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

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.

Quantitative Evaluation of Stromule Frequency at Hourly Intervals in <i>Arabidopsis</i> Stomatal Guard Cell Chloroplasts

“…We observed stomatal guard cell chloroplasts and counted the frequency of stromules in 1-and 2-day old cotyledons, taken as a developing leaf, and 5-day old cotyledons, as a developed leaf. Abnormal morphology of chloroplasts in stomata has not been reported in wild-type seedlings (Fujiwara et al 2018) and we observed normal morphology of the chloroplasts in stomata and pavement cells in this study (Fig. 1a).…”

Arabidopsis PARC6 Is Critical for Plastid Morphogenesis in Pavement, Trichome, and Guard Cells in Leaf Epidermis

“…Seeds of plastid division mutants, parc6-1 (SALK_100009; Glynn et al, 2009;Zhang et al, 2009;Ottesen et al, 2010;generated by Alonso et al, 2003), parc6-3 (Glynn et al, 2009), parc6-4 (SALK_138043; Zhang et al, 2009; generated by Alonso et al, 2003), arc6-3 (CS288; , and arc11-1 (CS281; Marrison et al, 1999) were obtained from the Arabidopsis Biological Resource Center (ABRC), Ohio State University, Columbus, OH, USA. Two transgenic Arabidopsis lines [FL4-4 and FL6-4; Columbia (Col) background] expressing organelle-targeted fluorescent proteins as well as offspring derived from crosses between the transgenic lines and mutants (parc6-1 × FL4-4, parc6-3 × FL4-4, parc6-4 × FL4-4, arc11-1 × FL4-4, and arc6-3 × FL6-4) were used (Chen et al, 2009;Itoh et al, 2010;Fujiwara et al, 2018;Itoh et al, 2018; see summary in Table 1). The parc6-5 (suba2) mutant was isolated from EMS-treated FL4-4 seeds carrying two nucleotide substitutions in the PARC6 coding sequence, resulting in G62R and W700stop mutations at the protein level (Itoh et al, 2018).…”

“…Basal parts of leaves including petioles were excised from Arabidopsis seedlings using tweezers. Based on previous observations (e.g., Fujiwara et al, 2004;Fujiwara et al, 2008;Fujiwara et al, 2009a;Fujiwara et al, 2009b;Itoh et al, 2010;Fujiwara et al, 2015;Fujiwara et al, 2017;Fujiwara et al, 2018;Itoh et al, 2018), the adaxial surface of the leaf epidermis in the upper petiole region was used to analyze leaf pavement, trichome, and guard cells in Arabidopsis, unless otherwise specified.…”

“…Stromule biogenesis also involves interactions with the cytoskeleton and other organelles (reviewed in Erickson and Schattat, 2018;Hanson and Hines, 2018). Furthermore, inhibition of chloroplast division causes excessive production of stromules in various tissues, as shown in tomato (Solanum lycopersicum) suffulta mutant and Arabidopsis arc3, arc5, arc6, crl, and atminE1 mutants (Forth and Pyke, 2006;Holzinger et al, 2008;Chen et al, 2009;Kojo et al, 2009;Fujiwara et al, 2015;Fujiwara et al, 2018). These studies indicate the importance of stromules in plant cells; however, the mechanism of the origin of stromules and their functions in plant cells remains largely unknown (Hanson and Hines, 2018).…”

“…In Arabidopsis, three different types of plastids have been reported, including pavement cell chloroplasts, trichome leucoplasts, and guard cell chloroplasts, all of which originate from proplastids in the shoot apical meristem, or more strictly, protodermal plastids with poor thylakoids (early differentiating chloroplasts) in leaf primordia Robertson et al, 1995;Barton et al, 2018). The number, shape, distribution, and dynamics of plastids have been relatively well-studied using pavement cell chloroplasts Schattat et al, 2012;Higa et al, 2014;Fujiwara et al, 2015;Erickson et al, 2017;Fujiwara et al, 2017;Fujiwara et al, 2018), while stomata physiology has long been investigated in guard cell chloroplasts (Lawson, 2009;Taiz et al, 2015). However, fewer studies have been conducted on the morphological aspects of epidermal plastids than on mesophyll chloroplasts.…”