Plastid chaperonin proteins Cpn60 alpha and Cpn60 beta are required for plastid division in Arabidopsis thaliana

Abstract

Background: Plastids arose from a free-living cyanobacterial endosymbiont and multiply by binary division as do cyanobacteria. Plastid division involves nucleus-encoded homologs of cyanobacterial division proteins such as FtsZ, MinD, MinE, and ARC6. However, homologs of many other cyanobacterial division genes are missing in plant genomes and proteins of host eukaryotic origin, such as a dynamin-related protein, PDV1 and PDV2 are involved in the division process. Recent identification of plastid division proteins has started to elucidate the similarities and differences between plastid division and cyanobacterial cell division. To further identify new proteins that are required for plastid division, we characterized previously and newly isolated plastid division mutants of Arabidopsis thaliana.

Results: Leaf cells of two mutants, br04 and arc2, contain fewer, larger chloroplasts than those of wild type. We found that ARC2 and BR04 are identical to nuclear genes encoding the plastid chaperonin 60 alpha (ptCpn60alpha) and chaperonin 60 beta (ptCpn60beta) proteins, respectively. In both mutants, plastid division FtsZ ring formation was partially perturbed though the level of FtsZ2-1 protein in plastids of ptcpn60beta mutants was similar to that in wild type. Phylogenetic analyses showed that both ptCpn60 proteins are derived from ancestral cyanobacterial proteins. The A. thaliana genome encodes two members of ptCpn60alpha family and four members of ptCpn60beta family respectively. We found that a null mutation in ptCpn60alpha abolished greening of plastids and resulted in an albino phenotype while a weaker mutation impairs plastid division and reduced chlorophyll levels. The functions of at least two ptCpn60beta proteins are redundant and the appearance of chloroplast division defects is dependent on the number of mutant alleles.

Conclusion: Our results suggest that both ptCpn60alpha and ptCpn60beta are required for the formation of a normal plastid division apparatus, as the prokaryotic counterparts are required for assembly of the cell division apparatus. Since moderate reduction of ptCpn60 levels impaired normal FtsZ ring formation but not import of FtsZ into plastids, it is suggested that the proper levels of ptCpn60 are required for folding of stromal plastid division proteins and/or regulation of FtsZ polymer dynamics.

Figure 1

Chloroplast division defects and mutation sites in plastid cpn60 mutants. (A-F) Chloroplasts in leaf mesophyll cells were observed by Nomarski optics. Since the background of ptcpn60β1-1 (br04) and ptcpn60β1–2 (SAIL_852_B03) is Col-0 and the background of ptcpn60α1-1 (arc2) is Ler, mutants were compared to their respective wild types. Scale bar = 10 μm. (G-H) Schematic diagram of ptCpn60β1 and ptCpn60α1. Mutation sites of ptcpn60β1-1 (br04) and ptcpn60α1-1 (arc2) are indicated by arrows and the positions of T-DNA insertions in ptcpn60β1–2 (SAIL_852_B03) and ptcpn60α1–2 (SALK_006606) are indicated by triangles. Exons are depicted as black boxes and UTRs are depicted as white boxes. bp, base pair. aa, amino acids.

Figure 2

Phylogenetic relationships among plastid chaperonin 60 proteins. A phylogenetic tree was constructed using the Maximum-likelihood and Bayesian methods. Sequences from Viridiplantae, Rhodophyta and other eukaryotic groups containing chloroplasts of red algal origin are shown in green, red, and blue, respectively. GI numbers or locus IDs of proteins are shown with names of species. Proteins highlighted by yellow boxes were examined in this study. Bootstrap values by RaxML [57] and posterior probability values by MrBayes [56] are indicated at the branch nodes. Only the clades containing cyanobacterial and plastid proteins are shown; the whole tree is shown in Additional file 1.

Figure 3

Comparison of phenotypes between two ptcpn60α1 mutants and in combinations with ptcpn60β1-1 and ptcpn60β2 mutants. (A-E) Seedlings, chloroplasts in leaf mesophyll cells, and chlorophyll contents of ptcpn60a1 mutants. Phenotypes of ptcpn60α1–2 (SALK_006606) were complemented by a ptCpn60α transgene (D). (F-H) The seedlings, chloroplasts in leaf mesophyll cells, and chlorophyll contents in plants with combinations of ptcpn60β1-1 and ptcpn60β2 mutations. +/+, wild type. +/-, heterozygous mutant. -/-, homozygous mutant. Scale bars = 2 mm (A-D, left panels), 10 μm (A-D, right panels), 2 mm (F), and 10 μm (G). Error bars represent the standard deviation (E, H). n.d., not determined (H).

Figure 4

Expression and localization of ptCpn60β and FtsZ in plastid cpn60 mutants. (A) Immunoblot analyses using anti-ptCpn60β and anti-FtsZ2-1 antibodies. Total proteins extracted from seedlings of wild type, ptcpn60β1-1, ptcpn60β2, ptcpn60β1-1 ptcpn60β2, and ftsZ2-1 in mesophyll cells were blotted. (B) RT-PCR analyses comparing transcript levels of ptCpn60β1 and ptCpn60β2. cDNA was prepared from total RNA extracted from the wild type, ptcpn60β1-1, and ptcpn60β2. (C) Localization of ptCpn60β in wild type and of FtsZ2-1 in wild type, ptcpn60β1-1, and ptcpn60α1-1 in mesophyll cells was examined by immunofluorescence microscopy. Scale bars = 10 μm.