Ichirou Karahara

Salinity tolerance mechanisms in glycophytes: An overview with the central focus on rice plants

Elevated Na+ levels in agricultural lands are increasingly becoming a serious threat to the world agriculture. Plants suffer osmotic and ionic stress under high salinity due to the salts accumulated at the outside of roots and those accumulated at the inside of the plant cells, respectively. Mechanisms of salinity tolerance in plants have been extensively studied and in the recent years these studies focus on the function of key enzymes and plant morphological traits. Here, we provide an updated overview of salt tolerant mechanisms in glycophytes with a particular interest in rice (Oryza sativa) plants. Protective mechanisms that prevent water loss due to the increased osmotic pressure, the development of Na+ toxicity on essential cellular metabolisms, and the movement of ions via the apoplastic pathway (i.e. apoplastic barriers) are described here in detail.

Casparian strip development and its potential function in salt tolerance

The root system is particularly affected by unfavourable conditions because it is in direct contact with the soil environment. Casparian strips, a specialised structure deposited in anticlinal walls, are characterised by the impregnation of the primary wall pores with lignin and suberin. The Casparian strips in the endo- and exodermis of vascular plant roots appear to play an important role in preventing the non-selective apoplastic bypass of salts into the stele along the apoplast under salt stress. However, only a few investigations have examined the deposition and function of these apoplastic barriers in response to salt stress in higher plants.

Role of ethylene signalling in the formation of constitutive aerenchyma in primary roots of rice

Although aerenchyma formed in the roots of some species can be promoted by ethylene, such roots also form a somewhat less extensive aerenchyma under well-aerated conditions. It has been unclear whether ethylene is involved in promoting this constitutive aerenchyma. To test this possibility a novel sandwich method was employed in rice roots. A more extensive aerenchyma was formed on the ACC-treated side. 1-MCP inhibited aerenchyma formation in the presence or absence of ACC. The results indicate that ethylene signaling is involved in aerenchyma development in rice roots and that this may include the regulation of constitutive aerenchyma.

Involvement of auxin dynamics in hypergravity-induced promotion of lignin-related gene expression in Arabidopsis inflorescence stems

Recent studies have shown that hypergravity enhances lignification through up-regulation of the expression of lignin biosynthesis-related genes, although its hormonal signalling mechanism is unknown. The effects of hypergravity on auxin dynamics were examined using Arabidopsis plants that were transformed with the auxin reporter gene construct DR5::GUS. Hypergravity treatment at 300 g significantly increased β-glucuronidase activity in inflorescence stems of DR5::GUS plants, indicating that endogenous auxin accumulation was enhanced by hypergravity treatment. The hypergravity-related increased expression levels of both DR5::GUS and lignin biosynthesis-related genes in inflorescence stems were suppressed after disbudding, indicating that the increased expression of lignin biosynthesis-related genes is dependent on an increase in auxin influx from the shoot apex.

Radial widening of the Casparian strip follows induced radial expansion of endodermal cells

Abstract. The Casparian strip, the barrier to apoplastic transport that is located at the endodermis in roots and stems, is formed by individual endodermal cells and is constructed as a highly organized mesh within the primary wall. Since little is known about the mechanism of formation of the strip, we tried to obtain morphological evidence for the existence, prior to suberization and lignification, of some regulatory system at the expected site of the strip. Endodermal cells in etiolated pea stems were induced to expand in the radial direction by piercing the stems through the cortex before formation of the strip. The radial width of the strip increased significantly with the expansion of the radial walls of these endodermal cells. The expansion of the cells occurred before the formation of the strip. However, strips that had already been formed when the stems were pierced did not increase in width despite an induced expansion of the radial walls. These observations suggest that some positional information exists in the radial wall of endodermal cells that defines the future site of formation of the strip and its width.

Micro-CT observations of the 3D distribution of calcium oxalate crystals in cotyledons during maturation and germination in Lotus miyakojimae seeds

The cotyledon of legume seeds is a storage organ that provides nutrients for seed germination and seedling growth. The spatial and temporal control of the degradation processes within cotyledons has not been elucidated. Calcium oxalate (CaOx) crystals, a common calcium deposit in plants, have often been reported to be present in legume seeds. In this study, micro-computed tomography (micro-CT) was employed at the SPring-8 facility to examine the three-dimensional distribution of crystals inside cotyledons during seed maturation and germination of Lotus miyakojimae (previously Lotus japonicus accession Miyakojima MG-20). Using this technique, we could detect the outline of the embryo, void spaces in seeds and the cotyledon venation pattern. We found several sites that strongly inhibited X-ray transmission within the cotyledons. Light and polarizing microscopy confirmed that these areas corresponded to CaOx crystals. Three-dimensional observations of dry seeds indicated that the CaOx crystals in the L. miyakojimae cotyledons were distributed along lateral veins; however, their distribution was limited to the abaxial side of the procambium. The CaOx crystals appeared at stage II (seed-filling stage) of seed development, and their number increased in dry seeds. The number of crystals in cotyledons was high during germination, suggesting that CaOx crystals are not degraded for their calcium supply. Evidence for the conservation of CaOx crystals in cotyledons during the L. miyakojimae germination process was also supported by the biochemical measurement of oxalic acid levels.

Extracting tissue and cell outlines of Arabidopsis seeds using refraction contrast X-ray CT at the SPring-8 facility

How biological form is determined is one of the important questions in developmental biology. Physical forces are thought to be the primary determinants of the biological forms, and several theories for this were proposed nearly a century ago. To evaluate how physical forces can influence biological forms, precise determination of cell and tissue shapes and their geometries is necessary. Computed tomography (CT) is useful for visualizing three-dimensional structures without destroying a sample. Because recent progress in micro-CT has enabled visualizing cells and tissues at the sub-micron level, we investigated if we could extract cell and tissue outlines of seeds using refraction contrast X-ray CT available at the SPring-8 synchrotron radiation facility. We used Arabidopsis seeds because Arabidopsis is a well-known model plant and its seed size is small enough to obtain whole images using the X-ray CT experimental system. We could trace the outlines of tissues in dry seeds using beamline BL20B2 (10 keV, ...

Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells

Actin–microtubule interactions have been postulated to play an important role in the formation of the preprophase microtubule band, which predicts the future division site in plants. Electron tomography reveals that microfilaments are used as bridging structures to draw widely spaced microtubules together during microtubule band formation.

High-Pressure Freezing and Low-Temperature Processing of Plant Tissue Samples for Electron Microscopy

Use of electron tomography methods improves image resolution of transmission electron microscopy especially in the z-direction, enabling determination of complicated 3D structures of organelles and cytoskeleton arrays. The increase in resolution necessitates preservation of cellular structures close to the native states with minimum artifacts. High-pressure freezing (HPF) that immobilizes molecules in the cell instantaneously has been used to avoid damages caused by convention chemical fixation. Despite the advantages of HPF, cells could still be damaged during dissection prior to HPF. Therefore, it is critical to isolate cells/tissues of interest quickly and carefully. The samples frozen by HPF are often processed by freeze substitution (FS), and FS should be carried out under appropriate conditions. Here we describe dissection, HPF, and FS methods that we have utilized to prepare plant samples for electron tomography/immuno-electron microscopy.

The Role of Endocytosis in the Creation of the Cortical Division Zone in Plants

Control of the pattern of cell division is essential for the proper development of multicellular organisms. In animal cells, cytokinesis is mediated by a contractile ring in which the cleavage force is produced by an acto-myosin system. Furthermore, the future site of cell division in animal cells (the site where contraction starts in the cell cortex) is determined by the position of the aster during the later stages of mitosis. In contrast, plant cytokinesis involves the assembly of a cell plate from Golgi-derived vesicles. The division site in plants (the cell cortex where the cell plate fuses with the parental cell walls) is defined by a band of cortical microtubules (MTs) – the preprophase band (PPB) of MTs – that mark the division site during prophase. The PPB MTs subsequently disassemble when the cells enter prometaphase. However, some positional information, or positional memory, is retained in the cell cortex/plasma membrane where the PPB of MTs was located, and the cell plate edge grows towards and fuses with this predetermined division site. Thus, how MTs demarcate the future division site during PPB development, and how the division site memory is created and maintained in the PPB region until the end of cytokinesis, are important questions related to the regulation of division plane positioning in plants.