). A 0.9-kb nptII gene was inserted into SylmG1 locus by homologous recombination. The insertion of the nptII gene was confirmed by PCR using the primer set indicated in the diagram. (B) Overexpression of SylmG1. Total RNA (3 μg) from exponential cells (OD₇₃₀ = 0.4) of the wild type or spectinomycin-resistant mutants (OX) was subjected to RNA-blot analysis with the SylmG1 specific probe. (C) Phenotype of the SylmG1 disruptant. Nucleoids of the wild type (WT) and the SylmG1 disruptant (ΔSylmG1) were stained with DAPI. Cells in the exponential phase were stained with DAPI and the images were obtained with the same exposure time. The blue is DAPI fluorescence showing the localization of DNA, and the autofluorescence of chlorophyll is red. Bars = 5 μm. (D) Nucleoids of the SylmG1 overexpresser (OX-SylmG1). The image was obtained by the same procedure as (c). The distribution patterns of the cell length of the wild type (WT) and the SylmG1 overexpresser, measured in the exponential phase, are shown in the histograms. The average of the cell length is shown in each graph along with the standard deviation. n = 50. Bar = 5 μm. (E) Relationship between the distribution of nucleoids and the localization of FtsZ in the wild type and the SylmG1 overexpresser. Localization of FtsZ was examined by immunofluorescence microscopy. The green fluorescence shows the localization of FtsZ. The blue is DAPI fluorescence which shows the localization of DNA, and the autofluorescence of chlorophyll is red. Merged images are also shown at the bottom. Bars = 5 μm.