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Hiroo Fukuda

Publications and source records attributed to Hiroo Fukuda.

11 recordsLinked to original sources
PubMed2002

Visualization by comprehensive microarray analysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells.

Plants have a unique transdifferentiation mechanism by which differentiated cells can initiate a new program of differentiation. We used a comprehensive analysis of gene expression in an in vitro zinnia (Zinnia elegans L.) culture model system to gather fundamental information about the gene regulation underlying the transdifferentiation of plant cells. In this model, photosynthetic mesophyll cells isolated from zinnia leaves transdifferentiate into xylem cells in a morphogenic process characterized by features such as secondary-wall formation and programmed cell death. More than 8,000 zinnia cDNA clones were isolated from an equalized cDNA library prepared from cultured cells transdifferentiating into xylem cells. Microarray analysis using these cDNAs revealed several types of unique gene regulation patterns, including: the transient expression of a set of genes during cell isolation, presumably induced by wounding; a rapid reduction in the expression of photosynthetic genes and the rapid induction of protein synthesis-associated genes during the first stage; the preferential induction of auxin-related genes during the subsequent stage; and the transient induction of genes closely associated with particular morphogenetic events, including cell-wall formation and degradation and programmed cell death during the final stage. This analysis also revealed a number of previously uncharacterized genes encoding proteins that function in signal transduction, such as protein kinases and transcription factors that are expressed in a stage-specific manner. These findings provide new clues to the molecular mechanisms of both plant transdifferentiation and wood formation.

Asteraceae↗
PubMed2002

Isolation of monoclonal antibodies recognizing rare and dominant epitopes in plant vascular cell walls by phage display subtraction.

A combination of phage display antibody technology and a subtraction method provides a powerful tool for the isolation of novel biomarkers. However, the dilemma that high stringency screening often reduces the diversity in the subtracted phage antibody repertoires and that it is difficult to isolate phage antibody against rare epitopes remain. Therefore, we carefully monitored the occupancy of differentiation-specific clones in a phage antibody library through an enrichment process, and succeeded in isolating monoclonal antibodies against rare and dominant epitopes in plant vascular cell walls. We also report that clones with stop and frameshift mutations significantly survived the enrichment process, owing to noncanonical translation mechanisms.

Amino Acid Sequence↗
PubMed2002

The advantages of cDNA microarray as an effective tool for identification of reproductive organ-specific genes in a model legume, Lotus japonicus.

To understand the molecular mechanisms intrinsic to reproductive organ development a cDNA microarray, fabricated from flower bud cDNA clones, was used to isolate genes, which are specifically expressed during the development of the anther and pistil in Lotus japonicus. Cluster analysis of the microarray data revealed 21 and 111 independent cDNA groups, which were specifically expressed in immature and mature anthers, respectively. RT-PCR was performed to provide a direct assessment of the accuracy and reproducibility of our approach. Confirmation of our results suggests that cDNA microarray technology is an effective tool for identification of novel reproductive organ-specific genes.

Cluster Analysis↗
PubMed2002

Activity of cell-wall degradation associated with differentiation of isolated mesophyll cells of Zinnia elegans into tracheary elements.

Cell walls were prepared from cultured mesophyll cells of Zinnia elegans L. that were transdifferentiating into tracheary elements and incubated in a buffer to undergo autolysis. The rate of autolysis of cell walls was determined by measuring the amount of carbohydrate released from the cell walls into the buffer during incubation. During the course of culture of mesophyll cells, the autolysis rate increased markedly at the time when thickenings of secondary cell walls characteristic of tracheary elements became visible (after 48-72 h of culture), and thereafter the rate remained at a high level. Comparative studies on the autolysis rate of cell walls using various control cultures, in which tracheary element differentiation did not take place, revealed a close relationship between the autolysis rate around the 60th hour of culture and differentiation. Sugar analysis by colorimetric assays and gas chromatography of carbohydrates released from the cell walls detected uronic acid, arabinose, galactose, glucose, xylose, rhamnose, fucose, and mannose. Among these sugars, uronic acid was the most abundant, and accounted for approximately half of the total released sugars. The decrease of acidic polysaccharides in the primary cell walls during tracheary element differentiation was visualized by staining cultured cells with alcian blue at pH 2.5. These results suggest that active degradation of components of primary cell walls, including pectin, is integrated into the program of tracheary element differentiation.

Alcian Blue↗
PubMed2002

Developmental programmed cell death in plants.

Mechanisms of plant developmental programmed cell death (PCD) have been intensively studied in recent years. Most plant developmental PCD is triggered by plant hormones, and the 'death signal' may be transduced by hormonal signaling pathways. Although there are some fundamental differences in the regulation of developmental PCD in various eukaryotes of different kingdoms, hormonal control and death signal transduction via pleiotropic signaling pathways constitute a common framework. However, plants possess a unique process of PCD execution that depends on vacuolar lytic function. Comparisons of the developmental PCD mechanisms of plants and other organisms are providing important insights into the detailed characteristics of developmental PCD in plants.

Apoptosis↗
PubMed2002

Analysis of early processes in wound-induced vascular regeneration using TED3 and ZeHB3 as molecular markers.

Interruption of the vascular bundles of Zinnia internodes induced transdifferentiation of cells into tracheary elements (TEs) or sieve elements (SEs) within 4 d of wounding. The early stage of the regeneration processes was analyzed using two molecular marker genes, TED3 and ZeHB3, which are expressed specifically in TE precursor cells and immature phloem cells, respectively. An increase in the numbers of TED3 and ZeHB3 mRNA-expressing cells always preceded an increase in the numbers of TEs and SEs formed. The earliest sign of vascular differentiation was the appearance 24 h after wounding of a layer(s) of TED3 mRNA-expressing cells in the inter- and intrafascicular cambial-like regions along the severed vascular bundles. In contrast, the number of ZeHB3 mRNA-expressing cells decreased dramatically along the severed bundles 24 h after wounding, and increased again 36 h after wounding. These results clearly indicate that xylem and phloem differentiation are not synchronized during vascular regeneration. Treatment with 10(-3) M colchicine abolished the expression of ZeHB3 mRNA in pith parenchyma, but not TED3 mRNA; this suggests that cell division is a prerequisite for the transdifferentiation of pith parenchymal cells into immature phloem cells expressing ZeHB3. In contrast, transdifferentiation of pith parenchymal cells to TE precursor cells does not require preceding cell division. However, the inhibition of cell division prevented the formation of both radial files of TEs and the cambial-like layer(s) of TED3 mRNA-expressing cells, and, ultimately, vascular regeneration altogether. These results imply that wound-induced cambial-like activity in and between severed vascular bundles is essential for vascular regeneration.

Asteraceae↗
PubMed2002

The use of multiple transcription starts causes the dual targeting of Arabidopsis putative monodehydroascorbate reductase to both mitochondria and chloroplasts.

Monodehydroascorbate reductase (MDAR) isoforms exist in mitochondria, chloroplasts, cytosol and microbodies. Two putative MDAR sequences with an extended N-terminal region are found in Arabidopsis. They differ in the length of the extension by 21 bp. We have shown that these two isoforms arise from a single gene by the use of multiple transcription starts. Green fluorescent protein was fused to each extension, revealing that the longer and shorter fusion proteins were imported into mitochondria and chloroplasts, respectively. These results demonstrate that putative MDAR is a dual-targeting protein transported into both mitochondria and chloroplasts. Although there have been several reports of dual targeting of proteins to mitochondria and chloroplasts, this is the first example in which the dual targeting of the protein to mitochondria and chloroplasts is achieved by the use of multiple transcription initiation sites.

Amino Acid Sequence↗
PubMed2002

Promotion of transcript accumulation of novel Zinnia immature xylem-specific HD-Zip III homeobox genes by brassinosteroids.

We isolated three novel homeobox genes (ZeHB-10, -11 and -12) from Zinnia elegans to elucidate the molecular mechanism underlying vascular system formation. ZeHB-10, -11 and -12 encode for HD-Zip proteins of the class III to which Arabidopsis Athb-8, -9, -14, -15 and IFL1 belong. In situ hybridization analysis demonstrated that the ZeHB-10, -11 and -12 mRNAs accumulated preferentially in procambium and immature xylem cells in 14-day-old plants. Transcripts for the three genes also accumulated in cultured Zinnia cells in a xylogenesis-specific manner. The accumulation of transcripts for all of ZeHB-10, -11 and -12 in cultured Zinnia cells was suppressed strongly by uniconazole, an inhibitor of brassinosteroid synthesis, and such suppression was reversed by the addition of brassinolide, a biologically active brassinosteroid. Thus the expression of ZeHB-10, -11 and -12 may be regulated by endogenous levels of brassinosteroids. Taken together with the fact that ZeHB-10, -11 and -12 proteins can bind to each other in yeast, the roles of HD-Zip III genes in vascular development are discussed.

Arabidopsis Proteins↗
PubMed2002

Preferential and asymmetrical accumulation of a Rac small GTPase mRNA in differentiating xylem cells of Zinnia elegans.

Rac-type small GTPases are known to function in some cellular processes in plants. To further understand the involvement of Rac type GTPases in plant development, we isolated from cultured Zinnia cells a gene (ZeRAC2) encoding a new Rac-type small GTPase. ZeRAC2 mRNA accumulates preferentially in xylogenic culture and transiently at the time when visible tracheary elements appear. Experiments with ZeRAC2 recombinant proteins demonstrated that ZeRAC2 binds to and hydrolyzes GTP. A GFP-ZeRAC2 fusion protein was localized to the plasma membrane. Together with the fact that ZeRAC2 possesses a putative geranylgeranylation site at the C-terminus, this suggests that ZeRAC2 acts on the plasma membrane. In situ hybridization indicated that ZeRAC2 mRNA accumulates preferentially in xylem parenchyma and tracheary element precursor cells, and surprisingly the accumulation is restricted to the site facing developing tracheary elements.

Amino Acid Sequence↗
PubMed2002

ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements.

Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the beginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in animal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease, Zinnia endonuclease 1 (ZEN1), functions directly in nuclear DNA degradation during programmed cell death (PCD) of TEs. In-gel DNase assay demonstrated the presence of a 24-kD Ca(2+)/Mg(2+)-dependent nuclease and a 40-kD Zn(2+)-dependent nuclease as well as ZEN1 in 60-h-cultured cells that included differentiating TEs. Such cell extracts possessed the ability to degrade the nuclear DNA isolated from Zinnia elegans cells in the presence of Zn(2+), and its activity was suppressed by an anti-ZEN1 antibody, indicating that ZEN1 is a central DNase responsible for nuclear DNA degradation. The introduction of the antisense ZEN1 gene into Zinnia cells cultured for 40 h specifically suppressed the degradation of nuclear DNA in TEs undergoing PCD but did not affect vacuole collapse. Based on these results, a common mechanism between animal and plant PCD is discussed.

Antibodies↗