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C 4 plants have C 4 photosynthetic cycle, a CO 2 -concentrating pump that functions across mesophyll (M) and bundle sheath (BS) cells. M chloroplasts aggregate toward BS cells in response to environmental stress, which would contribute to adjustment in C 4 photosynthetic cycle. However, it remains unclear whether M chloroplast movement is an intercellular response mediated by BS cells. One major challenge to resolve this is the difficulty in observing chloroplast movement due to scattering and absorption of observation light in live-leaf tissues. We established a live leaf-section imaging technique that enables the long-term observation of sections of chemically unfixed leaf blades, with which we quantitatively analyzed M chloroplast movements. Another challenge in clarifying the contribution of BS cells to M chloroplast movement is the selective ablation of BS cells without impairing M cell function. To investigate the necessity of BS cells for M chloroplast movement, we developed a method to remove BS cells only based on differences in shape between M and BS cells. We also found that chloroplasts in M cells without adjacent BS cell contents did not show typical aggregative movement. This indicates that the M chloroplast aggregative movement occurs during communication with BS cells.

Chloroplasts, mitochondria, and peroxisomes are consistently positioned in close proximity with physical contact in mesophyll cells, as shown by two-dimensional transmission electron microscopy observations. However, the three-dimensional arrangement of these organelles has not yet been investigated in rice. In this study, we investigated the positions and proximity of the chloroplasts, mitochondria, and peroxisomes in rice mesophyll cells three-dimensionally. Chloroplasts aligned along the cell walls, and mitochondria and peroxisomes were located on the intracellular side of the cell rather than in the peripheral chloroplasts. In total, 345 mitochondria were included in nine replicated cells, and 89.4% of the mitochondria formed binary complexes with peroxisomes. Binary complexes were located between the chloroplasts, forming clusters in the three organelles. Among the 134 chloroplasts and 210 peroxisomes detected in the nine mesophyll cells, only one chloroplast and one peroxisome were not included in the clusters. The clustering of chloroplasts, mitochondria, and peroxisomes at a high frequency has not been observed in other plants; therefore, this arrangement is considered to be unique to rice mesophyll cells, likely due to the smaller and denser cells than those of other plants. In the clusters, the three organelles were in contact with each other and in close membrane proximity. Considering the high correlation between the total volumes of mitochondria and peroxisomes and the area of membrane proximity among the three organelles, it is suggested that the proximity areas in rice mesophyll cells are determined by the size of the binary complexes within the clusters.

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.

Rice leaf blades have intricate-shaped mesophyll cells (MCs) with a large volume of chloroplasts enhancing gas exchange between stroma and intercellular airspace (IAS). Since the rice MCs do not form palisade or spongy tissue cells and are considered monotypic cells, the structural analysis of MCs in the middle part of the leaf tissue has been done, neglecting the various shapes of MCs can be observed on the cross-section of rice leaves. Moreover, the middle MC layer is sandwiched between the upper and lower layers and is more restricted in its demand for light and CO2 entering from the outside. Therefore, the different layers of MCs may differ in their sensitivity to salt stress that causes structural changes in cells. This study aims to elucidate the intra- and extra-cellular structures of MC in different layers of leaf tissue and determine how salinity affects the MC structure in each layer. The mesophyll tissue was divided into adaxial, middle and abaxial layers, and eight MCs and chloroplast regions were selected from each layer and reconstructed into three-dimensional (3D) representations. The whole leaf anatomical and physiological parameters were measured to find the effects of salinity stress on the MC structures. As a result, the 3D analysis of rice leaf tissue revealed the different structures of MCs with greater diversity in the adaxial and abaxial layers than in the middle layer. Salinity stress reduced the size and height of the MCs and coverage of the chloroplast on the cytoplasm periphery of the adaxial and abaxial layers, as well as the chloroplast size of adaxial MCs. Overall, these results reveal the variation of rice MC in leaf tissue and suggest the higher sensitivity to salt stress in the adaxial mesophyll among the layers, which may partly account for the decrease in photosynthetic capacity. This study focuses on the diverse structures of rice leaf mesophyll cells (MCs) across different layers and investigates their response to salinity stress. While middle-layer MCs are traditionally studied, our three-dimensional analysis reveals greater diversity in adaxial and abaxial layers. Salinity stress disproportionately affects adaxial MCs, reducing size and chloroplast coverage. These findings emphasize layer-specific variations in structural sensitivity, providing insights into the nuanced responses of rice MCs to environmental stressors.

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.

2D sections of rice (Oryza sativa L.) mesophyll cells are complex and the cell interior contains a large volume of chloroplasts. Furthermore, the structure of chloroplasts changes in response to salt stress. In this study, we used a 3D reconstruction method based on serial section light microscopy to analyze a wide range of structures in leaf tissues and compared the intracellular structure of mesophyll cells of rice under normal and salt-treated conditions. The 3D reconstructed models revealed that mesophyll cells appeared as ellipsoid discs with several lobes around the cell periphery, with the volumes showing no significant difference between control and salt-treated plants. The chloroplast structure in the whole mesophyll cell was altered under salt stress, showing a reduced coverage area, whereas their volumes did not differ between treatments. These findings suggest that 3D reconstruction based on serial light micrographs can reveal the morphology of cells and chloroplasts in plant tissue.

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 F 2 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.

Chloroplast protrusions (CPs), large stromal areas lacking thylakoid membranes, are present before the formation of Rubisco-containing bodies (RCBs). RCBs are round bodies found within the cytoplasm and transported to the vacuole via the autophagy pathway. In the present study, we observed CPs and RCBs in rice mesophyll cells under polyethylene glycol-induced osmotic stress following 3D reconstruction of transmission electron microscopy images of serial sections. Osmotic stress induced the formation of CPs and RCBs whose features were similar to those observed under salt stress, suggesting that osmotic effect contributes to the formation of CPs and RCBs. The 3D image of CPs revealed that some CPs were formed far from the main chloroplast body, and one of the CP was in physical contact with a mitochondrion and a peroxisome, which were located far from the main chloroplast body. Thus, the CPs may aid the association with mitochondria and peroxisomes far from the main chloroplast body. Additionally, a CP with a connection to the main chloroplast body by a narrow structure was observed three-dimensionally. Since the volume of CPs was similar to that of RCBs, the CP with the narrow structure may be a precursor structure just before the release of an RCB into the cytoplasm. In the present study, the volumes of two CPs among 24 CPs were markedly larger than the volumes of other CPs and RCBs. Thus, the large body of the CP could be an unviable structure that fails to release RCBs.

Background Sleep apnea headache is a major symptom accompanying obstructive sleep apnea (OSA), but relatively little evidence has been reported on the magnitude of its negative effects on patients or the evaluation of therapeutic effects. We quantitatively assessed sleep apnea headache using the Epworth sleepiness scale (ESS) and headache impact test (HIT)-6. Methods The first part of this study enrolled 86 patients (72 male; mean [± standard deviation] age 53.2 ± 13.8 years) who had been diagnosed with OSA by polysomnography in our sleep center and investigated the prevalence and characteristics of sleep apnea headache. The second part enrolled 21 patients (12 male; mean age, 47.5 ± 13.0 years) diagnosed with sleep apnea headache by polysomnography and/or peripheral arterial tonometry and evaluated the effects of OSA therapy on headache. Medical records including ESS, HIT-6, and polysomnographic data were retrospectively analyzed. Results The prevalence of sleep apnea headache among OSA patients was 22.1%, and was higher in female (50.0%) than in male (16.7%). The proportion of N3 and HIT-6 score showed a significant negative correlation (Pearson’s R = -0.51, p  < 0.05). In female, median apnea–hypopnea index (AHI) was significantly lower in patients with headache (26.1 /h [interquartile range 21.4–29.6 /h]) than in patients without (54.2 /h [41.3–73.9 /h], p  < 0.05, Wilcoxon rank-sum test). HIT-6 and ESS scores improved from 56.4 ± 9.7 to 45.9 ± 8.4 and from 9.0 ± 4.4 to 5.3 ± 4.2 in patients with oral appliance (OA), and from 54.3 ± 10.7 to 44.6 ± 6.1 and from 10.0 ± 4.0 to 4.9 ± 2.9 with continuous positive airway pressure (CPAP). In patients with good CPAP adherence, these scores improved from 58.1 ± 10.8 to 44.0 ± 6.0 and from 9.6 ± 3.8 to 3.6 ± 1.7. Conclusion Among patients with OSA, prevalence of sleep apnea headache was higher in female than in male. In female, headache was associated with relatively mild OSA. OA showed substantial effects on headache in mild to moderate OSA patients. CPAP adherence was important for improving the headache. HIT-6 score appears useful for the diagnosis and management of sleep apnea headache.

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-localizedmalate 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.