Hiromitsu Nakanishi

The PLASTID DIVISION1 and 2 Components of the Chloroplast Division Machinery Determine the Rate of Chloroplast Division in Land Plant Cell Differentiation

In most algae, the chloroplast division rate is held constant to maintain the proper number of chloroplasts per cell. By contrast, land plants evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts change along with their respective cellular function by regulation of the division rate. Here, we show that PLASTID DIVISION (PDV) proteins, land plant–specific components of the division apparatus, determine the rate of chloroplast division. Overexpression of PDV proteins in the angiosperm Arabidopsis thaliana and the moss Physcomitrella patens increased the number but decreased the size of chloroplasts; reduction of PDV levels resulted in the opposite effect. The level of PDV proteins, but not other division components, decreased during leaf development, during which the chloroplast division rate also decreased. Exogenous cytokinins or overexpression of the cytokinin-responsive transcription factor CYTOKININ RESPONSE FACTOR2 increased the chloroplast division rate, where PDV proteins, but not other components of the division apparatus, were upregulated. These results suggest that the integration of PDV proteins into the division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.

Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by dynamin proteins

Chloroplasts have evolved from a cyanobacterial endosymbiont and been retained for more than 1 billion years by coordinated chloroplast division in multiplying eukaryotic cells. Chloroplast division is performed by ring structures at the division site, encompassing both the inside and the outside of the two envelopes. A part of the division machinery is derived from the cyanobacterial cytokinetic activity based on the FtsZ protein. In contrast, other parts of the division machinery involve proteins specific to eukaryotes, including a member of the dynamin family. Each member of the dynamin family is involved in the division or fusion of a distinct eukaryotic membrane system. To gain insight into the kind of ancestral dynamin protein and eukaryotic membrane activity that evolved to regulate chloroplast division, we investigated the functions of the dynamin proteins that are most closely related to chloroplast division proteins. These proteins in the amoeba Dictyostelium discoideum and Arabidopsis thaliana localize at the sites of cell division, where they are involved in cytokinesis. Our results suggest that the dynamin for chloroplast division is derived from that involved in eukaryotic cytokinesis. Therefore, the chloroplast division machinery is a mixture of bacterial and eukaryotic cytokinesis components, with the latter a key factor in the synchronization of endosymbiotic cell division with host cell division, thus helping to establish the permanent endosymbiotic relationship.

Plant-Specific Protein MCD1 Determines the Site of Chloroplast Division in Concert with Bacteria-Derived MinD

Chloroplasts evolved from a cyanobacterial endosymbiont, and chloroplast division requires the formation of an FtsZ division ring, which is descended from the cytokinetic machinery of cyanobacteria. As in bacteria, the positioning of the chloroplast FtsZ ring is regulated by the proteins MinD and MinE. However, chloroplast division also involves mechanisms invented by the eukaryotic host cell. Here we show that a plant-specific protein MULTIPLE CHLOROPLAST DIVISION SITE 1 (MCD1) regulates FtsZ ring positioning in Arabidopsis thaliana chloroplasts. Our analyses show that both MCD1 and MinD are required for chloroplast division, localizing at the division sites and punctate structures dispersed on the inner envelope. MinD overexpression inhibited FtsZ ring formation whereas MCD1 overexpression did not. Localization studies suggest that MCD1 is required for MinD localization to regulate FtsZ ring formation. Furthermore, the interaction between MCD1 and MinD in yeast two-hybrid assays suggests that MCD1 recruits MinD by direct interaction. These results point out differences in the MinD localization mechanism between chloroplasts and bacterial model systems and suggest that the plant cell evolved a component to modulate the cyanobacteria-derived Min system so as to regulate chloroplast FtsZ ring positioning.

Plastid chaperonin proteins Cpn60α and Cpn60β are required for plastid division in Arabidopsis thaliana

BackgroundPlastids 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.ResultsLeaf 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 60α (ptCpn60α) and chaperonin 60β (ptCpn60β) proteins, respectively. In both mutants, plastid division FtsZ ring formation was partially perturbed though the level of FtsZ2-1 protein in plastids of ptcpn60β 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 ptCpn60α family and four members of ptCpn60β family respectively. We found that a null mutation in ptCpn60α 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 ptCpn60β proteins are redundant and the appearance of chloroplast division defects is dependent on the number of mutant alleles.ConclusionOur results suggest that both ptCpn60α and ptCpn60β 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.

Structure, regulation, and evolution of the plastid division machinery.

Plastids have evolved from a cyanobacterial endosymbiont, and their continuity is maintained by the plastid division and segregation which is regulated by the eukaryotic host cell. Plastids divide by constriction of the inner- and outer-envelope membranes. Recent studies revealed that this constriction is performed by a large protein and glucan complex at the division site that spans the two envelope membranes. The division complex has retained certain components of the cyanobacterial division complex along with components developed by the host cell. Based on the information on the division complex at the molecular level, we are beginning to understand how the division complex has evolved and how it is assembled, constricted, and regulated in the host cell. This chapter reviews the current understanding of the plastid division machinery and some of the questions that will be addressed in the near future.

The YlmG protein has a conserved function related to the distribution of nucleoids in chloroplasts and cyanobacteria

BackgroundReminiscent of their free-living cyanobacterial ancestor, chloroplasts proliferate by division coupled with the partition of nucleoids (DNA-protein complexes). Division of the chloroplast envelope membrane is performed by constriction of the ring structures at the division site. During division, nucleoids also change their shape and are distributed essentially equally to the daughter chloroplasts. Although several components of the envelope division machinery have been identified and characterized, little is known about the molecular components/mechanisms underlying the change of the nucleoid structure.ResultsIn order to identify new factors that are involved in the chloroplast division, we isolated Arabidopsis thaliana chloroplast division mutants from a pool of random cDNA-overexpressed lines. We found that the overexpression of a previously uncharacterized gene (AtYLMG1-1) of cyanobacterial origin results in the formation of an irregular network of chloroplast nucleoids, along with a defect in chloroplast division. In contrast, knockdown of AtYLMG1-1 resulted in a concentration of the nucleoids into a few large structures, but did not affect chloroplast division. Immunofluorescence microscopy showed that AtYLMG1-1 localizes in small puncta on thylakoid membranes, to which a subset of nucleoids colocalize. In addition, in the cyanobacterium Synechococcus elongates, overexpression and deletion of ylmG also displayed defects in nucleoid structure and cell division.ConclusionsThese results suggest that the proper distribution of nucleoids requires the YlmG protein, and the mechanism is conserved between cyanobacteria and chloroplasts. Given that ylmG exists in a cell division gene cluster downstream of ftsZ in gram-positive bacteria and that ylmG overexpression impaired the chloroplast division, the nucleoid partitioning by YlmG might be related to chloroplast and cyanobacterial division processes.

Effect of Environmental Conditions on the α-Glucosidase Inhibitory Activity of Mulberry Leaves

Mulberry leaves have been used as the sole food for silkworms in sericulture, and also as a traditional medicine for diabetes prevention. Mulberry leaf components, for example 1-deoxynojirimycin (1-DNJ), inhibit the activity of α-glucosidase and prevent increased blood glucose levels, and they are highly toxic to caterpillars other than silkworms. The α-glucosidase inhibitory activity of mulberry leaves changes with the season, but it is unknown which environmental conditions influence the α-glucosidase inhibitory activity. We investigated in this study the relationship between the α-glucosidase inhibitory activity and environmental conditions of temperature and photoperiod. The results demonstrate that low temperatures induced decreasing α-glucosidase inhibitory activity, while the induction of newly grown shoots by the scission of branches induced increasing α-glucosidase inhibitory activity. These results suggest that the α-glucosidase inhibitory activity was related to the defense mechanism of mulberry plants against insect herbivores.

Development of a novel evaluation method for air particles using surface plasmon resonance spectroscopy analysis

The aim of this study was to develop a novel evaluation method for air particles using surface plasmon resonance spectroscopy (SPR) analysis. An L1 sensor chip modified with immobilized liposome was used as a model of the membrane of epithelial cells in organs of respiration. A test suspension of dispersed air particles was flowed onto the sensor chip. The interaction between the surface of the sensor chip and particulates in the sample solution was detected by SPR. It is deduced that the SPR measurement provides information about the adsorption/desorption behavior of the particles on the membrane. Environmentally certified reference materials, diesel particulate matter, vehicle exhaust particulates, urban particulate matter, coal fly ash, and rocks, were used as air particulate samples. Filtrates of suspensions of these samples were analyzed by SPR. Each sample revealed characteristic SPR sensor-gram patterns. For example, diesel particulate matter strongly interacted with the lipid bilayer, and was hardly dissociated. On the other hand, coal fly ash and rock particles interacted poorly with the membrane. The presented method could be used to evaluate or characterize air particles.

Conservation and differences of the Min system in the chloroplast and bacterial division site placement.

Chloroplasts are descended from a cyanobacterial endosymbiont and divide by binary fission. Reminiscent of the process in their bacterial ancestor, chloroplast division involves a part of cyanobacteria-derived division machineries in addition to those acquired during chloroplast evolution.1,2 In both bacterial and chloroplast division, formation of the FtsZ ring at the mid position is required for subsequent constriction and fission at the mid division site.1-4 As in bacteria, positioning of the FtsZ ring at the mid-chloroplast is mediated by the Min system.1,2 Recently, we identified the MCD1 protein, a plant-specific component of the Min system in Arabidopsis thaliana chloroplasts.5 Unlike other division components that have been acquired after endosymbiosis and function outside of the chloroplasts (i.e. in/on the outer envelope membrane),6-9 MCD1 functions inside the chloroplast. Since we already discussed about the function and significance of MCD1 as a division component of plant origin,5 here we focus on and discuss about the diversity and evolution of the Min system.

Evaluation of the Interaction between Pesticides and a Cell Membrane Model by Surface Plasmon Resonance Spectroscopy Analysis

A surface plasmon resonance (SPR) spectroscopy analysis was used for the characterization of the interaction between pesticides and a cell membrane model. A liposome was immobilized onto the surface of the SPR sensor chip (L1), and the lipid bilayer membrane formed on the sensor chip was regarded as the cell membrane model. The solution containing a pesticide was flowed onto the sensor chip, and an SPR sensorgram, which reflected the interaction between the pesticide and the lipid bilayer membrane, was obtained. As the results, the pattern and strength of the interaction of the pesticides with the cell membrane model were visualized and quantified. Triflumizole, hexythiazox, and pentachlorophenol showed a strong interaction with the lipid bilayer. It is well-known that triflumizole and pentachlorophenol interact with the membrane and reveal toxicities for cell membranes. In addition, there was a tendency for higher residual ratios to be observed when the no observable adverse effect level (NOAEL) values for chronic toxicity (1 year toxicity study in dogs) were lower. We suggest that a novel parameter for the evaluation or presumption of the behaviors and chronic toxicities of pesticides is obtained by the presented method.