Daisuke Tamaoki

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.

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, ...

Nicotinamide mononucleotide and related metabolites induce disease resistance against fungal phytopathogens in Arabidopsis and barley

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), is known to act as a functional molecule in animals, whereas its function in plants is largely unknown. In this study, we found that NMN accumulated in barley cultivars resistant to phytopathogenic fungal Fusarium species. Although NMN does not possess antifungal activity, pretreatment with NMN and related metabolites enhanced disease resistance to Fusarium graminearum in Arabidopsis leaves and flowers and in barley spikes. The NMN-induced Fusarium resistance was accompanied by activation of the salicylic acid-mediated signalling pathway and repression of the jasmonic acid/ethylene-dependent signalling pathways in Arabidopsis. Since NMN-induced disease resistance was also observed in the SA-deficient sid2 mutant, an SA-independent signalling pathway also regulated the enhanced resistance induced by NMN. Compared with NMN, NAD and NADP, nicotinamide pretreatment had minor effects on resistance to F. graminearum. Constitutive expression of the NMNAT gene, which encodes a rate-limiting enzyme for NAD biosynthesis, resulted in enhanced disease resistance in Arabidopsis. Thus, modifying the content of NAD-related metabolites can be used to optimize the defence signalling pathways activated in response to F. graminearum and facilitates the control of disease injury and mycotoxin accumulation in plants.

Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.

Plasma membrane-anchored chloroplasts are necessary for the gravisensing system of Ceratopteris richardii prothalli

The prothalli of the fern Ceratopteris richardii exhibit negative gravitropism when grown in darkness. However, no sedimentable organelles or substances have been detected in the prothallial cells, suggesting that a non-sedimentable gravisensor exists. We investigated whether chloroplasts are involved in the gravisensing system of C. richardii prothalli. We used a clumped-chloroplast mutant, clumped chloroplast 1 (cp1), in which the chloroplasts are detached from the plasma membrane and clustered around the nucleus likely because of a partial deletion in the KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 gene. The cp1 mutation resulted in prothalli that had a significantly diminished gravitropic response, while the phototropic response occurred normally. These results suggest that plasma membrane-anchored chloroplasts in prothallial cells function as one of the gravisensors in C. richardii prothalli.