Takaaki Miyaji

As a staple food for half the world’s population, rice is an important dietary source of magnesium (Mg), an essential mineral for human health. Enhanced Mg accumulation in rice grains has also been linked to eating quality. However, the mechanisms underlying Mg transport to the grains remains poorly understood. Here, we report that OsMGR2, a member belonging to Magnesium Release (MGR) family, is required for Mg accumulation in rice grains. OsMGR2 encodes a plasma membrane-localized transporter that mediates Mg efflux. OsMGR2 is constitutively and highly expressed in the stele tissues of roots, the phloem region of both enlarged and diffused vascular bundles in nodes, and the ovular vascular trace of caryopses. Knockout of this gene results in decreased root-to-shoot translocation and altered distribution of Mg to different organs; less Mg is allocated to the second newest leaf with high Mg requirement for active photosynthesis. The osmgr2 mutants exhibit decreased Mg accumulation in the grain, which are smaller, lighter, and shriveled, but show increased accumulation in the husk. The eating quality of the mutant grains is significantly decreased compared with the wild-type rice. These results indicate that OsMGR2 plays multiple roles within the rice; facilitating the root-to-shoot Mg translocation, mediating phloem-to-xylem Mg transfer at nodes for preferential distribution to the most active leaf, and exporting Mg from maternal vascular tissues of the caryopsis to the grains, processes essential for grain development and eating quality in rice.

Intracellular zinc (Zn2+) homeostasis is essential for physiological and pathological processes and is strictly regulated by Zn2+ transporters. Zinc transporter 1 (ZnT1) is a ubiquitously expressed plasma membrane-localized Zn transporter that exports Zn2+ from the cytoplasm to the extracellular space. However, the functional transport properties regarding kinetics and driving forces of ZnT1 remain debatable. In this study, we established a cell-free proteoliposome assay system and demonstrated that ZnT1 transports Zn2+ with high affinity in pH-dependent and pH-independent manners. The Km and Vmax of pH-dependent Zn2+ transport were 0.40 μM and 15.13 nmol/min/mg protein, and those of pH-independent Zn2+ transport were 0.52 μM and 8.88 nmol/min/mg protein (low concentrations of Zn2+), 3.02 μM and 17.59 nmol/min/mg protein (high concentrations of Zn2+), respectively, suggesting biphasic kinetic components of Zn2+ transport. Even without pH gradient formation, ZnT1 exhibits potent Zn2+ transport activity. In pH dependency, Zn2+ transport activity was higher at an inside pH of 6.0 than at 6.5–7.5 for proteoliposomes, despite the same ΔpH of 0.5–1.5. The Zn2+ transport activity decreased at an outside pH of 8.0, despite an increase in ΔpH. Although previous studies have proposed that ZnT1-mediated Zn2+ transport activity is driven by a calcium (Ca2+) gradient and not by a pH gradient, Ca2+ does not enhance Zn2+ transport activity in the presence or absence of a pH gradient. These results strongly suggest that ZnT1 protein transports Zn2+ optimally at a specific pH and exports excess intracellular Zn2+ even without ΔpH.

Iron (Fe) is an essential micronutrient for plant growth and development. It plays crucial roles in various organs and tissues of plants, but the molecular mechanisms governing its distribution to the above-ground parts after root uptake remain unclear. In this study, we identify OsIET1 (Oryza sativa Iron Efflux Transporter 1), a rice gene highly expressed in the nodes. OsIET1 encodes a plasma membrane-localized protein, which shows efflux transport activity for ferrous iron. It is predominantly expressed in the xylem regions of diffuse vascular bundles, and its expression is upregulated under high Fe conditions. Disruption of OsIET1 impairs Fe allocation, reducing Fe transport to developing tissues (young leaves and grains), while increasing accumulation in nodes and older leaves. This misdistribution causes chlorosis in young leaves and decreases grain yield, especially under Fe-deficient conditions. Furthermore, we detect excessive Fe deposition around the xylem of diffuse vascular bundles in the nodes. Given the pivotal role of nodes in mineral distribution, our results indicate that OsIET1 mediates inter-vascular Fe transfer by facilitating Fe loading into the xylem of diffuse vascular bundles. This process ensures preferential Fe delivery to developing tissues, thereby promoting optimal plant growth and productivity.

Biochemical applications are increasingly utilized in paleontological studies, especially for detecting ancient proteins in fossil samples. Histopathological staining techniques have been applied, but they have yet to specifically target type I collagen, the primary bone matrix protein and the most significant protein of interest in paleoproteomic research. Moreover, these staining methods are often applied to demineralized fossils, which remove the original microstructure of the bone matrix and increase the risk of contamination. To address these limitations, this study aimed to test the effectiveness of special staining methods for detecting collagen using Pleistocene-aged fossil specimens. Trials on demineralized and nondemineralized modern bone samples, as well as nondemineralized fossil samples, demonstrated Van Gieson's staining method as the most suitable for visualizing collagen distribution in hard tissue matrices. Colorimetric analysis, electrophoresis, and subsequent mass spectrometry of extracts further confirmed the endogenous nature of the collagen in the fossil samples. Future studies may benefit from employing Van Gieson's staining on nondemineralized bone samples to detect collagen in fossils, advancing our understanding of ancient protein preservation.

Glomerular podocytes act as a part of the filtration barrier in the kidney. The activity of this filter is regulated by ionotropic and metabotropic glutamate receptors. Adjacent podocytes can potentially release glutamate into the intercellular space; however, little is known about how podocytes release glutamate. Here, we demonstrated vesicular glutamate transporter 3 (VGLUT3)-dependent glutamate release from podocytes. Immunofluorescence analysis revealed that rat glomerular podocytes and an immortal mouse podocyte cell line (MPC) express VGLUT1 and VGLUT3. Consistent with this finding, quantitative RT-PCR revealed the expression of VGLUT1 and VGLUT3 mRNA in undifferentiated and differentiated MPCs. In addition, the exocytotic proteins vesicle-associated membrane protein 2, synapsin 1, and synaptophysin 1 were present in punctate patterns and colocalized with VGLUT3 in MPCs. Interestingly, approximately 30% of VGLUT3 colocalized with VGLUT1. By immunoelectron microscopy, VGLUT3 was often observed around clear vesicle-like structures in differentiated MPCs. Differentiated MPCs released glutamate following depolarization with high potassium levels and after stimulation with the muscarinic agonist pilocarpine. The depletion of VGLUT3 in MPCs by RNA interference reduced depolarization-dependent glutamate release. These results strongly suggest that VGLUT3 is involved in glutamatergic signalling in podocytes and may be a new drug target for various kidney diseases.

Glomerular podocytes act as a part of filtration barrier in the kidney. The activity of this filter is regulated by ionotropic and metabotropic glutamate receptors. Adjacent podocytes can potentially release glutamate into intercellular space; however, little is known about how podocytes release glutamate. Here, we demonstrated vesicular glutamate transporter 3 (VGLUT3)-dependent glutamate release from podocytes. Immunofluorescence analysis revealed that rat glomerular podocytes and an immortal mouse podocyte cell line (MPCs) express VGLUT1 and VGLUT3. Consistent with this finding, quantitative RT-PCR revealed the expression of VGLUT1 and VGLUT3 mRNA in undifferentiated and differentiated MPCs. In addition, the exocytotic proteins vesicle-associated membrane protein 2, synapsin 1, and synaptophysin 1 were present in punctate patterns, and colocalised with VGLUT3 in MPCs. Interestingly, approximately 30% of VGLUT3 colocalised with VGLUT1. By immunoelectron microscopy, VGLUT3 was often observed around clear vesicle-like structures in differentiated MPCs. Differentiated MPCs released glutamate following depolarization with high potassium levels and after stimulation with the muscarinic agonist pilocarpine. Depletion of VGLUT3 in MPCs by RNA interference reduced depolarization-dependent glutamate release. These results strongly suggest that VGLUT3 is involved in glutamatergic signaling in podocytes and maybe a new drug target for various kidney diseases.

Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design.

Summary: Iron (Fe) needs to be delivered to different organs and tissues of above‐ground parts for playing its multiple physiological functions once it is taken up by the roots. However, the mechanisms underlying Fe distribution are poorly understood. We functionally characterized OsOPT7, a member of oligo peptide transporter family in terms of expression patterns, localization, transport activity and phenotypic analysis of knockdown lines. OsOPT7 was highly expressed in the nodes, especially in the uppermost node I, and its expression was upregulated by Fe‐deficiency. OsOPT7 transports ferrous iron into the cells coupled with proton. Immunostaining revealed that OsOPT7 is mainly localized in the xylem parenchyma cells of the enlarged vascular bundles in the nodes and vascular tissues in the leaves. Knockdown of OsOPT7 did not affect the Fe uptake, but altered Fe distribution; less Fe was distributed to the new leaf, upper nodes and developing panicle, but more Fe was distributed to the old leaves. Furthermore, knockdown of OsOPT7 also resulted in less Fe distribution to the leaf sheath, but more Fe to the leaf blade. Taken together, OsOPT7 is involved in the xylem unloading of Fe for both long‐distance distribution to the developing organs and local distribution within the leaf in rice.

During Drosophila aversive olfactory conditioning, aversive shock information needs to be transmitted to the mushroom bodies (MBs) to associate with odor information. We report that aversive information is transmitted by ensheathing glia (EG) that surround the MBs. Shock induces vesicular exocytosis of glutamate from EG. Blocking exocytosis impairs aversive learning, whereas activation of EG can replace aversive stimuli during conditioning. Glutamate released from EG binds to N -methyl- d -aspartate receptors in the MBs, but because of Mg ²⁺ block, Ca ²⁺ influx occurs only when flies are simultaneously exposed to an odor. Vesicular exocytosis from EG also induces shock-associated dopamine release, which plays a role in preventing formation of inappropriate associations. These results demonstrate that vesicular glutamate released from EG transmits negative valence information required for associative learning.

Silicon (Si) is the most abundant mineral element in the earth’s crust. Some plants actively accumulate Si as amorphous silica (phytoliths), which can protect plants from stresses. Here, we report a gene (SIET4) that is required for the proper accumulation and cell-specific deposition of Si in rice and show that it is essential for normal growth. SIET4 is constitutively expressed in leaves and encodes a Si transporter. SlET4 polarly localizes at the distal side of epidermal cells and cells surrounding the bulliform cells (motor cells) of the leaf blade, where Si is deposited. Knockout of SIET4 leads to the death of rice in the presence but not absence of Si. Further analysis shows that SIET4 knockout induces abnormal Si deposition in mesophyll cells and the induction of hundreds of genes related to various stress responses. These results indicate that SIET4 is required for the proper export of Si from leaf cells to the leaf surface and for the healthy growth of rice on land.