Applicability of self-consistent reaction field (SCRF) method to DFT estimation of hydrogen isotope separation factors in reversible processes.

In earlier quantum chemical calculations of isotope effects, chemical species in the liquid phase were generally treated as existing in the gas phase. In recent years, however, advances in computational programs have made it easier for the self-consistent reaction field (SCRF) method to handle chemical species in the liquid phase, and as a result, it has become easier to apply the SCRF method to isotope effect calculations. This paper concerns the scope of application of the DFT-SCRF method to reversible processes for hydrogen isotope enrichment. It is found that the applicability of the method depends on the type of the intermolecular interaction in the liquid phase and the degree of hydrogen isotope effect (separation factor) on which the process is based. When the magnitude of the isotope effect of the separation system is greater than 10-1, the simple SCRF method is fully applicable; when the magnitude is around 10-2, SCRF with a dimer model, in which the monomer is replaced by a dimer, is applicable for the analysis of the liquid phase with relatively strong intermolecular interactions. Anharmonic correction to the separation factor calculated based on harmonic frequencies may be effective to systems with the liquid phase with weak intermolecular interactions.

Cytochrome c oxidase subunit I gene in Thalassiosira nordenskioeldii strains inhabiting in cold and warm sea waters.

From the biota beneath the sea ice in Lake Saroma, which is adjacent to Sea of Okhotsk, a diatom culture of Saroma 16 was isolated. Strutted processes and a labiate process in Saroma 16 were characteristic of those in Thalassiosira nordenskioeldii. Similarity search analysis showed that the 826-bp rbcL-3P region sequence of this strain was 100% identical to multiple sequences registered as T. nordenskioeldii in a public database. The 4305-bp PCR-amplified mitochondrial cytochrome c oxidase subunit I (COI) gene (COI)-5P region of Saroma 16 included a 1060-bp open reading frame (ORF), which was interrupted by 934-bp and 2311-bp introns that included frame-shifted ORFs encoding reverse-transcriptase (RTase)-like proteins. Previous reports showed that a strain of the same species, CNS00052, originating from the East China Sea included no introns in the COI, whereas North Atlantic Ocean strains of the same species, such as CCMP992, CCMP993, and CCMP997, included a 2.3-kb intron in the same position as Saroma 16.

DFT analysis of nitrogen isotopic reduction partition function ratios in proton exchange equilibria of glutamic acid species in water and its application to the estimation of nitrogen isotope fractionation.

L-Glutamic acid (Glu) plays a pivotal role in amino acid metabolism in living organisms. Theoretical knowledge of how metabolism propagates the nitrogen isotope composition (δ15N) is essential for elucidating the mechanism of amino acid metabolism in vivo. In this paper, to estimate the nitrogen isotope fractionation involving Glu and its protonated/deprotonated species, their nitrogen isotopic reduction partition function ratios (RPFRs) were calculated at the apfd/6-311+G(2d,p) level of theory by the SCRF method. The results show that the RPFR values of the eight possible Glu species at 25 °C are: cation > zwitterion with α-COOH and γ-COO- > zwitterion with α-COO- and γ-COOH > anion with -NH3+ ≫ uncharged molecule > anion with -NH2 and γ-COO- > anion with -NH2 and α-COO- > di-anion. Several correlations between RPFR and bond distances were found. Most importantly, it was found that the shorter the distance of the (C-)N⋯H(-O) hydrogen bond (HB), the greater the RPFR value for that species. This correlation indicates that the tri-coordinate nitrogen atom of the amino group may take on the character of the tetra-coordinate nitrogen atom to some extent by forming a HB of this type. The nitrogen isotope exchange equilibrium between Glu and glycine/serine was evaluated at a typical physiological pH of 7.4. Calculations suggest that the equilibrium constant for the former is an increasing function of temperature from 0 to 100 °C, while the latter is a decreasing function of temperature over the same temperature range.

Triterpenoids in aerenchymatous phellem contribute to internal root aeration and waterlogging adaptability in soybeans.

Soybeans (Glycine max) develop newly differentiated aerenchymatous phellem (AP) in response to waterlogging stress. AP is formed in the hypocotyl and root, thus contributing to internal aeration and adaptation to waterlogging for several legumes. Extensive accumulation of triterpenoids - lupeol and betulinic acid - has been identified in AP. However, their physiological roles in plants remain unclarified. Lupeol is converted from 2,3-oxidosqualene by lupeol synthase (LUS) and oxidized to betulinic acid. Notably, soybeans have two LUS genes (GmLUS1 and GmLUS2). Functional analysis was performed to reveal the biological and physiological functions of triterpenoids in AP using lus mutants. The AP cells of lus1 mutant lacked triterpenoid accumulation and epicuticular wax. Lupeol and betulinic acid were the major components of epicuticular wax and contributed to tissue hydrophobicity and oxygen transport to the roots. Tissue porosity in AP was lower in the lus1 mutant than in the wild-type, which resulted in reduced oxygen transport to the roots via AP. This reduction in oxygen transport resulted in shallow root systems under waterlogged conditions. Triterpenoid accumulation in AP contributes to effective internal aeration and root development for adaptation to waterlogging, suggesting the significance of triterpenoids in improving waterlogging tolerance.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

3-D reconstruction of rice leaf tissue for proper estimation of surface area of mesophyll cells and chloroplasts facing intercellular airspaces from 2-D section images.

BACKGROUND AND AIMS: The surface area of mesophyll cells (Smes) and chloroplasts (Sc) facing the intercellular airspace (IAS) are important parameters for estimating photosynthetic activity from leaf anatomy. Although Smes and Sc are estimated based on the shape assumption of mesophyll cells (MCs), it is questionable if the assumption is correct for rice MCs with concave-convex surfaces. Therefore, in this study, we establish a reconstruction method for the 3-D representation of the IAS in rice leaf tissue to calculate the actual Smes and Sc with 3-D images and to determine the correct shape assumption for the estimation of Smes and Sc based on 2-D section images. METHODS: We used serial section light microscopy to reconstruct 3-D representations of the IAS, MCs and chloroplasts in rice leaf tissue. Actual Smes and Sc values obtained from the 3-D representation were compared with those estimated from the 2-D images to find the correct shape-specific assumption (oblate or prolate spheroid) in different orientations (longitudinal and transverse sections) using the same leaf sample. KEY RESULTS: The 3-D representation method revealed that volumes of the IAS and MCs accounted for 30 and 70 % of rice leaf tissue excluding epidermis, respectively, and the volume of chloroplasts accounted for 44 % of MCs. The shape-specific assumption on the sectioning orientation affected the estimation of Smes and Sc using 2-D section images with discrepancies of 10-38 %. CONCLUSIONS: The 3-D representation of rice leaf tissue was successfully reconstructed using serial section light microscopy and suggested that estimation of Smes and Sc of the rice leaf is more accurate using longitudinal sections with MCs assumed as oblate spheroids than using transverse sections with MCs as prolate spheroids.

Extracellular Vesicle-Mediated Secretion of Protochlorophyllide in the Cyanobacterium Leptolyngbya boryana.

Protochlorophyllide (Pchlide) reduction in the late stage of chlorophyll a (Chl) biosynthesis is catalyzed by two enzymes: light-dependent Pchlide oxidoreductase (LPOR) and dark-operative Pchlide oxidoreductase (DPOR). The differential operation of LPOR and DPOR enables a stable supply of Chl in response to changes in light conditions and environmental oxygen levels. When a DPOR-deficient mutant (YFC2) of the cyanobacterium Leptolyngbya boryana is grown heterotrophically in the dark, Pchlide accumulates in the cells and is secreted into the culture medium. In this study, we demonstrated the extracellular vesicle-mediated secretion of Pchlide. Pchlide fractions were isolated from the culture medium using sucrose density gradient centrifugation. Mass spectrometry analysis revealed that the Pchlide fractions contained porin isoforms, TolC, and FG-GAP repeat-containing protein, which are localized in the outer membrane. Transmission electron microscopy revealed extracellular vesicle-like structures in the vicinity of YFC2 cells and the Pchlide fractions. These findings suggested that the Pchlide secretion is mediated by extracellular vesicles in dark-grown YFC2 cells.

Salt tolerance in relation to elemental concentrations in leaf cell vacuoles and chloroplasts of a C4 monocotyledonous halophyte.

Halophytes accumulate and sequester high concentrations of salt in vacuoles while maintaining lower levels of salt in the cytoplasm. The current data on cellular and subcellular partitioning of salt in halophytes are, however, limited to only a few dicotyledonous C3 species. Using cryo-scanning electron microscopy X-ray microanalysis, we assessed the concentrations of Na, Cl, K, Ca, Mg, P and S in various cell types within the leaf-blades of a monocotyledonous C4 halophyte, Rhodes grass (Chloris gayana). We also linked, for the first time, elemental concentrations in chloroplasts of mesophyll and bundle sheath cells to their ultrastructure and photosynthetic performance of plants grown in nonsaline and saline (200 mM NaCl) conditions. Na and Cl accumulated to the highest levels in xylem parenchyma and epidermal cells, but were maintained at lower concentrations in photosynthetically active mesophyll and bundle sheath cells. Concentrations of Na and Cl in chloroplasts of mesophyll and bundle sheath cells were lower than in their respective vacuoles. No ultrastructural changes were observed in either mesophyll or bundle sheath chloroplasts, and photosynthetic activity was maintained in saline conditions. Salinity tolerance in Rhodes grass is related to specific cellular Na and Cl distributions in leaf tissues, and the ability to regulate Na and Cl concentrations in chloroplasts.

Evaluation of the validity of large-scale serial sectioning TEM for three-dimensional reconstruction of rice mesophyll cells and chloroplasts.

Serial sectioning transmission electron microscopy (ssTEM) is a classical method of 3D reconstruction using serial sections obtained with an ultramicrotome. However, producing a long ribbon with homogeneity is difficult. Here, ultramicrotome movement was suspended after producing a ribbon of 15-30 serial sections (cutting intervals, 100 nm), and then, the ribbon was mounted on an individual one-slot grid. However, as this ssTEM method may include influencing factors such as incorrect intervals of section thickness and distortion of sections, which is produced by cutting sections using a diamond knife and beam interaction under TEM observation, qualitative and quantitative data on rice mesophyll cells and chloroplasts were compared with those obtained from a focused ion beam scanning electron microscopy (FIB-SEM) (cutting intervals, 50 nm). No structural distortion in 3D models was observed. In addition, no significant differences in the volume and surface area were observed between the two methods. The surface to volume ratio was significantly affected by the increase in section thickness, but not the difference of methodologies. Our method was useful for observing large volumes of plant cells and organelles, leading to the identification of various sizes and types of chloroplasts. The formation of a chloroplast pocket, which is a structure surrounding other intracellular compartments, was confirmed in rice leaves grown under moderate growth conditions using the ssTEM method. As only four out of 90 chloroplasts formed pocket structures, the formation was considered to be rare under the applied moderate growth conditions.

A density functional theory (DFT) study on reduced partition function ratios of oxygen species adsorbed on a Pt19 cluster and oxygen isotope effects.

A density functional theory (DFT) computation on oxygen species adsorbed on platinum (Pt) catalyst surfaces has been carried out to elucidate oxygen isotope fractionation observed at the cathode of a polymer electrolyte membrane fuel cell (PEMFC). The Pt(111) catalyst surface was modelled by a Pt19 cluster, and O, OH, OHH, OO, OOH, OHOH and HOHOH were assumed to be the oxygen species adsorbed on the Pt(111) surface. The oxygen isotope reduced partition function ratios (RPFRs) of the adsorbed species were calculated using the vibrational frequencies obtained by normal mode analyses performed on the optimized structures. Various oxygen isotope exchange equilibria among the adsorbed oxygen species and oxygen and water molecules in the gas phase were examined using their RPFRs. Experimental observation that the lighter 16O is enriched in water molecules exhausted from the cathode is explainable in a satisfactory manner by assuming oxygen isotope exchange equilibria of O2 molecule with O, OH, OO and OOH adsorbed on the Pt(111) surface that appear in the first half of the conversion reaction from O2 to H2O and those of H2O molecule with the adsorbed oxygen species, OHH, OHOH and HOHOH, formed in the latter half of the conversion reaction.

Effect of flutianil on the morphology and gene expression of powdery mildew.

Flutianil, a fungicide effective only on powdery mildew, was previously reported to affect the host cell's haustorial formation and nutrient absorption. Studies were conducted to investigate flutianil's primary site of action on Blumeria graminis morphology using transmission electron microscope (TEM) observation and RNA sequencing (RAN-seq) techniques. TEM observation revealed that flutianil caused the extra-haustorial matrix and fungal cell wall to be obscured, without remarkable changes of other fungal organelles. RNA-seq analysis indicated that, unlike other powdery-mildew fungicides, flutianil did not significantly affect the constantly expressed genes for the survival of B. graminis. Genes whose expression is up- or downregulated by flutianil were found; these are the three sugar transporter genes and various effector genes, mainly expressed in haustoria. These findings indicate that the primary site of action of flutianil might be in the haustoria.

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia.

Rice orange leaf phytoplasma (ROLP) causes clear orange to yellowish leaf discoloration and severe stunting in rice seedlings. The ecological and biological characteristics of ROLP are largely unknown because the disease has not widely caused serious problems in rice cultivated areas, thereby leading to the low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognised in other Asian countries, such as Vietnam, Thailand and the Philippines. Here, we observed the occurrence of ROLP in paddies of the Southeast Asian counties (Cambodia, Vietnam and the Philippines) and found that the isolates in the Philippines and Vietnam were monophyletic, while those in India, Thailand and Cambodia were more diverse, suggesting their potential origins. In Cambodia, it was revealed that following polymerase chain reaction (PCR) detection, the known ROLP-insect vectors, N. virescens Distant and Recilia dorsalis Motchulsky, were ROLP-positive, indicating their roles in pathogen dispersal. Moreover, fluorescent and scanning electron microscopy revealed the intensive accumulation of the phytoplasma in phloem tissues and massive accumulation of storage starch in vascular bundle sheath and parenchyma. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen's biological impact on rice tissue.

The role of Oryza sativa L. 'Milyang 44' husks on the resistance to two rice stink bugs.

To elucidate the resistance mechanisms of the rice (Oryza sativa L.) cultivar 'Milyang 44' against rice stink bugs, we compared the number of stylet sheaths, husk perforations, and feeding marks on the surface of the grains caused by Leptocorisa chinensis and Cletus punctiger on Milyang 44 and the control cultivar, i.e., 'Aichinokaori SBL'. We also examined the cross-sectional structure of the rice husks. We found that the number of stylet sheaths per panicle was higher in Milyang 44 than in Aichinokaori SBL for both rice stink bug species, except in one test involving C. punctiger. However, Milyang 44 had significantly less damage per number of stylet sheaths than Aichinokaori SBL, resulting in a lower percentage rates of pecky rice grains in Milyang 44. Interestingly, there was no difference in the percentage rates of pecky rice between the two cultivars after removing one third of the husks. Histological analysis showed that the sclerenchymatous cell wall containing lignin of husk was thicker in Milyang 44 than in Aichinokaori SBL, suggesting that the husk of Milyang 44 plays an important role in its resistance to these two rice stink bug species.

Failure of Pollen Attachment to the Stigma Triggers Elongation of Stigmatic Papillae in Arabidopsis thaliana.

Pollination is one of key determinants of yield production in important crops, such as grains and beans in which seeds are utilized as agricultural products. Thus, to fulfil food demand for growing world population, it is necessary to elucidate the mechanisms that regulate pollination, leading to increase in yield production. In this study, we compared detailed morphological characteristics of reproductive organs in Arabidopsis thaliana grown under control conditions or subjected to heat stress. Shorter length of anthers, filaments, and petals were observed in plants subjected to heat stress compared to those under control conditions. In contrast, heat stress resulted in enlargement of stigma via elongation of stigmatic papillae. Classification of stigmas based on patterns of pollen attachment indicated that pollen attachment to stigma clearly decreased under heat stress. In addition, artificial pollination experiment demonstrated that stigma shrank when pollen attached, but, continued to enlarge in the absence of pollen. Such modulation of stigma size depending on the presence or absence of pollen was observed both under control and heat stressed conditions. Taken together, these results suggest that elongation of stigmatic papillae is associated with failure of pollen attachment to the stigma, rather than heat stress. Furthermore, histochemical staining experiments suggest that Ca2+ derived from pollen together with O2 - might be associated with morphological alteration of stigma depending on the patterns of pollen attachment.

Estimation of water seepage rate in the active crater lake system of Kusatsu-Shirane volcano, Japan, using FDNPP-derived radioactive cesium as a hydrological tracer.

We describe here a first attempt to estimate the water seepage rate of an active crater lake using radioactive cesium dispersed into the environment by the Fukushima Dai-ichi Nuclear Power Plant accident in March 2011 as a hydrological tracer. Kusatsu-Shirane volcano is one of the most active volcanoes in Japan and has an active crater lake named Yugama. There is no outflow such as a river from Yugama crater lake. The content level of stable cesium (133Cs) in Yugama water was almost constant at 15-20 nM during the sampling period of Nov. 2011 to Nov. 2014. In contrast to 133Cs, however, the radioactive cesium (134Cs and 137Cs) concentrations in Yugama water decreased at a more rapid rate than expected by radioactive decay. Based on the decreasing rates of activity concentrations of 134Cs and 137Cs in Yugama water during the three years between 2012 and 2014, it is estimated that 700-800 m3 of the Yugama water, which corresponds approximately to 0.1% of the total volume, leaks through the lakebed per day. In the estimation, balance between leakage of radioactive cesium contaminated water though lake bed and geothermal inflow of radioactive cesium-free water in the volcano was taken into account. Consequently, the water seepage rate of the Yugama crater lake was calculated to be 8.1-9.3 L s-1.

Nitrogen Isotopic Reduced Partition Function Ratios of Glycine and Serine in Water.

The nitrogen isotopic reduced partition function ratio (RPFR) of glycine (Gly) and serine (Ser) in water has been evaluated by a couple of methods. The experimental fact that those amino acids are present in the zwitterionic form in water has been best reproduced when trihydrated Gly and Ser molecules were treated by the discrete microsolvation method coupled with the self-consistent reaction field (SCRF) approach. The RPFR values of Gly and Ser in water at 25 °C are evaluated as 1.11152 and 1.11070, respectively, and the equilibrium constant of the nitrogen isotope exchange reaction between the two amino acids is calculated as 1.0007 ± 0.0008 at 25 °C. Equivalent results may be obtained by the SCRF method for non- and monohydrated molecules in the zwitterionic form.

Benzoquinone synthesis-related genes of Tribolium castaneum confer the robust antifungal host defense to the adult beetles through the inhibition of conidial germination on the body surface.

Insects fight against invading microbial pathogens through various immune-related measures that comprise 'internal', 'external' as well as 'social' immunities. The defenses by external immunity associated with the cuticular integument are supposed to be of particular importance in repelling entomopathogenic fungi that infect host insects transcutaneously. Among such integument-related defenses, external secretions of benzoquinone derivatives typical of tenebrionid beetles have been suggested to play important roles in the antimicrobial defenses. In the present study, by utilizing the experimental infection system composed of the red flour beetle Tribolium castaneum and generalist ascomycete entomopathogens Beauveria bassiana and Metarhizium anisopliae, we performed the functional assays of the three T. castaneum genes whose involvement in benzoquinone synthesis in the adults has been reported, namely GT39, GT62 and GT63. Observations by scanning electron microcopy (SEM) revealed that the conidia of the two fungal species did not germinate on the wild-type adult body surface but did on the pupae. The expression analyses demonstrated that the levels of GT39 and GT62 mRNA increased from middle pupae and reached high in early adults while GT63 did not show a clear adult-biased expression pattern. The RNA interference-based knockdown of any of the three genes in pupae resulted in the adults compromised to the infection of the both fungal species. SEM observations revealed that the gene silencing allowed the conidial germination on the body surface of the knockdown beetles, thereby impairing the robust antifungal defense of adult beetles. Thus, we have provided direct experimental evidence for the functional importance in vivo of these benzoquinone synthesis-related genes that support the antifungal defense of tenebrionid beetles.

Three-dimensional ultrastructural change of chloroplasts in rice mesophyll cells responding to salt stress.

BACKGROUND AND AIMS: Excess salinity inhibits the metabolism of various systems and induces structural changes, especially in chloroplasts. Although the chloroplast body seems to swell under salinity stress as observed by conventional transmission electron microscopy, previous studies are limited to 2-D data and lack quantitative comparisons because specimens need to be sliced into ultrathin sections. This study shows three-dimensionally the structural changes in a whole mesophyll cell responding to salinity stress by serial sectioning with a focused ion beam scanning electron microscope (FIB-SEM) and compares the differences in chloroplast structures based on reconstructed models possessing accurate numerical voxel values. METHODS: Leaf blades of rice plants treated with 100 mm NaCl or without (control) for 4 d were fixed chemically and embedded in resin. The specimen blocks were sectioned and observed using the FIB-SEM, and then the sliced image stacks were reconstructed into 3-D models by image processing software. KEY RESULTS: On the transverse sections of rice mesophyll cells, the chloroplasts in the control leaves appeared to be elongated meniscus lens shaped, while those in the salt-treated leaves appear to be expanded oval shaped. The 3-D models based on serial sectioning images showed that the chloroplasts in the control cells spread like sheets fitted to the shape of the cell wall and in close contact with the adjacent chloroplasts. In contrast, those in the salt-stressed cells curled up into a ball and fitted to cell protuberances without being in close contact with adjacent chloroplasts. Although the shapes of chloroplasts were clearly different between the two treatments, their volumes did not differ. CONCLUSIONS: The 3-D reconstructed models of whole rice mesophyll cells indicated that chloroplasts under salt stress conditions were not swollen but became spherical without increasing their volume. This is in contrast to findings of previous studies based on 2-D images.

Observation of 18O/16O isotope effects at the cathode of a polymer electrolyte membrane fuel cell.

18O/16O isotope effects were observed at the cathode of a polymer electrolyte membrane fuel cell at 25 and 35°C. Results of experiments in which the 18O/16O isotope ratios of the oxygen gases supplied to and exhausted from the cell were measured revealed that the lighter isotope 16O reacted more preferentially to form water molecules at the cathode than the heavier one, 18O. The value of the oxygen isotope separation factor, S1, defined as the ratio of the 18O/16O isotope ratios of the oxygen gases supplied to and exhausted from the cell, ranged from 1.0030 to 1.0139, and tended to decrease with decreasing rate of oxygen utilisation (θ) and with increasing flow rate of the feed oxygen gas (DF). The value of another separation factor, S2, defined as the ratio of the 18O/16O isotope ratios of the exhausted oxygen gas and oxygen having reacted to form water molecules at the cathode, ranged from 1.0049 to 1.0304. The S2 value was much less affected by the change in θ and DF than the S1 value with the majority of the S2 value being in the range of 1.0240-1.0304.

Three-dimensional ultrastructure of chloroplast pockets formed under salinity stress.

We investigated the invagination structure of a chloroplast that surrounds organelles such as mitochondria and peroxisomes within a thin layer of chloroplast stroma, which is called a chloroplast pocket. In this study, chloroplast pockets were observed in rice plants subjected to salinity stress but not under moderate growth condition. They included cytosol, transparent structure, lipid bodies, mitochondria, and peroxisomes. We constructed the three-dimensional architecture of chloroplast pockets by using serial images obtained by transmission electron microscopy and focused ion beam-scanning electron microscopy. Three types of chloroplast pockets were observed by transmission electron microscopy: Organelles were completely enclosed in a chloroplast pocket (enclosed type), a chloroplast pocket with a small gap in the middle part (gap type), and a chloroplast pocket with one side open (open type). Of the 70 pockets observed by serial imaging, 35 were enclosed type, and 21 and 14 were gap and open types, respectively. Mitochondria and peroxisomes were often in contact with the chloroplast pockets. Focused ion beam-scanning electron microscopy revealed chloroplasts with a sheet structure partially surrounding peroxisomes. This fact suggests that chloroplasts might construct large sheet structures that would be related to the formation of chloroplast pockets.