Tomoo Shimada

Bio: Tomoo Shimada is an academic researcher from Kyoto University. The author has contributed to research in topics: Endoplasmic reticulum & Vacuole. The author has an hindex of 47, co-authored 105 publications receiving 6403 citations. Previous affiliations of Tomoo Shimada include National Institute for Basic Biology, Japan & Kobe University.

Stomagen positively regulates stomatal density in Arabidopsis

Abstract: As the portals through which carbon dioxide is exchanged between the epidermal leaf tissue of plants and the atmosphere, the stomata are key elements in plant physiology. As such they are the focus of genetic and environmental regulation in plants, but until now no positive signalling factor of stomata development has been identified. Now a protein with stomata-inducing properties has been identified in Arabidopsis. Termed stomagen, it is a cysteine-rich peptide found in the inner tissue (mesophyll) layers of young leaves where it initiates the stomatal lineage possibly by binding to the cell-surface receptor TMM. This discovery raises the prospect that stomagen might be used to develop crops and trees with high stomatal densities and hence high absorption capacity for CO2, not only by genetic engineering, but also by spraying with synthetic stomagen or related synthetic peptides. Stomata are specialized structures in the epidermal layer of leaves that regulate the exchange of gases between the plant and the atmosphere. One or more positive intercellular signalling factors are assumed to be involved in stomatal development, but their identities remain elusive. A novel secretory protein — named stomagen — is now shown to be just such a factor; it is conserved among vascular plants and positively regulates stomatal density. Stomata in the epidermal tissues of leaves are valves through which passes CO2, and as such they influence the global carbon cycle1. The two-dimensional pattern and density of stomata in the leaf epidermis are genetically and environmentally regulated to optimize gas exchange2. Two putative intercellular signalling factors, EPF1 and EPF2, function as negative regulators of stomatal development in Arabidopsis, possibly by interacting with the receptor-like protein TMM3,4,5,6. One or more positive intercellular signalling factors are assumed to be involved in stomatal development, but their identities are unknown7. Here we show that a novel secretory peptide, which we designate as stomagen, is a positive intercellular signalling factor that is conserved among vascular plants. Stomagen is a 45-amino--rich peptide that is generated from a 102-amino-acid precursor protein designated as STOMAGEN. Both an in planta analysis and a semi-in-vitro analysis with recombinant and chemically synthesized stomagen peptides showed that stomagen has stomata-inducing activity in a dose-dependent manner. A genetic analysis showed that TMM is epistatic to STOMAGEN (At4g12970), suggesting that stomatal development is finely regulated by competitive binding of positive and negative regulators to the same receptor. Notably, STOMAGEN is expressed in inner tissues (the mesophyll) of immature leaves but not in the epidermal tissues where stomata develop. This study provides evidence of a mesophyll-derived positive regulator of stomatal density. Our findings provide a conceptual advancement in understanding stomatal development: inner photosynthetic tissues optimize their function by regulating stomatal density in the epidermis for efficient uptake of CO2.

Transport of Storage Proteins to Protein Storage Vacuoles Is Mediated by Large Precursor-Accumulating Vesicles

Abstract: Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and had diameters of 200 to 400 nm. They contained an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. Immunocytochemical analysis revealed numerous electron-dense aggregates of storage proteins within the endoplasmic reticulum. It is likely that these aggregates develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis also showed that complex glycans are associated with the peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles.

Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells

Abstract: Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ∼3.5 μm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.

A rapid and non‐destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana

Abstract: The creation of transgenic plants has contributed extensively to the advancement of plant science. Establishing homozygous transgenic lines is time-consuming and laborious, and using antibiotics or herbicides to select transformed plants may adversely affect the growth of some transgenic plants. Here we describe a novel technology, which we have named FAST (fluorescence-accumulating seed technology), that overcomes these difficulties. Although this technology was designed for use in Arabidopsis thaliana, it may be adapted for use in other plants. The technology is based on the expression of a fluorescent co-dominant screenable marker FAST, under the control of a seed-specific promoter, on the oil body membrane. The FAST marker harbors a fusion gene encoding either GFP or RFP with an oil body membrane protein that is prominent in seeds. The marker protein was only expressed in a specific organ (i.e. in dry seeds) and at a specific time (i.e. during dormancy), which are desirable features of selectable and/or screenable markers. This technique provides an immediate and non-destructive method for identifying transformed dry seeds. It identified the heterozygous transformed seeds among the T(1) population and the homozygous seeds among the T(2) population with a false-discovery rate of <1%. The FAST marker reduces the length of time required to produce homozygous transgenic lines from 7.5 to 4 months. Furthermore, it does not require sterilization, clean-bench protocols or the handling of large numbers of plants. This technology should greatly facilitate the generation of transgenic Arabidopsis plants.

Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana

Abstract: The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1/AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 12S globulin in a Ca2+-dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.

The new frontier of genome engineering with CRISPR-Cas9

Abstract: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

How Matrix Metalloproteinases Regulate Cell Behavior

Abstract: ▪ Abstract The matrix metalloproteinases (MMPs) constitute a multigene family of over 25 secreted and cell surface enzymes that process or degrade numerous pericellular substrates. Their targets include other proteinases, proteinase inhibitors, clotting factors, chemotactic molecules, latent growth factors, growth factor–binding proteins, cell surface receptors, cell-cell adhesion molecules, and virtually all structural extracellular matrix proteins. Thus MMPs are able to regulate many biologic processes and are closely regulated themselves. We review recent advances that help to explain how MMPs work, how they are controlled, and how they influence biologic behavior. These advances shed light on how the structure and function of the MMPs are related and on how their transcription, secretion, activation, inhibition, localization, and clearance are controlled. MMPs participate in numerous normal and abnormal processes, and there are new insights into the key substrates and mechanisms responsible for regula...

A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.

Abstract: Genome sequencing has resulted in the identification of a large number of uncharacterized genes with unknown functions It is widely recognized that determination of the intracellular localization of the encoded proteins may aid in identifying their functions To facilitate these localization experiments, we have generated a series of fluorescent organelle markers based on well-established targeting sequences that can be used for co-localization studies In particular, this organelle marker set contains indicators for the endoplasmic reticulum, the Golgi apparatus, the tonoplast, peroxisomes, mitochondria, plastids and the plasma membrane All markers were generated with four different fluorescent proteins (FP) (green, cyan, yellow or red FPs) in two different binary plasmids for kanamycin or glufosinate selection, respectively, to allow for flexible combinations The labeled organelles displayed characteristic morphologies consistent with previous descriptions that could be used for their positive identification Determination of the intracellular distribution of three previously uncharacterized proteins demonstrated the usefulness of the markers in testing predicted subcellular localizations This organelle marker set should be a valuable resource for the plant community for such co-localization studies In addition, the Arabidopsis organelle marker lines can also be employed in plant cell biology teaching labs to demonstrate the distribution and dynamics of these organelles

Redox Regulation in Photosynthetic Organisms: Signaling, Acclimation, and Practical Implications

Abstract: Reactive oxygen species (ROS) have multifaceted roles in the orchestration of plant gene expression and gene-product regulation. Cellular redox homeostasis is considered to be an “integrator” of information from metabolism and the environment controlling plant growth and acclimation responses, as well as cell suicide events. The different ROS forms influence gene expression in specific and sometimes antagonistic ways. Low molecular antioxidants (e.g., ascorbate, glutathione) serve not only to limit the lifetime of the ROS signals but also to participate in an extensive range of other redox signaling and regulatory functions. In contrast to the low molecular weight antioxidants, the “redox” states of components involved in photosynthesis such as plastoquinone show rapid and often transient shifts in response to changes in light and other environmental signals. Whereas both types of “redox regulation” are intimately linked through the thioredoxin, peroxiredoxin, and pyridine nucleotide pools, they ...

Acyl-Lipid Metabolism

Abstract: Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.