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.

Relationships between respiratory and oromotor events differ between motor phenotypes in patients with obstructive sleep apnea.

Purpose: The present study investigated the relationship between sleep bruxism (SB) and obstructive sleep apnea (OSA) in relation to the sleep architecture. Methods: We conducted a cross-sectional study. Polysomnographic recordings were performed on 36 patients. Sleep, respiratory, and oromotor variables, such as rhythmic masticatory muscle activity (RMMA) and non-specific masticatory muscle activity (NSMA), were compared between OSA patients with or without SB. A correlation analysis of the frequency of respiratory and oromotor events in NREM and REM sleep was performed. The frequency of oromotor events following respiratory events was also assessed. Results: The proportion of REM sleep was higher in OSA patients with SB than in those without SB (p = 0.02). The apnea-hypopnea index (AHI) did not significantly differ between the two groups; however, AHI was approximately 8-fold lower during REM sleep in OSA patients with SB (p = 0.01) and the arousal threshold was also lower (p = 0.04). Although the RMMA index was higher in OSA patients with than in those without SB (p < 0.01), the NSMA index did not significantly differ. The percentage of RMMA following respiratory events was significantly higher in OSA patients with than in those without SB, whereas that of NSMA did not significantly differ. The frequency of oromotor events throughout the whole night positively correlated with AHI. However, regardless of the sleep state, AHI did not correlate with the RMMA index, but positively correlated with the NSMA index. Conclusion: In consideration of the limitations of the present study, the results obtained indicate that OSA patients with SB have a unique phenotype of OSA and also emphasize the distinct relationship of respiratory events with RMMA and NSMA.

QTL analysis for sodium removal ability in rice leaf sheaths under salinity using an IR-44595/318 F2 population.

Over-accumulation of salt in rice plants is an effect of salt stress which decreases growth and grain yield. Salt removal ability in leaf sheaths is a tolerance mechanism to decrease salt entry and accumulation in leaf blades and maintain photosynthesis under salinity. In this study, a QTL analysis of removal ability of sodium ions (Na+) in leaf sheaths and Na+ accumulation-related traits, was conducted using F2 population between two rice varieties, IR-44595 with superior Na+ removal ability, and 318 with contrasting Na+ removal ability in leaf sheaths under salinity. Suggestive QTLs for Na+ removal ability in leaf sheaths were found on chromosomes 4 and 11. The suggestive QTL on chromosome 11 overlapped with other significant QTLs for Na+ concentration in shoots, leaf blades and leaf sheaths, and Na+/K+ ratio in leaf blades. Correlation analysis indicated that Na+ removal ability in leaf sheaths is important in reducing Na+ accumulation in leaf blades. The varietal difference of Na+ removal ability in leaf sheaths at the whole plant level was greater at lower NaCl concentrations and became smaller as the treatment NaCl concentration increased. Although the Na+ removal ability in leaf sheath was comparable between IR-44595 and 318 under high salinity at the whole plant level, the younger leaves of IR-44595 still showed a higher Na+ sheath-blade ratio than 318, which implied the Na+ removal ability functions in the younger leaves in IR-44595 to reduce Na+ entry in young leaf blades even under high salinity.

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.

DCT4-A New Member of the Dicarboxylate Transporter Family in C4 Grasses.

Malate transport shuttles atmospheric carbon into the Calvin-Benson cycle during NADP-ME C4 photosynthesis. Previous characterizations of several plant dicarboxylate transporters (DCT) showed that they efficiently exchange malate across membranes. Here, we identify and characterize a previously unknown member of the DCT family, DCT4, in Sorghum bicolor. We show that SbDCT4 exchanges malate across membranes and its expression pattern is consistent with a role in malate transport during C4 photosynthesis. SbDCT4 is not syntenic to the characterized photosynthetic gene ZmDCT2, and an ortholog is not detectable in the maize reference genome. We found that the expression patterns of DCT family genes in the leaves of Zea mays, and S. bicolor varied by cell type. Our results suggest that subfunctionalization, of members of the DCT family, for the transport of malate into the bundle sheath plastids, occurred during the process of independent recurrent evolution of C4 photosynthesis in grasses of the PACMAD clade. We also show that this subfunctionalization is lineage independent. Our results challenge the dogma that key C4 genes must be orthologues of one another among C4 species, and shed new light on the evolution of C4 photosynthesis.

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.

Rate of augmentation and risk factors with long-term follow-up in Japanese patients with restless legs syndrome.

OBJECTIVES: Preventing augmentation is the critical clinical issue for RLS treatment. As for augmentation in Asian RLS patients, there have been only four studies and the follow-up durations of these studies were not long. We investigated Japanese RLS patients with longer duration of treatment in a clinical setting. METHODS: This study is a retrospective assessment of 42 RLS patients with follow-up durations of longer than 18 months (78.4 ± 29.2, range 19-139) at two urban sleep centers in Osaka, Japan from May 2004 to April 2014. RESULTS: The mean age of first visit was 63.5 ± 14.1 years old and the estimated age of RLS onset was 47.9 ± 16.5 years old. Twenty-eight out of 42 patients were female. At initial evaluation, the mean International Restless Legs Scale score (IRLS score) was 22.0 ± 5.9. Thirty-one of 42 had already visited other clinics before coming to our sleep centers, and the number of clinics visited was 1.3 ± 0.6. Augmentation developed in two patients (4.8%), and the dosage of dopamine equivalent in patients with and without augmentation was 12.5 and 18.8 mg vs. 15.8 ± 17.7 mg. In the two RLS patients with augmentation, ferritin was 113.1 and 114.1 ng/mL, respectively, and the number of clinics before coming to our sleep centers was both three. CONCLUSIONS: The augmentation rate of Japanese RLS patients from our study is low compared with previous Western and Asian studies. It might be attributable to racial difference, lower dosage of dopaminergic treatment, and the level of ferritin.

[A case of severe obstructive sleep apnea mimicking REM sleep behavior disorder].

The rapid eye movement (REM) sleep behavior disorder (RBD) is a parasomnia characterized by dream-enacting behaviors related to the loss of the normal generalized skeletal muscle atonia during REM sleep, and shows REM sleep without atonia (RWA) during polysomnography (PSG). Patients with idiopathic RBD have been known to have a siginificantly increased risk of developing one of the α-synucleiopathies later in life, therefore the diagnosis of RBD is very important and must be dealt with carefully. A 51-year-old man was identified presenting dream-enacting behaviors and unpleasant dreams suggesting the diagnosis of RBD, in addition to snoring and excessive daytime sleepiness. Attended video-PSG excluded RBD showing REM sleep with atonia and without increased phasic EMG activity, and diagnosed with severe obstructive sleep apnea (OSA) with an apnea-hypopnea index of 30.1 demonstrating that the reported abnormal sleep behaviors occurred only during respiratory event-induced arousals. Continuous positive airway pressure therapy eliminated the abnormal behaviors, unpleasant dreams, snoring and daytime hypersomnolence. This case shows that severe OSA mimic the symptoms of RBD and that attended video-PSG is necessary to establish the diagnosis of RBD, and identify or exclude other causes of dream-enacting behaviors.

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.

Three-dimensional intracellular structure of a whole rice mesophyll cell observed with FIB-SEM.

Background and Aims: Ultrathin sections of rice leaf blades observed two-dimensionally using a transmission electron microscope (TEM) show that the chlorenchyma is composed of lobed mesophyll cells, with intricate cell boundaries, and lined with chloroplasts. The lobed cell shape and chloroplast positioning are believed to enhance the area available for the gas exchange surface for photosynthesis in rice leaves. However, a cell image revealing the three-dimensional (3-D) ultrastructure of rice mesophyll cells has not been visualized. In this study, a whole rice mesophyll cell was observed using a focused ion beam scanning electron microscope (FIB-SEM), which provides many serial sections automatically, rapidly and correctly, thereby enabling 3-D cell structure reconstruction. Methods: Rice leaf blades were fixed chemically using the method for conventional TEM observation, embedded in resin and subsequently set in the FIB-SEM chamber. Specimen blocks were sectioned transversely using the FIB, and block-face images were captured using the SEM. The sectioning and imaging were repeated overnight for 200-500 slices (each 50 nm thick). The resultant large-volume image stacks ( x = 25 µm, y = 25 µm, z = 10-25 µm) contained one or two whole mesophyll cells. The 3-D models of whole mesophyll cells were reconstructed using image processing software. Key Results: The reconstructed cell models were discoid shaped with several lobes around the cell periphery. The cell shape increased the surface area, and the ratio of surface area to volume was twice that of a cylinder having the same volume. The chloroplasts occupied half the cell volume and spread as sheets along the cell lobes, covering most of the inner cell surface, with adjacent chloroplasts in close contact with each other. Conclusions: Cellular and sub-cellular ultrastructures of a whole mesophyll cell in a rice leaf blade are demonstrated three-dimensionally using a FIB-SEM. The 3-D models and numerical information support the hypothesis that rice mesophyll cells enhance their CO 2 absorption with increased cell surface and sheet-shaped chloroplasts.

Subjective oropharyngeal symptoms for abnormal swallowing in Japanese patients with obstructive sleep apnea syndrome: a descriptive questionnaire study.

OBJECTIVES: This study aimed to investigate the prevalence of clinical symptoms related to abnormal swallowing in a large sample of obstructive sleep apnea syndrome (OSAS) patients. METHODS: Oropharyngeal symptoms for abnormal swallowing were assessed by a self-administered questionnaire in 507 consecutive patients (females: 65, males: 442; mean age: 49.6 ± 12.6 years old) with clinical symptoms of OSAS, enrolled for cardiorespiratory evaluation. RESULTS: Overall, 16.2% of patients (82/507) had at least one symptom for abnormal swallowing and 6.3% (32/507) had two or more symptoms. The most frequent symptom was difficulty with coughing up phlegm during or after a meal (8.3%). Demographic, sleep, and clinical variables did not differ between the patients with and without abnormal symptoms. CONCLUSIONS: The results of the current study showed that 16% of middle-aged OSAS patients reported pharyngeal symptoms related to abnormal swallowing, regardless of the severity of OSAS.

Interactions of C4 Subtype Metabolic Activities and Transport in Maize Are Revealed through the Characterization of DCT2 Mutants.

C4 photosynthesis in grasses requires the coordinated movement of metabolites through two specialized leaf cell types, mesophyll (M) and bundle sheath (BS), to concentrate CO2 around Rubisco. Despite the importance of transporters in this process, few have been identified or rigorously characterized. In maize (Zea mays), DCT2 has been proposed to function as a plastid-localized malate transporter and is preferentially expressed in BS cells. Here, we characterized the role of DCT2 in maize leaves using Activator-tagged mutant alleles. Our results indicate that DCT2 enables the transport of malate into the BS chloroplast. Isotopic labeling experiments show that the loss of DCT2 results in markedly different metabolic network operation and dramatically reduced biomass production. In the absence of a functioning malate shuttle, dct2 lines survive through the enhanced use of the phosphoenolpyruvate carboxykinase carbon shuttle pathway that in wild-type maize accounts for ∼ 25% of the photosynthetic activity. The results emphasize the importance of malate transport during C4 photosynthesis, define the role of a primary malate transporter in BS cells, and support a model for carbon exchange between BS and M cells in maize.

Salinity induces membrane structure and lipid changes in maize mesophyll and bundle sheath chloroplasts.

The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress.

The soybean mycorrhiza-inducible phosphate transporter gene, GmPT7, also shows localized expression at the tips of vein endings of senescent leaves.

GmPT7 was originally identified as an arbuscular mycorrhiza-inducible gene of soybean that encodes a member of subfamily I in the PHOSPHATE TRANSPORTER 1 family. In the present study, we established conditions under which a number of dwarf soybean plants complete their life cycles in a growth chamber. Using this system, we grew transgenic soybean with a GmPT7 promoter-ß-glucuronidase fusion gene and evaluated GmPT7 expression in detail. GmPT7 was highly expressed in mature, but not in collapsed, arbuscule-containing cortical cells, suggesting its importance in the absorption of fungus-derived phosphate and/or arbuscule development. GmPT7 was also expressed in the columella cells of root caps and in the lateral root primordia of non-mycorrhizal roots. The expression of GmPT7 occurred only in the late stage of phosphorus translocation from leaves to seeds, after water evaporation from the leaves ceased, and later than the expression of GmUPS1-2, GmNRT1.7a and GmNRT1.7b, which are possibly involved in nitrogen export. GmPT7 expression was localized in a pair of tracheid elements at the tips of vein endings of senescent leaves. Transmission electron microscopy revealed that the tip tracheid elements in yellow leaves were still viable and had intact plasma membranes. Thus, we think that GmPT7 on the plasma membranes transports phosphate from the apoplast into the tip elements. GmPT7 knockdown resulted in no significant effects, the function of GmPT7 remaining to be clarified. We propose a working model in which phosphate incorporated in vein endings moves to seeds via xylem to phloem transfer.