Kiyoshi Masuda

Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture.

Abstract. Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 × 10−4 M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis.

Sugar signalling mediates cluster root formation and phosphorus starvation-induced gene expression in white lupin

Cluster root (CR) formation contributes much to the adaptation to phosphorus (P) deficiency. CR formation by white lupin (Lupinus albus L.) is affected by the P-limiting level in shoots, but not in roots. Thus, shoot-derived signals have been expected to transmit the message of P-deficiency to stimulate CR formation. In this study, it is shown that sugars are required for a response to P starvation including CR formation and the expression of P starvation-induced genes. White lupin plants were grown in vitro on P-sufficient or P-deficient media supplemented with sucrose for 4 weeks. Sucrose supply stimulated CR formation in plants on both P-sufficient and P-deficient media, but no CR appeared on the P-sufficient medium without sucrose. Glucose and fructose also stimulated CR formation on the P-sufficient medium. On the medium with sucrose, a high concentration of inorganic phosphate in leaves did not suppress CR formation. Because sorbitol or organic acid in the media did not stimulate CR formation, the sucrose effect was not due to increased osmotic pressure or enriched energy source, that is, sucrose acted as a signal. Gene transcription induced by P starvation, LaPT1 and LaPEPC3, was magnified by the combination of P limitation and sucrose feeding, and that of LaSAP was stimulated by sucrose supply independently of P supply. These results suggest that at least two sugar-signalling mediating systems control P starvation responses in white lupin roots. One system regulates CR formation and LaSAP expression, which acts even when P is sufficient if roots receive sugar as a signal. The other system controls LaPT1 and LaPEPC3 expression, which acts when P is insufficient.

Accumulation of raffinose and stachyose in shoot apices of Lonicera caerulea L. during cold acclimation

Abstract Seasonal changes were examined in the freezing tolerance, water content and soluble sugar composition of shoot apices of Lonicera caerulea L. var. emphyllocaryx Nakai. Shoot apices were prepared from field-grown plants between April 1993 and August 1994. The LT50, the temperature at which 50% of frozen shoot tips survived, decreased from −18 to −40°C between October and December, it remained below −40°C throughout the winter and increased again from March to May. The composition of soluble sugars changed conspicuously throughout the year. Raffinose and stachyose accumulated rapidly from September to November, while the levels of total soluble sugars and sucrose gradually increased from June to September, prior to the substantial decrease in LT50. The levels of glucose and fructose were lower than the levels of the others and were independent of seasonal changes in temperature. These results suggest that raffinose and stachyose might be involved in the increase in freezing tolerance of the shoot apices of L. caerulea.

Lamin-like analogues in plants: the characterization of NMCP1 in Allium cepa

The nucleoskeleton of plants contains a peripheral lamina (also called plamina) and, even though lamins are absent in plants, their roles are still fulfilled in plant nuclei. One of the most intriguing topics in plant biology concerns the identity of lamin protein analogues in plants. Good candidates to play lamin functions in plants are the members of the NMCP (nuclear matrix constituent protein) family, which exhibit the typical tripartite structure of lamins. This paper describes a bioinformatics analysis and classification of the NMCP family based on phylogenetic relationships, sequence similarity and the distribution of conserved regions in 76 homologues. In addition, NMCP1 in the monocot Allium cepa characterized by its sequence and structure, biochemical properties, and subnuclear distribution and alterations in its expression throughout the root were identified. The results demonstrate that these proteins exhibit many similarities to lamins (structural organization, conserved regions, subnuclear distribution, and solubility) and that they may fulfil the functions of lamins in plants. These findings significantly advance understanding of the structural proteins of the plant lamina and nucleoskeleton and provide a basis for further investigation of the protein networks forming these structures.

Differential nuclear envelope assembly at the end of mitosis in suspension-cultured Apium graveolens cells

NMCP1 is a plant protein that has a long coiled-coil domain within the molecule. Newly identified NMCP2 of Daucus carota and Apium graveolens showed similar peripheral localization in the interphase nucleus, and the sequence spanning the coiled-coil domain exhibited significant similarity with the corresponding region of NMCP1. To better understand disassembly and assembly of the nuclear envelope (NE) during mitosis, subcellular distribution of NMCP1 and NMCP2 was examined using A. graveolens cells. AgNMCP1 (NMCP1 in Apium) disassembled at prometaphase, dispersed mainly within the spindle, and accumulated on segregating chromosomes, while AgNMCP2 (NMCP2 in Apium), following disassembly at prometaphase with timing similar to that of AgNMCP1, dispersed throughout the mitotic cytoplasm at metaphase and anaphase. The protein accumulated at the periphery of reforming nuclei at telophase. A probe for the endomembrane indicated that the nuclear membrane (NM) disappears at prometaphase and begins to reappear at early telophase. Growth of the NM continued after mitosis was completed. NMCP2 in the mitotic cytoplasm localized in vesicular structures that could be distinguished from the bulk endomembrane system. These results suggest that NMCP1 and NMCP2 are recruited for NE assembly in different pathways in mitosis and that NMCP2 associates with NM-derived vesicles in the mitotic cytoplasm.

Assembly and disassembly of the peripheral architecture of the plant cell nucleus during mitosis

Abstract. The architecture of the nuclei of higher plants includes a structure similar to the nuclear lamina of vertebrates. Changes in this structure were monitored during mitosis in carrot (Daucus carota L.) and celery (Apium graveolens L.) cells by immunofluorescence microscopy using an antibody that recognized the nuclear-matrix protein NMCP1. This protein has been shown to be localized exclusively at the periphery of the nucleus (K. Masuda et al. 1997, Exp Cell Res 232: 173–187). Immunofluorescence was recognized throughout cells in mitotic metaphase, although it was distributed predominantly in the mitotic spindle zone. At late anaphase or telophase, the immunofluorescence was localized around each set of daughter chromosomes. Immunofluorescence in newly formed daughter nuclei was restricted to the periphery of nuclei. This behavior was very similar to that of the nuclear lamina of vertebrates, suggesting that the structure located between the nuclear envelope and the chromosomes in plants disassembles and assembles in parallel with the disintegration and re-formation of the nuclear envelope.

Development of nano-surgery system for cell organelles

A nano-surgery system has been developed for operation of minute organelles in a cell. such as chloroplast and mitochondria. For the operation, nano-pipettes, nano-manipulators, sample stage, and bellows pump were fabricated. It has been succeeded to prick a chromosphere in a plant cell and the membrane just under the nucleus of an animal cell with the tip of nano-pipette and to inject fluorescent dye into these organelles.

Explants of Ri-transformed hairy roots of spinach can develop embryogenic calli in the absence of gibberellic acid, an essential growth regulator for induction of embryogenesis from non-transformed roots

Abstract Hairy roots of spinach (Spinacia oleracea L.) were obtained by inoculation of cotyledon explants with wild-type Agrobacterium rhizogenes A13. Integration of the transfer DNA (T-DNA) of a root-inducing (Ri) plasmid of A. rhizogenes into the plant DNA was confirmed by the polymerase chain reaction. Somatic embryogenesis from explants of hairy roots were induced even when the callus-induction (CI) medium did not contain gibberellic acid (GA3), which is considered to be a key growth regulator that is critical for induction of somatic embryogenesis in spinach. Proteins extracted from hairy root-derived calli and from non-transformed root-derived calli generated on medium that contained 10 μM naphthaleneacetic acid (NAA), without GA3, were analyzed by polyacrylamide gel electrophoresis. The proteins from the former calli included appreciably higher levels of a 31-kDa basic protein (BP31) (pI=8.8) than proteins from the latter. This 31-kDa protein also accumulated in embryogenic calli derived from non-transformed roots. Somatic embryos from hairy root explants, grown on medium without growth regulators, yielded regenerated plantlets with actively growing root systems and shoots that did not bolt.

Differential expression of ribosome-inactivating protein genes during somatic embryogenesis in spinach (Spinacia oleracea)

Root segments from spinach (Spinacia oleracea L. cv. Jiromaru) seedlings form embryogenic callus (EC) that responded to exogenous GA(3) by accumulating a 31-kDa glycoprotein [BP31 or S. oleracea ribosome-inactivating protein (EC 3.2.2.22) (SoRIP1)] in association with the expression of embryogenic potential. Microsequencing of this protein revealed significant similarity with type 1 RIPs. We identified cDNAs for SoRIP1 and S. oleracea RIP2 (SoRIP2), a novel RIP having a consensus shiga/ricin toxic domain and performed a comparative analysis of the expression of SoRIPs during somatic embryogenesis. Western blotting and quantitative polymerase chain reaction analyses revealed that the expression of SoRIP1 in calli increased remarkably in association with the acquisition of embryogenic potential, although the expression in somatic embryos decreased moderately with their development. However, the expression of SoRIP2 in calli remained low and constant but increased markedly with the development of somatic embryos. Treatment of callus with GA(3) and/or ABA for 24 h, or with ABA for a longer period, failed to stimulate the expression of either gene. Immunohistochemistry showed that SoRIP1 preferentially accumulated in the proembryos and peripheral meristem of somatic embryos early in development. Appreciable expression of SoRIP2 was not detected in the callus, but intense expression was found in the epidermis of somatic embryos. These results suggest that the expression of spinach RIP genes is differentially regulated in a development-dependent fashion during somatic embryogenesis in spinach.

Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain

Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS) motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1) and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.