Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space.

Chloroplast division involves the coordination of protein complexes from the stroma to the cytosol. The Min system of chloroplasts includes multiple stromal proteins that regulate the positioning of the division site. The outer envelope protein PLASTID DIVISION1 (PDV1) was previously reported to recruit the cytosolic chloroplast division protein ACCUMULATION AND REPLICATION OF CHLOROPLAST5 (ARC5). However, we show here that PDV1 is also important for the stability of the inner envelope chloroplast division protein PARALOG OF ARC6 (PARC6), a component of the Min system. We solved the structure of both the C-terminal domain of PARC6 and its complex with the C terminus of PDV1. The formation of an intramolecular disulfide bond within PARC6 under oxidized conditions prevents its interaction with PDV1. Interestingly, this disulfide bond can be reduced by light in planta, thus promoting PDV1-PARC6 interaction and chloroplast division. Interaction with PDV1 can induce the dimerization of PARC6, which is important for chloroplast division. Magnesium ions, whose concentration in chloroplasts increases upon light exposure, also promote the PARC6 dimerization. This study highlights the multilayer regulation of the PDV1-PARC6 interaction as well as chloroplast division.

Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology.

The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A-type granules and small spherical B-type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat (Triticum turgidum) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A- and B-type granules than those of the wild-type. The mutant had increased A- and B-type granule size in mature grains, and its A-type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest TtPARC6 can complement disrupted TtARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.

A Mutation in GIANT CHLOROPLAST Encoding a PARC6 Homolog Affects Spikelet Fertility in Rice.

Chloroplasts are not generated de novo but proliferate from a pre-existing population of plastids present in meristematic cells. Chloroplast division is executed by the co-ordinated action of at least two molecular machineries: internal machinery located on the stromal side of the inner envelope membrane and external machinery located on the cytosolic side of the outer envelope membrane. To date, molecular studies of chloroplast division in higher plants have been limited to several species such as Arabidopsis. To elucidate chloroplast division in rice, we performed forward genetics and isolated a mutant displaying large chloroplasts among an ethyl methanesulfonate (EMS)-mutagenized Oryza sativa spp japonica Nipponbare population. Using a map-based approach, this mutation, termed giant chloroplast (gic), was allocated in a gene that encodes a protein that is homologous to Paralog of ARC6 (PARC6), which is known to play a role in chloroplast division. GIC is unique in that it has a long C-terminal extension that is not present in other PARC6 homologs. Characterization of gic phenotypes in a rice field showed that gic exhibited defective growth in seed setting, suggesting that the gic mutant negatively affects the reproductive stage. This report is the first describing a chloroplast division mutant in monocotyledons and its effect on plant development.

Adaptive evolution of chloroplast division mechanisms during plant terrestrialization.

Despite extensive research, the origin and evolution of the chloroplast division machinery remain unclear. Here, we employ recently sequenced genomes and transcriptomes of Archaeplastida clades to identify the core components of chloroplast division and reconstruct their evolutionary histories, respectively. Our findings show that complete division ring structures emerged in Charophytes. We find that Glaucophytes experienced strong selection pressure, generating diverse variants adapted to the changing terrestrial environments. By integrating the functions of chloroplast division genes (CDGs) annotated in a workflow developed using large-scale multi-omics data, we further show that dispersed duplications acquire more species-specific functions under stronger selection pressures. Notably, PARC6, a dispersed duplicate CDG, regulates leaf color and plant growth in Solanum lycopersicum, demonstrating neofunctionalization. Our findings provide an integrated perspective on the functional evolution of chloroplast division machinery and highlight the potential of dispersed duplicate genes as the primary source of adaptive evolution of chloroplast division.

ORHis, a Natural Variant of OR, Specifically Interacts with Plastid Division Factor ARC3 to Regulate Chromoplast Number and Carotenoid Accumulation.

Chromoplasts are colored plastids that synthesize and store massive amounts of carotenoids. Chromoplast number and size define the sink strength for carotenoid accumulation in plants. However, nothing is known about the mechanisms controlling chromoplast number. Previously, a natural allele of Orange (OR), ORHis, was found to promote carotenoid accumulation by activating chromoplast differentiation and increasing carotenoid biosynthesis, but cells in orange tissues in melon fruit and cauliflower OR mutant have only one or two enlarged chromoplasts. In this study, we investigated an ORHis variant of Arabidopsis OR, genetically mimicking the melon ORHis allele, and found that it also constrains chromoplast number in Arabidopsis calli. Both in vitro and in vivo experiments demonstrate that ORHis specifically interacts with the Membrane Occupation and Recognition Nexus domain of ACCUMULATION AND REPLICATION OF CHLOROPLASTS 3 (ARC3), a crucial regulator of chloroplast division. We further showed that ORHis interferes with the interaction between ARC3 and PARALOG OF ARC6 (PARC6), another key regulator of chloroplast division, suggesting a role of ORHis in competing with PARC6 for binding to ARC3 to restrict chromoplast number. Overexpression or knockout of ARC3 in Arabidopsis ORHis plants significantly alters total carotenoid levels. Moreover, overexpression of the plastid division factor PLASTID DIVISION 1 greatly enhances carotenoid accumulation. These division factors likely alter carotenoid levels via their influence on chromoplast number and/or size. Taken together, our findings provide novel mechanistic insights into the machinery controlling chromoplast number and highlight a potential new strategy for enhancing carotenoid accumulation and nutritional value in food crops.