Kaien Fujino

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.

Expansion of potato cells in response to jasmonic acid

Abstract Since jasmonic acid (JA) has strong potato tuber-inducing activity and tuberization of potato plants is initiated mainly by expansion of cells, it is highly likely that JA is capable of inducing expansion of potato cells. When disks cut from potato tubers were cultured on medium that contained JA, the disks began to swell markedly after 1 day in culture. Within 5 days in culture, the fresh weight of the disks doubled in the presence of JA at 3 × 10 −5 M. Light microscopy revealed that the swelling was due to the expansion and not the division of cells. JA exhibited this expansion-inducing activity at concentrations above 10 −5 M. Air-borne methyl jasmonate (JA-Me) also exhibited this activity. The cells that expanded in response to JA in the medium or to airborne JA-Me were localized on the lower side of each disk. The localization seemed to be a result of the greater availability of water. Sucrose in the culture medium was not necessary for the expansion of cells. The expansion-inducing activity appeared to be specific to JA and related compounds, since various plant hormones and a precursor of ethylene had no appreciable effects on the size of cells. Abscisic acid at concentrations above 10 −5 M and benzyl adenine at concentrations above 10 −4 M markedly inhibited the JA-induced expansion of cells. The expansion-inducing activities of JA and JA-Me seem to be associated with their tuber-inducing activities.

Involvement of the accumulation of sucrose and the synthesis of cell wall polysaccharides in the expansion of potato cells in response to jasmonic acid

Jasmonic acid (JA) is capable of inducing the expansion of cells in potato tubers. Changes in the levels of soluble sugars and starch, as well as changes in the levels of cell wall polysaccharides, during the JA-induced expansion of cells were studied. JA caused a considerable increase in the level of sucrose in the cells, while it did not affect levels of glucose and fructose. JA also increased the levels of cell wall polysaccharides. An inhibitor of cellulose synthesis, 2,6-dichlorobenzonitrile (DCB), almost completely inhibited the JA-induced expansion of cells. The results suggest that the cellulose synthesis is necessary for the JA-induced expansion of cells. Cytoskeletal inhibitors also strongly inhibited the JA-induced expansion of cells, suggesting the involvement of cytoskeletal structures, namely, microtubules and microfilaments, in the JA-induced expansion of cells.

Molecular basis of a shattering resistance boosting global dissemination of soybean

Significance Pod dehiscence is a critical step in the seed dispersal (shattering) of legume and crucifer crops and can cause significant yield losses. Upon drying, pod walls are dehisced by two factors: the reduction of pod-wall binding strength and the generation of dehiscing forces. Although the previously reported shattering-resistant mutants maintained binding strength, here, we show a gene regulating the dehiscing force. The gene, Pdh1, encodes a dirigent family protein, known to be involved in lignification, which increases dehiscing forces by promoting torsion of dried pod walls. The loss-of-function pdh1 gene has been widely used as a shattering-resistance gene in soybean breeding. This knowledge could be useful in improving other legume and crucifer crops, as well as soybean breeding. Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world’s leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement.

Molecular characterization of a 10‐kDa buckwheat molecule reactive to allergic patients’ IgE

Background:  Using the sera from buckwheat (BW)‐allergic patients, several putative causative molecules were reported. However, few molecules were determined on the molecular structure. We demonstrated in 2000 that the major allergen with 24 kDa (BW24KD) is a legumin‐like storage protein.

Temperature controls nuclear import of Tam3 transposase in Antirrhinum

It has been proposed that environmental stimuli can activate transposable elements (TEs), whereas few substantial mechanisms have been shown so far. The class-II element Tam3 from Antirrhinum majus exhibits a unique property of low-temperature-dependent transposition (LTDT). LTDT has proved invaluable in developing the gene isolation technologies that have underpinned much of modern plant developmental biology. Here, we reveal that LTDT involves differential subcellular localization of the Tam3 transposase (TPase) in cells grown at low (15°C) and high (25°C) temperatures. The mechanism is associated with the nuclear import of Tam3 TPase in Antirrhinum cells. At high temperature, the nuclear import of Tam3 TPase is severely restricted in Antirrhinum cells, whereas at low temperature, the nuclear localization of Tam3 TPase is observed in about 20% of the cells. However, in tobacco BY-2 and Allium cepa (onion) cells, Tam3 TPase is transported into most nuclei. In addition to three nuclear localization signals (NLSs), the Tam3 TPase is equipped with a nuclear localization inhibitory domain (NLID), which functions to abolish nuclear import of the TPase at high temperature in Antirrhinum. NLID in Tam3 TPase is considered to interact with Antirrhinum-specific factor(s). The host-specific regulation of the nuclear localization of transposase represents a new repertoire controlling class-II TEs.

Fine mapping and development of DNA markers for the qPDH1 locus associated with pod dehiscence in soybean.

Pod dehiscence (shattering) is a major cause of yield loss in mechanical harvesting of soybeans. To develop useful selection markers, we conducted a high-resolution mapping of a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1. The progeny of a residual heterozygous line, which was a recombinant inbred line segregating only for the genomic region around qPDH1, was screened for flanking markers to obtain various recombinants in the vicinity of the QTL. Analysis of the relationship between degree of pod dehiscence and graphical genotype of these lines confined the location of qPDH1 to a 134-kb region on chromosome 16 (formerly linkage group J), where ten putative genes were predicted to be present. None of these genes showed significant sequence homology with the Arabidopsis genes that have previously been reported to be associated with pod dehiscence, suggesting the presence of a novel gene and mechanism underlying pod dehiscence in soybean. Sequencing analysis of the parental shattering-resistant and -susceptible cultivars for the candidate genes revealed a high-frequency nucleotide polymorphism in this genomic region between the cultivars. Three markers were developed using insertion/deletion variations in the region. Polymorphism at these marker loci was basically conserved between diverse shattering-resistant and -susceptible cultivars/lines, suggesting the versatility and usefulness of these markers for marker-assisted selection.

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.

Jasmonic acid-inducible gene expression of a Kunitz-type proteinase inhibitor in potato tuber disks.

Messenger RNAs of a potato (Solanum tuberosum L.) Kunitz-type proteinase inhibitor(s) (PKPI) were present in potato disks excised from tubers stored for 14 months (old tubers) or 2 months (young tubers) after harvest, and disappeared during the aseptic culture. The PKPI mRNA accumulation was found to be induced in potato disks from the old tubers by the addition of jasmonic acid (JA) [3-oxo-2-(2′-cis-pentenyl)-cyclopentane-1-acetic acid].

A Major Soybean QTL, qPDH1, Controls Pod Dehiscence without Marked Morphological Change

Abstract Pod dehiscence (shattering) is a major source of yield loss in the mechanically harvested soybean. We examined near-isogenic lines (NILs) for a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1, to reveal the mechanism underlying the effect of this QTL on shattering resistance. The degree of shattering resistance differed among the NILs; as pod dehiscence percentage after 3 hr heat treatment was under 50% and over 90% for the genotypes resistant to shattering and those susceptible to shattering, respectively. On the other hand, there were no significant differences in the length, width and thickness of pods among the NILs. Anatomical analysis of the dorsal sutures of pods, at which pod dehiscence was found to commence most frequently, revealed no marked differences between the NILs. These results suggest that qPDH1 controls pod dehiscence without markedly changing the morphology of the pods.