The Arabidopsis arc5 and arc6 mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis.

Makoto T Fujiwara,Shigeo Yoshida,Yasuo Niwa,Kei H Kojo,Tomoko Abe,Mana Yasuzawa,Takeshi Nakano,Ryuuichi D Itoh,Ryozo Imai

doi:10.1371/journal.pone.0192380

Abstract

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.

Highlights

Chloroplasts are double membrane-bound organelles that form a homogeneous population with respect to shape and size in photosynthetic cells
Plastid image data were obtained from wild type (WT), arc5, and arc6 plants in which stroma were labeled with YFP and detected by confocal laser scanning microscopy (CLSM)
The phenotypes of plastids in arc5 and arc6 leaf petioles were essentially the same as those in leaf blade mesophyll cells observed with differential interference contrast (DIC) optics [5,6] and with GFP fused to a transit peptide [19,22]

Summary

Introduction

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. 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 [13], PDV2–ARC6 in the intermembrane space [14,15], and ARC6–FtsZ2 in the stroma [12]. In addition to these protein-protein interactions, the chloroplast phenotypes of arc and arc 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)

Methods

Results

Conclusion