Date Published: February 21, 2018
Publisher: Public Library of Science
Author(s): Makoto T. Fujiwara, Mana Yasuzawa, Kei H. Kojo, Yasuo Niwa, Tomoko Abe, Shigeo Yoshida, Takeshi Nakano, Ryuuichi D. Itoh, Ryozo Imai.
Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells. In Arabidopsis thaliana, the division apparatus (or division ring) of mesophyll chloroplasts includes an inner envelope transmembrane protein ARC6, a cytoplasmic dynamin-related protein ARC5 (DRP5B), and members of the FtsZ1 and FtsZ2 families of proteins, which co-assemble in the stromal mid-plastid division ring (FtsZ ring). FtsZ ring placement is controlled by several proteins, including a stromal factor MinE (AtMinE1). During leaf mesophyll development, ARC6 and AtMinE1 are necessary for FtsZ ring formation and thus plastid division initiation, while ARC5 is essential for a later stage of plastid division. Here, we examined plastid morphology in leaf epidermal pavement cells (PCs) and stomatal guard cells (GCs) in the arc5 and arc6 mutants using stroma-targeted fluorescent proteins. The arc5 PC plastids were generally a bit larger than those of the wild type, but most had normal shapes and were division-competent, unlike mutant mesophyll chloroplasts. The arc6 PC plastids were heterogeneous in size and shape, including the formation of giant and mini-plastids, plastids with highly developed stromules, and grape-like plastid clusters, which varied on a cell-by-cell basis. Moreover, unique plastid phenotypes for stomatal GCs were observed in both mutants. The arc5 GCs rarely lacked chlorophyll-bearing plastids (chloroplasts), while they accumulated minute chlorophyll-less plastids, whereas most GCs developed wild type-like chloroplasts. The arc6 GCs produced large chloroplasts and/or chlorophyll-less plastids, as previously observed, but unexpectedly, their chloroplasts/plastids exhibited marked morphological variations. We quantitatively analyzed plastid morphology and partitioning in paired GCs from wild-type, arc5, arc6, and atminE1 plants. Collectively, our results support the notion that ARC5 is dispensable in the process of equal division of epidermal plastids, and indicate that dysfunctions in ARC5 and ARC6 differentially affect plastid replication among mesophyll cells, PCs, and GCs within a single leaf.
Chloroplasts are double membrane-bound organelles that form a homogeneous population with respect to shape (round or ellipsoidal) and size (usually 3–10 μm in diameter) in photosynthetic cells. Such homogeneity in chloroplast shape and size is attained via several cycles of symmetric binary fission of the organelle, which eventually generate up to several hundred chloroplasts per cell. The most popular model for studying chloroplast division in higher plants is leaf mesophyll cells [1–3]. The discovery of Arabidopsis thaliana chloroplast number mutants (accumulation and replication of chloroplasts; arc), which was the first successful example of mutant screening to identify subcellular structure mutants in plants, was achieved through the observation of leaf mesophyll cells . Among the arc mutants, the recessive arc5 and arc6 mutants are the most well characterized. The arc5 mutant has a reduced number of chloroplasts, all of which are arrested at a late stage of division, in mature mesophyll cells [5,6]. The arc6 mutant exhibits a more extreme phenotype, with each mesophyll cell containing only one or two expanded chloroplasts . ARC5 encodes a cytoplasmic dynamin-related protein (DRP5B), while ARC6 encodes an inner envelope-spanning protein that anchors the prokaryotic tubulin-like GTPase FtsZ to promote its self-assembly into a stromal division ring (FtsZ ring) [8,9]. Ferns and seed plants encode two subfamilies of FtsZ proteins, FtsZ1 and FtsZ2 . FtsZ1 and FtsZ2 can form heteropolymers (protofilaments), resembling those assembled from α- and β-tubulins within microtubules, which further bundle into a contractile ring . ARC6 interacts specifically with FtsZ2  and could have a role in tethering the FtsZ ring to the inner envelope. ARC5/DRP5B and FtsZ form distinct concentric rings at the chloroplast division site and on the cytoplasmic and stromal surface of the envelope, respectively. Spatial coordination of those two rings across two membranes (the outer and inner envelopes) is achieved by a chain of protein interactions, namely ARC5–PDV1/PDV2 (PLASTID DIVISION1/2; bitopic outer envelope membrane proteins homologous to each other) in the cytosol , PDV2–ARC6 in the intermembrane space [14,15], and ARC6–FtsZ2 in the stroma . In addition to these protein-protein interactions, the chloroplast phenotypes of arc5 and arc6 and the properties of ARC5 and ARC6 proteins provide valuable insights into the molecular components of the division machinery on both sides of the envelope, the evolutionary process by which such machinery was established, and the orderly progression of events that occurs during division: division initiation (involving ARC6), membrane constriction (FtsZ), and final separation of the daughter chloroplasts (ARC5).