Publishing type：Research paper (scientific journal)  

© 2017 American Society of Plant Biologists. All rights reserved. Many land plants evolved tall and sturdy growth habits due to specialized cells with thick lignified cell walls: tracheary elements that function in water transport and fibers that function in structural support. The objective of this study was to define how and when diverse cell populations contribute lignin precursors, monolignols, to secondary cell walls during lignification of the Arabidopsis (Arabidopsis thaliana) inflorescence stem. Previous work demonstrated that, when lignin biosynthesis is suppressed in fiber and tracheary element cells with thickened walls, fibers become lignin-depleted while vascular bundles still lignify, suggesting that nonlignifying neighboring xylem cells are contributing to lignification. In this work, we dissect the contributions of different cell types, specifically xylary parenchyma and fiber cells, to lignification of the stem using cell-type-specific promoters to either knock down an essential monolignol biosynthetic gene or to introduce novel monolignol conjugates. Analysis of either reductions in lignin in knockdown lines, or the addition of novel monolignol conjugates, directly identifies the xylary parenchyma and fiber cell populations that contribute to the stem lignification and the developmental timing at which each contribution is most important.

DOI： 10.1104/pp.17.00434

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase. FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fo1-abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.

DOI： 10.3389/fpls.2016.01326

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) donates GlcNAc for various glycans and glycoconjugates. We previously found that GlcNAc supplementation increases the UDP-GlcNAc content in Arabidopsis; however, the metabolic pathway was undefined. Here, we show that the homolog of human GlcNAc kinase (GNK) is conserved in land plants. Enzymatic assays of the Arabidopsis homologous protein (AtGNK) revealed kinase activity that was highly specific for GlcNAc. We also demonstrate the role of AtGNK in plants by using its knockout mutant, which presents lower UDP-GlcNAc contents and is insensitive to GlcNAc supplementation. Moreover, our results demonstrate the presence of a GlcNAc salvage pathway in plants. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2015.09.011

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Language：Japanese   Publisher：環境放射能除染学会  

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

We investigated the effect of elicitors on xylem differentiation and lignification using a Zinnia elegans xylogenic culture system. Water-soluble chitosan and a fungal elicitor derived from Botrytis cinerea were used as elicitors. Elicitor addition at the start of culturing inhibited tracheary element (TE) differentiation in a concentration-dependent manner, and 30 mu g mL(-1) of chitosan or 16.7 mu g mL(-1) of the fungal elicitor strikingly inhibited TE differentiation and lignification. Addition of chitosan (at 50 mu g mL(-1)) or the fungal elicitor (at 16.7 mu g mL(-1)) during the culturing period also inhibited TE differentiation without inhibiting cell division, except for immature TEs undergoing secondary wall thickening. Elicitor addition after immature TE appearance also caused the accumulation of an extracellular lignin-like substance. It appears that elicitor addition at the start of culturing inhibits the process by which dedifferentiated cells differentiate into xylem cell precursors. Elicitor addition during culturing also appears to inhibit the transition from xylem cell precursors to immature TEs, and induces xylem cell precursors or xylem parenchyma cells to produce an extracellular stress lignin-like substance.

DOI： 10.1007/s10265-013-0568-0

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

To study the regulatory mechanisms underlying lignin biosynthesis, we isolated and characterized lignescens (lig), a previously undescribed temperature-sensitive mutant of Arabidopsis thaliana that exhibits ectopic lignin deposition and growth defects under high-temperature conditions. The lig mutation was identified as a single base transition in GNA1 encoding glucosamine-6-phosphate N-acetyltransferase (GNA), a critical enzyme of UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. lig harbors a glycine-to-serine substitution at residue 68 (G68S) of GNA1. Enzyme activity assays of the mutant protein (GNA1(G68S)) showed its thermolability relative to the wild-type protein. The lig mutant exposed to the restrictive temperature contained a significantly smaller amount of UDP-GlcNAc than did the wild type. The growth defects and ectopic lignification of lig were suppressed by the addition of UDP-GlcNAc. Since UDP-GlcNAc is an initial sugar donor of N-glycan synthesis and impaired N-glycan synthesis is known to induce the unfolded protein response (UPR), we examined possible relationships between N-glycan synthesis, UPR, and the lig phenotype. N-glycans were reduced and LUMINAL BINDING PROTEIN3, a typical UPR gene, was expressed in lig at the restrictive temperature. Furthermore, treatment with UPR-inducing reagents phenocopied the lig mutant. Our data collectively suggest that impairment of N-glycan synthesis due to a shortage of UDP-GlcNAc leads to ectopic lignin accumulation, mostly through the UPR.

DOI： 10.1105/tpc.112.102806

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：VERSITA  

Lignin is synthesized not only during morphogenesis of vascular plants but also in response to various stresses. Isolated Zinnia elegans mesophyll cells can differentiate into tracheary elements (TEs), and deposit lignin into cell walls in TE-inductive medium (D medium). Meanwhile isolated mesophyll cells cultured in hormone-free medium (Co medium) accumulate stress lignin-like substance during culture. Therefore this culture system is suitable for study of lignin and lignin-like substance formation.
In D medium lignin was deposited in TEs, but in Co medium, extracellular lignin-like substance accumulated. Analysis of the culture media indicated the presence of dilignols in D culture, but not in Co culture. To investigate the fate of lignin precursors, we added coniferyl alcohol (CA) in each culture. In Co medium, CA was polymerized into dilignols rapidly but they were present only temporarily, and in D medium CA was polymerized into dilignols relatively slowly but their content increased continually.
Meanwhile, in Co culture, peroxidase activity in the medium was much higher than the peroxidase activity bound ionically to the cell walls. In D culture, ionically bound peroxidase activity was higher than that in the medium. These results may suggest that lignin deposition in TEs is related to ionically bound peroxidases in D culture, and lignin-like substance deposition in the medium is related to peroxidases in the medium in Co culture.

DOI： 10.2478/s11756-010-0130-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Evans Blue staining indicated that actively growing tobacco BY-2 cells in the exponential phase died more rapidly than quiescent cells in the stationary phase when the cells cultured under agitation were placed under still conditions. Fifty percent cell death was induced at about 18, 26, 80 and 140 h for early, mid, late exponential- and stationary-phase cells, respectively. Actively growing cells became TUNEL (transferase-mediated dUTP nick end labelling)-positive more rapidly than quiescent cells, suggesting that the cell death evaluated by Evans Blue is accompanied by DNA cleavages. Electrophoresis of genomic DNA showed a typical 'DNA laddering' pattern formed by multiples of about 200 bp internucleosomal units. Chromatin condensation was first detected at least within 24 h by light microscopy, and then cell shrinkage followed. These findings suggest that the death of BY-2 cells induced by still conditions is POD (programmed cell death).

DOI： 10.1042/CBI20090003

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

We have investigated the mechanism of lignification during tracheary element (TE) differentiation using a Zinnia elegans xylogenic culture. In the process, we isolated ZPO-C, a peroxidase gene of Z. elegans that is expressed specifically in differentiating TEs. ZPO-C is suggested to be involved in lignification of Z. elegans TEs in vivo and in vitro. Furthermore, a peroxidase gene of Arabidopsis thaliana (AtPrx66), which is homologous to ZPO-C, was identified. The expression profile and functions of the gene in planta remain to be investigated. In this study, we performed promoter::beta-glucuronidase (GUS) assays to investigate the expression profiles and functions of the ZPO-C-like peroxidases in A. thaliana. We generated transgenic A. thaliana lines carrying AtPrx66, AtPrx47 or AtPrx64 (peroxidases showing high sequence similarity to AtPrx66) promoter::GUS reporter gene fusions. The GUS activities of AtPrx66, AtPrx47 and AtPrx64 promoter::GUS lines were arranged concentrically from the center to the periphery in the roots of seedlings. Furthermore, histochemical GUS assays using inflorescence stems showed that AtPrx66, AtPrx47 and AtPrx64 promoter-driven GUS were mainly expressed in the differentiating vessels, xylem parenchyma and sclerenchyma, respectively. These results suggest that the gene expressions of these three peroxidases, which showed high sequence similarity to one another, are differentially regulated in various tissues and organs. In addition, our results suggest that while AtPrx66 and AtPrx47 are associated with lignification of vessels, AtPrx64 is associated with lignification of sclerenchyma.

DOI： 10.1111/j.1399-3054.2009.01233.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

The cellular structures of statocytes implicated in gravisensing in primary and lateral roots of Vigna angularis were compared. The statocytes of lateral roots already had small amyloplasts immediately after they emerged from the primary root. Although these amyloplasts sedimented, the lateral roots showed much weaker gravitropism than primary roots, at least until they reached a length of about 30 mm. The nuclei were usually positioned in the upper end of the statocytes in both types of roots. Electron microscopic surveys showed that many tubular elements of endoplasmic reticulum (ER) were frequently localized in the lower end of the statocyte and they sometimes diverged or curved, suggesting that the ER forms a large reticulate complex. It is worth noting that statocytes with a large ER complex were found much more frequently in primary roots than in lateral roots. The amyloplasts were not always settled on this complex but were very frequently under it, especially in the primary roots. In lateral roots, they were usually localized under the ER complex when they were present. Thus, it is suggested that the differential development and organization of the amyloplast-ER complex system is involved in the differential gravitropism of the two types of roots.

DOI： 10.1007/s00709-006-0188-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Fibrillarin is known to play an important role in precursor ribosomal RNA processing and ribosome assembly. The present study describes a fibrillarin homolog gene isolated from tobacco BY-2 cells and its expression during the cell cycle. The cDNA for a fibrillarin homolog, named NtFib1, was first cloned in Nicotiana tabacum with degenerate primers. It encodes 314 amino acids and the deduced amino acid sequence has some highly conserved functional domains, such as the glycine and arginine-rich (GAR) domain for nucleolar localization and the RNA-binding motif. The C-terminal region is highly conserved and has 7 beta-sheets and 7 alpha-helices which are peculiar to fibrillarin. Thus, it is suggested that the fibrillarin homolog of this plant species functions in the same way as the fibrillarin already known from human and yeast cells. Northern blot analysis of BY-2 cells synchronized with aphidicolin or a combination of aphidicolin and propyzamide showed that the histone H4 gene was specifically expressed in the S phase but NtFib1 mRNA remained at high levels during the cell cycle. Examination of the localization of NtFib1 protein tagged with green-fluorescent protein (GFP) suggested that some persisting in the mitotic apparatus was eventually incorporated into reconstructed nucleoli in late telophase. Newly synthesized GFP-tagged NtFib1 protein in the cytoplasm was added to the recycled protein in early mitosis. Highly concentrated actinomycin D completely inhibited the transcription of genes coding for rRNA (rDNA) but did not significantly suppress the amount of either NtFib1 mRNA or protein, although the NtFib1 protein was reversibly dislocated from nucleoli. Although hypoxic shock completely prohibited rDNA transcription, NtFib1 mRNA remained at the same level as in the control experiment, even after the 4 h treatment. These results indicate that the transcription of NtFib1 mRNA is not related to rDNA transcription and NtFib1 mRNA is resistant to disrupting factors during the cell cycle.

DOI： 10.1007/s00709-006-0183-1

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

We previously showed that eight laccase genes (Lac 1-Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147-155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO4 in the reaction mixtures increased in the range from 1 to 100 mu M. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.

DOI： 10.1007/s10265-006-0020-9

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Gibberellin (GA) is considered an important growth regulator involved in many aspects of plant development. However, little is known about the relationship between GA and lignification. In this study, we analyzed the role of GA in tracheary element (TE) differentiation and lignification using a Zinnia elegans xylogenic culture. When gibberellic acid-3 (GA(3)) was exogenously supplied, a slight increase in the frequency of TE differentiation and a remarkable increase in lignin content were observed. Computer image analysis of individual TEs showed that the lignification level of each TE was significantly increased in the culture treated with GA(3) compared with those of the control. In contrast, suppression of TE differentiation and lignification was observed when GA biosynthesis was inhibited by ancymidol, paclobutrazol, or uniconazole. This suppression was restored by the addition of GA(3). These results suggest that GA plays an important role in TE differentiation, and even more so in lignification. When conditioned medium obtained after 120 h of control culture was analyzed by high-performance liquid chromatography, many lignin precursors were detected. However, these lignin precursors were greatly reduced in the GA-treated culture. This result suggests that GA promotes lignification by activating the polymerization of lignin precursors. Gibberellin (GA) is considered an important growth regulator involved in many aspects of plant development. However, little is known about the relationship between GA and lignification. In this study, we analyzed the role of GA in tracheary element (TE) differentiation and lignification using a Zinnia elegans xylogenic culture. When gibberellic acid-3 (GA(3)) was exogenously supplied, a slight increase in the frequency of TE differentiation and a remarkable increase in lignin content were observed. Computer image analysis of individual TEs showed that the lignification level of each TE was significantly increased in the culture treated with GA(3) compared with those of the control. In contrast, suppression of TE differentiation and lignification was observed when GA biosynthesis was inhibited by ancymidol, paclobutrazol, or uniconazole. This suppression was restored by the addition of GA(3). These results suggest that GA plays an important role in TE differentiation, and even more so in lignification. When conditioned medium obtained after 120 h of control culture was analyzed by high-performance liquid chromatography, many lignin precursors were detected. However, these lignin precursors were greatly reduced in the GA-treated culture. This result suggests that GA promotes lignification by activating the polymerization of lignin precursors.

DOI： 10.1007/s00709-006-0180-4

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In an attempt to elucidate the regulatory mechanism of vessel lignification, we isolated ZPO-C, a novel peroxidase gene of Zinnia elegans that is expressed specifically in differentiating tracheary elements (TEs). The ZPO-C transcript was shown to accumulate transiently at the time of secondary wall thickening of TEs in xylogenic culture of Zinnia cells. In situ hybridization indicated specific accumulation of the ZPO-C transcript in immature vessels in Zinnia seedlings. Immunohistochemical analysis using anti-ZPO-C antibody showed that the ZPO-C protein is abundant in TEs, especially at their secondary walls. For enzymatic characterization of ZPO-C, 6xHis-tagged ZPO-C was produced in tobacco cultured cells and purified. The ZPOC:6xHis protein had a peroxidase activity preferring sinapyl alcohol as well as coniferyl alcohol as a substrate, with a narrow pH optimum around 5.25. The peroxidase activity required calcium ion and was elevated by increasing Ca2+ concentration in the range of 0-10 mM. An Arabidopsis homolog of ZPO-C, At5g51890, was examined for expression patterns with transgenic plants carrying a yellow fluorescent protein (YFP) gene under the control of the A15g51890 promoter. The YFP fluorescence localization demonstrated vessel-specific expression of A15g51890 in the Arabidopsis roots. Taken collectively, our results strongly suggest that ZPO-C and its homologs play an important role in lignifcation of secondary cell walls in differentiating TEs.

DOI： 10.1093/pcp/pcj016

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Language：English   Publisher：SPRINGER TOKYO  

The nucleolus is the most obvious structure in the eukaryotic nucleus. It is known to be a ribosome-producing apparatus where ribosomal (r) DNA is transcribed and the primary rRNA transcripts are processed to produce three of the four rRNA species. Electron microscopy has shown that the nucleolus consists of three major components, a dense fibrillar component (DFC), a granular component (GC) and a fibrillar center (FC). The DFC and FCs are integrated into a fundamental nucleolar substructure called the nucleolonema. The DFC corresponds to the matrix of the nucleolonema, and the FC is an electron microscopic counterpart of argyrophobic lacunae localized in the nucleolonema. The spherical FCs are intermittently arranged along the length of the nucleolonema in actively growing cells but are fused with each other to form tubular FCs when rDNA transcription is hampered. The RNase-gold complex does not bind to the FC but to the DFC and the GC, suggesting that rDNA transcription does not occur in the FC although both fluorescence in situ hybridization (FISH) and electron microscopic in situ hybridization reveal that the rDNA is specifically localized in the FCs. Immunogold-labeling after bromo-UTP (BrUTP) incorporation shows that rDNA transcription takes place in the boundary region between the FC and the DFC, and primary rRNA transcripts are expected to be processed outward within the DFC. Data have accumulated suggesting that the nucleolonema is a fundamental substructure of the nucleolus, and its skeleton is the tandem arrangement of the FCs, which are resting harbors or storages of rDNA. Ibis paper proposes that the transversal structural organization of the nucleolonema is centrifugally built up by several structural and functional domains: condensed and/or loosened rDNA, rDNA transcription zone, and transcript processing and ribosome assembly zones.

DOI： 10.1007/s10265-005-0204-8

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

During differentiation of isolated Zinnia mesophyll cells into tracheary elements (TEs), lignification on TEs progresses by supply of monolignols not only from TEs themselves but also from surrounding xylem parenchymalike cells through the culture medium. However, how lignin polymerizes from the secreted monolignols has not been resolved. In this study, we analyzed phenol compounds in culture medium with reversed-phase HPLC, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry, and found 12 phenolic compounds including coniferyl alcohol and four dilignols, i.e. erythroguaiacylglycerol-beta-coniferyl ether, threo-guaiacylglycerol-beta-coniferyl ether, dehydrodiconiferyl alcohol and pinoresinol, in the medium in which TEs were developing. Coniferyl alcohol applied to TE-inductive cultures during TE formation rapidly disappeared from the medium, and caused a sudden increase in dilignols. Addition of the dilignols promoted lignification of TEs in which monolignol biosynthesis was blocked by an inhibitor of phenylalanine anmmonia-lyase (PAL), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP). These results suggested that dilignols can act as intermediates of lignin polymerization.

DOI： 10.1093/pcp/pci017

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In order to understand cell-cell interactions involved in xylem differentiation, we studied intercellular molecules in an in vitro Zinnia xylogenic culture system, where single mesophyll cells transdifferentiate into tracheary elements (TEs) and xylem parenchyma cells. We found that UV-absorbing substances accumulated predominantly in xylogenesis - inducing medium and kept increasing even after the TEs died. This accumulation was inhibited by L- α -aminooxy- β - phenylpropionic acid (AOPP), an inhibitor of phenylalanine ammonia-lyase, and also by brefeldin A, an inhibitor of vesicle transport. These results indicated that living non-TE cells, probably xylem parenchyma cells, secrete some kinds of phenylpropanoids via a vesicle transport system. Further experiment showed that inhibition of brassinosteroid biosynthesis by uniconazole suppressed TE differentiation, but not the secretion of UV-absorbing substances into the medium, implying that differentiation of xylem parenchyma cells might not be strongly affected by the depletion of endogenous brassinosteroids.

DOI： 10.5511/plantbiotechnology.21.205

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Tracheary element (TE) differentiation is a typical example of programmed cell death (PCD) in higher plants, and maturation of TEs is completed by degradation of all cell contents. However, lignification of TEs progresses even after PCD. We investigated how and whence monolignols are supplied to TEs which have undergone PCD during differentiation of isolated Zinnia mesophyll cells into TEs. Higher densities of cell culture induced greater lignification of TEs. Whereas the continuous exchanging of culture medium suppressed lignification of TEs, further addition of coniferyl alcohol into the exchanging medium reduced the suppression of lignification. Analysis of the culture medium by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol accumulated in TE inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall thickening, then increased again. These results indicated that lignification on TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro.

DOI： 10.1093/pcp/pce124

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BOTANICAL SOC JAPAN  

Eight cDNA clones for laccase (Lac 1-Lac 8) were isolated and characterized for analysis of the role of laccases in xylem formation by molecular and genetic approaches. Five of them (Lac 1-5) were isolated from cDNA libraries derived from differentiating xylem through a cDNA sequencing project in loblolly pine xylem (Allona of al. 1998) and other three (Lac 6-8) were isolated using a probe from a tobacco blue copper oxidase. Analysis of the cDNAs revealed that the proteins predicted had N-terminal signal sequences, 8-21 glycosylation sites, and four copper binding sites. The putative mature laccases range between 59.2 and 61.7 kD. The predicted isoelectric points vary between 7.3 and 9.9. Phylogenetic analysis shows these laccase cDNAs form three clusters in the cationic plant laccase group. Northern analysis indicated that all eight of the laccase transcripts are most abundant in differentiating xylem of the six tissues and organs tested. The Lad transcript is also detectable in immature pollen cones, and the Lac 7 transcript is detectable in several organs tested. The predominance of transcript abundance in differentiating xylem suggests that laccases play an important role in pine xylem development, consistent with previous hypotheses that laccases are involved in lignin synthesis in differentiating xylem.

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT PHYSIOLOGISTS  

Changes in the enzymatic activity of cinnamyl alcohol dehydrogenase (CAD) and in the expression of a gene for CAD during tracheary element (TE) differentiation were investigated in cultures of single cells isolated from the mesophyll of zinnia (Zinnia elegans). In cultures in which TE differentiation was induced (TE-inductive cultures), CAD activity increased from h 36 after the start of culture (12 h before the start of thickening of the secondary cell wall) and peaked at h 72, when lignin was actively being deposited. In control cultures in which TE differentiation was not induced, CAD activity remained at a very low level for 5 d. Some isoforms of CAD were detected only in the TE-inductive cultures by native gel electrophoresis and subsequent staining for CAD activity. A cDNA clone for CAD, ZCAD1, was isolated from Z. elegans using a cDNA clone for CAD from Aralia cordata as the probe. RNA gel-blot analysis revealed that in the TE-inductive cultures the level of ZCAD1 mRNA increased from h 36 and peaked at h 38 to 60. No such increases were observed in control cultures. These results indicated that both the gene expression and the activity of CAD are strictly regulated, in association with lignification, during TE differentiation in Z. elegans.

DOI： 10.1104/pp.113.2.425

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BOTANICAL SOC JAPAN  

Cell wall-bound and tracheary element-specific peroxidase isoenzymes, designated P5A and P5B, were shown previously to be associated with lignification during the differentiation into tracheary elements of single cells isolated from the mesophyll of Zinnia elegans (Sate et al. Planta 189: 584-589, 1993; Planta 196: 141-147, 1995). Isoenzymes corresponding to P5 (RP5A and RP5B) were present at a relatively high level in the roots of Zinnia elegans. These isoenzymes were purified from the Zinnia roots by several column-chromatographic steps. Both RP5A and RP5B had molecular masses of 35 kDa. purified RP5A and RP5B were cleaved by CNBr and the partial amino acid sequences of these isoenzymes were determined.

DOI： 10.1007/BF02344235

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca2+ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed. © 1995, Springer-Verlag. All rights reserved.

DOI： 10.1007/BF00193227

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) at 10 muM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11. 1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.

DOI： 10.1007/BF00198223

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

Web of Science

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Language：English   Publisher：Japanese Society of Plant Physiologists  

CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184291

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CiNii Books

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CiNii Books

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CiNii Books

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

CiNii Books

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Event date： 2023.9

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Event date： 2023.5

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Event date： 2023.5

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Event date： 2022.5

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Event date： 2022.3

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Event date： 2021.11

Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Event date： 2021.3

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Event date： 2020.3

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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