The Positron Emitting Tracer Imaging System (PETIS) is a non-invasive tool to capture a serial image of distribution of positron-emitting radioactive tracers in an intact plant body. This technique has been employed in studies of transportation of nutrients in living plants. In this study, we focused on cadmium which contamination of crops is one of the most important problems in the world.
¹⁰⁷Cd (half-life: 6.5 hours) was prepared as the radiotracer, with ion beam bombardment and chemical purification. The tracer was fed to four-week-old rice plants from hydroponic culture, and PETIS imaging of aerial part of the plants was performed for 36 hours. A serial image of cadmium transport was obtained, and time-activity curves in some regions of interest were extracted from the data. We analyzed the time-activity curves using kinetic modeling and successfully estimated physiological parameters, e.g. transit times of cadmium transport from the culture to the aerial portion.

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Sorghum, which is one of the major crop plants, attracts researchers' interests as plants used for removing Cd from polluted soils. In order to practicalize this phytoremediation system, it is essential to transport Cd absorbed by root systems to the shoot effectively. In this study, we tried to investigate Cd accumulation and Cd transport via xylem of sorghum plants to elucidate the mechanism of Cd transport from root to shoot.
Four varieties of sorghum were grown hydroponically and treated with 0.1μM Cd for about two weeks. Effects of Cd on plant growth were not observed. However, about three times of differences among varieties were found in amounts of Cd accumulated in plants. A difference in Cd concentration in the xylem sap was also seen among the varieties. Our results indicated that Cd accumulation in the shoot organs is related closely to the xylem transport of Cd in sorghum plants.

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Molybdenum (Mo) is an essential trace element of plants, but molecular mechanisms of Mo transport have not been understood. We reported identification of the responsible gene (MOT1, stands for Mo transporter 1) that make the difference of Mo contents between two accessions of Arabidopsis thaliana, and subcellular localization of MOT1-GFP fusion protein, at the plasma membrane, previously.
Recent our investigations of MOT1 revealed that the growth of shoots of the mot1 mutant was much poorer than that of wild-type plants under conditions of limited Mo supply. Accumulation of MOT1 transcripts was induced by limited Mo supply. The reduction of Mo concentrations in mot1 was greater in shoots than in roots. MOT1 was mainly expressed in the petioles of the shoot and the pericycle of the roots. MOT1 was capable of transporting molybdate against the concentration gradients. The MOT1 protein represents the first eukaryotic Mo transporter to be identified.

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Arabidopsis BOR1 and BOR2 are efflux transporters of B for xylem loading and required for root cell elongation under B limitation. We studied roles of BOR3, BOR4 and BOR5, BOR1 paralogs, in B transport. Transcripts of BOR3, BOR4 and BOR5 were all detected both in shoots and roots. Using transgenic A. thaliana lines carrying promoter-GUS constructs, we found that BOR3 is predominately expressed in cortex in roots, trichome and guard cells in shoots: BOR4 in endodermis in roots: BOR5 in epidermis at root tips and steles in roots. Expression of BOR3-5 decreased B concentration of yeast cells, suggesting that they encode functional efflux-type B transporters. T-DNA insertion mutants for BOR3-5 did not show apparent phenotype, but bor1-3/bor2-1/bor3-1, triple T-DNA insertion mutant showed more severe root growth retardation under B deficiency compared to the double mutant bor1-3/bor2-1, suggesting that BOR3 was supportive of BOR1-2 in roots.

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We previously demonstrated that OsBOR3 is an efflux transporter of B, necessary for normal growth in rice plants under B-limiting condition. In the present study, we determined B concentration in plants carrying Tos17 in the OsBOR3 gene. B concentration in roots and leaves was not different between the mutant and wild type plants both under the normal and B-limiting conditions. However, considering the difference in the growth, total B taken-up and transported were lower in the mutant plants than the wild type plants. Several independent lines of transgenic plants carrying Promoter:GUS fusion gene were generated and patterns of GUS activities were observed. In root tips, GUS proteins were found in exodermis, while in the elongation zones, it was found both in endodermis. These data suggested that localization of OsBOR3 changes depending on growth stages of roots and the physiological functions may also be changed in the course of root development.

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Under phosphate deficiency, phosphate re-translocation from older organs to younger organs is one of the important adaptive mechanisms. In the former study, we measured phosphate distribution in shoot which was hydroponically cultured and determined source and sink leaves. Then, we searched potential genes involving phosphate re-translocation in Arabidopsis using microarray analysis of m-RNA isolated from source and sink leaves. Finally we identified some possible genes which changed transcriptional levels between source and sink leaves.
In the present study, we further repeated microarray analysis and confirmed expression levels of genes related to source-sink status with quantitative PCR method. We are now measuring phosphate distribution in T-DNA insertion lines of those genes, and discuss the possible roles of source-sink related genes in phosphate re-translocation.

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We developed real-time measuring system for [¹⁵O]water, which is a positron emitter with an extremely short half-life, 2 minutes. Using a pair of BGO (Bi₄Ge₃O₁₂) which was set as close as possible to a stem of a soybean plant, it was possible to measure a trace amount of water, μl level, within the 1cm length of the stem. Through this system, water in xylem vessels was found to exchange with the water outside of the vessels. To analyze water exchange manner more in detail, water evaporation rate as well as water translocation rate was measured under different humidity condition. The water exchange rate was kept the same when humidity was changed from 55% to 80%, when water evaporation amount was drastically reduced. The water leaking site of the xylem vessels was now under investigation using ³H-labeled water with imaging plate.

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The transition from vegetative to reproductive phase in plants is regulated by environmental stimuli such as light and temperature. In Arabidopsis, it is well known that phytochrome, a red/far-red light photoreceptor, and cryptochrome, a blue light photoreceptor, are the major photoreceptor to regulate flowering. However little is known about the sites where these photoreceptors act. We have reported that phytochrome B acts in mesophyll cells of cotyledons to regulate flowering (Endo et al., 2005). In the present study, we produced several transgenic lines that express cryptochrome 2- (cry2-) GFP fusion protein in organ/tissue specific manners. Analyses of these lines revealed that only cry2 in vascular bundles (probably that in the phloem) regulated flowering. Further analysis suggested that cry2 in vascular bundle regulated flowering by altering the expression of a key flowering regulator gene, FLOWERING LOCUS T, in a cell-autonomous manner.

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Cryptochromes (CRYs) are blue-light receptors found in both plants and animals. In Arabidopsis, a long-day (LD) plant, cry1 and cry2 are implicated in numerous blue light-dependent responses, including inhibition of hypocotyl elongation, pigment biosynthesis, entrainment of circadian clock, and control of flowering time. Recent studies revealed that these photoreceptors act not only in the light input to the circadian clock but also in the post-transcriptional regulation of CONSTANS (CO). To elucidate the role of the CRY genes in short-day (SD) flowering, we isolated CRY genes in Pharbitis, namely, PnCRY1 and PnCRY2. Immunoblot analyses using specific antibodies prepared against C-termini of each protein revealed that both PnCRY1 and PnCRY2 underwent rapid blue light-induced degradation. Transgenic plants overexpressing either PnCRY1 or PnCRY2 showed shorter hypocotyls when grown under white light. We are currently investigating whether PnCRY1 and PnCRY2 are involved in the photoperiodic induction of flowering in Pharbitis.

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In Arabidopsis, FT is one of the genes which are located most downstream in regulatory cascade of flowering. FT encodes a protein of the PEBP/RKIP family and functions interdependently with another flowering-time gene FD. FD encodes a bZIP transcription factor and is expressed in shoot apex. FT and FD proteins interact in vitro and in yeast cells. FT is expressed in vascular tissues of cotyledons and leaves. However, when expressed in shoot apex, FT can complement ft loss-of-function mutant phenotype. These facts suggest that FT acts with FD in shoot apex. Fluorescence from functional FT:EGFP fusion protein was observed in both nucleus and cytoplasm, and EYFP:FD fluorescence was observed only in nucleus.
We analyzed protein interaction in planta by BiFC and observed 35S::FT:GR plant phenotype. These results suggest that FT functions in the nucleus with FD to regulate expression of downstream genes.

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In the absolute short-day (SD) plant Pharbitis nil, flowering can be induced by a single dark period just after cotyledons are fully opened. It has been postulated that during the inductive dark period, floral stimulus is produced in the cotyledons, and then transported to the shoot apex (SA). Recent studies in Arabidopsis indicated that FT has critical roles for flowering. We had isolated Pharbitis two FT homologs, LEAF-TYPE (PnFTL) and APICAL-TYPE (PnFTA), whose expression was observed in leaf and SA, respectively, only under SD conditions. Moreover, they were repressed by night-break treatment. Ectopic expression of PnFTL caused flower bud formation directly on unorganized callus. RNAi plants that showed reduced PnFTL and PnFTA expression abolished the 1SD induction of flowering. Our results indicate that apical type FT in addition to leaf type FT may be involved in flowering in Pharbitis nil.

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TWIN SISTER OF FT (TSF) is the closest homolog of FT in the PEBP/RKIP family, suggesting that TSF plays a similar role as a floral promoter. TSF expression was regulated by CO and showed diurnal oscillation. tsf mutation enhanced the late-flowering phenotype of ft. TSF and FT shared similar mode of regulation by FLC, an integrator of autonomous and vernalization pathways. In addition, TSF overexpression caused a precocious flowering phenotype independent of photoperiod, CO and FLC, but dependent of FD. These observations suggest that TSF plays a role as a floral pathway integrator and promotes flowering in a manner similar to FT. Interestingly, TSF and FT expression pattern did not overlap, though both were expressed in the phloem. This finding indicates that the phloem is the site where multiple regulatory pathways are integrated at the transcriptional regulation of FT and TSF.
Yamaguchi et al. (2005) Plant Cell Physiol. 46: 1175-1189

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Dominant late-flowering mutant fwa is an epigenetic mutant that ectopically expresses FWA due to promoter hypomethylation. We have shown that ectopically-expressed FWA inhibits floral transition by interfering with the FT function through protein interaction. We further tested the possibility that FWA inhibits flowering through the transcriptional mis-regulation of its target genes. We performed microarray analysis using two fwa epialleles and 35S::FWA. The transcriptional profiles of these plants suggest that late-flowering phenotype of fwa is unlikely due to the mis-regulation of transcription. We also investigated the site of action of FT protein using FWA protein as a specific inhibitor of the FT function. We examined the tissue where FWA can exert its negative effect on flowering. Results of these analyses will be presented.

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In Arabidopsis thaliana, many biological processes are rhythmically controlled through the circadian clock, which is also involved in the photoperiodic control of flowering time that is regulated by the downstream factors such as CO and FT. We previously proposed that the pseudo-response regulators (PRR3, PRR5, PRR7, PRR9), including PRR1/TOC1, play essential roles close to the central oscillator. As the result, misexpressions and/or mutational lesions of any one of these genes result in a marked phenotype of flowering time. To gain insight into this particular issue, here we characterized certain prr multiple mutants with reference to the expression of the flowering genes, CO and FT. To see a link with the light signal transduction, a red-light photoreceptor mutant (phyB) was also integrated into such genetic analyses. Together with morphological characteristics of these prr mutants, the results will be discussed about the link between the PRR clock function(s) and the photosignal-dependent flowering.

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The circadian clock coordinates plant physiological events with the environmental day/night cycle. Resent studies have revealed the central mechanism of the plant circadian clock, but the out put pathways from the clock are not well understood.
To explain how the clock regulates out put events, we carried out transcriptome analysis in prr9/prr7/prr5 clock mutant after 18-day under the continuous light condition. We obtained genes expressed 2-fold high in the mutants compared with the wild. Moreover, these genes were transcribed at the manner of the diurnal rhythm under the LD cycle, and the transcript peaks were seen between ZT0 to ZT9. And PRRs had ability to repress the promoter activity of the genes in T87 cultured cells. These results suggested that PRR9/PRR7/PRR5 are involved in not only the clock core mechanism, but also some out put events directly during the day phase.

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In higher plants, many biological processes are rhythmically controlled through the circadian clock. For the model plant Arabidopsis thaliana, a certain number of clock-asscociated genes (e.g., CCA1/LHY, ELF4, LUX) were already reported. We also proposed that a set of pseudo-response regulators (PRRs) play essential roles close to the central oscillator. In our previous studies, however, we examined the expression profiles of these clock-associated PRR genes mainly at the level of transcription. Thus, better clarification of the molecular functions of PRRs must await examination of PRRs at the level of protein (translation and degradation). To this end, here we constructed and characterized a set of transgenic plants, which expressed epitope-tagged PRRs (e.g., TAP-PRR9), demonstrating that they indeed showed robust rhythms with reference to the protein profile. Based on these results, the possibility was also examined whether a clock-complex containing PRRs could be biochemically isolated.

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Arabidopsis thaliana has a small family of pseudo-response regulators (PRRs), consisting of five members, among which PRR1/TOC1 is believed to be a clock component. PRRs are nuclear-localized proteins, otherwise nothing is known about their molecular function(s). In their amino acid sequences, they share an intriguing structural design, in which each contains an N-terminal pseudo-receiver domain, followed by a common C-terminal CCT motif. To gain insight into the function(s) of the PRR family of clock components by employing the PRR1 and PRR5 genes, here we constructed a set of transgenic plants, each of which was designed so as to constitutively express each structural domain separately (e.g., pseudo-receiver domain, CCT motif). These transgenic plants were extensively characterized with regard to the clock-associated phenotypes (e.g., rhythm, flowering, photo-sensitivity), and these results will be discussed with reference to the current idea of the clock-associated functions of PRRs.

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In higher plants, many biological processes are rhythmically controlled through the circadian clock. For the model plant Arabidopsis thaliana, a certain number of genes that encode clock-associated genes (e.g., CCA1/LHY, ELF4) were already reported. A set of pseudo-response regulators (PRRs), including PRR1/TOC1, also play essential roles close to the central oscillator. On the other hand, recent results from plant genomics made it possible to conduct a comparative genomics with other model plants. Based on this idea, in the hope of understanding the clock function in the crop, Oryza sativa, here we intensively inspected a set of rice genes, which appear to be orthologous to the well-documented Arabidopsis clock-associated genes (e.g., OsTOC1, OsPRRs, OsCCA1, OsZTL, OsLUX). The results showed that certain clock-associated genes are highly conserved both in the dicot and monocot. The results will also be discussed with reference to the conserved clock mechanism in higher plants.

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Greening of etiolated seedlings is an important process of photomorphogenic responses, during which the chlorophyll synthesis must be finely controlled because certain intermediates of chlorophyll synthesis act as light induced oxidative reactants, which might cause harmful effects on plants. The regulatory mechanism underlying the chlorophyll synthesis during de-etiolation (or greening) is not fully understood. In this respect, it was recently reported that a phytochrome-interacting bHLH transcription factor, named PIL5 (PIF1), serves as a negative regulator of the chlorophyll synthesis in etiolated seedlings. We previously identified PIL5 as a factor that directly interacts with the Arabidopsis clock-component PRR1/TOC1 in yeast two-hybrid analyses. This fact led us to envisage that the clock-associated factors, such as PRRs and CCA1, might also implicated in the control of the chlorophyll synthesis. Here we report several lines evidence that strongly support this intriguing idea.

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The central circadian oscillator, kaiABC genes, has been identified in cyanobacterium Synechococcus elongatus PCC 7942. The bona-fide central clock proteins occurs autonomous oscillation in vitro, and it was proposed that the transcriptional feedback loop involving kaiABC expression provides a global circadian expression. As a fact, we have never found non-oscillatory genes from cyanobacteria using luxAB reporter system. In this study, we therefore examined how cyanobacterial genes get such a global expression pattern. A microarray analysis exhibited that only 30% genes had a possible oscillation in the mRNA accumulation level. Additionally, these genes showed negative feedback regulation against excess KaiC but not the others. Thus, the global oscillation of gene expression is not generated in transcriptional level, suggesting it is given in post-transcriptional level like a translation process. According these aspects, we will finally propose new model for circadian output cascade in cyanobacteria.

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To determine essential nuclear-genes for chloroplast development, we systematically analyzed albino or pale green Arabidopsis thaliana mutants by using a two-component transposon system based on the Ac/Ds element of maize as mutagen.
In this meeting, we present phenotypes of the apg4 mutant, and discuss functions of the APG4 gene in chloroplast development. The apg4 mutant has white cotyledons and yellow or green variegated true leaves, fertility. We showed that apg4 has Ds inserted in promoter region of ribosome binding factor (RBFA) homologue. The mRNA expression of RBFA was not detected in apg4 by RT-PCR analysis. White cotyledons had abnormal plastids that lacked internal membrane's structures and highly vacuolated. But, their true leaves had normal plastids contained developed lamella structure and abnormal ones. Moreover, we found that the APG4 is involved in the processing of the pre-rRNA to form the mature 23S and 4.5S rRNAs.

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The rice virescent-1 (v₁) mutants is temperature-conditional and develops chloroplast-deficient leaves under the restrictive temperature. We previously speculated that the V₁ gene functions at an early stage of leaf development (P4), and are involved in the regulation of chloroplast translation/transcription capacity. Positional cloning revealed that V₁ encodes a novel 32 kDa protein, and is deduced to be localized to chloroplast. Western blot analysis indicated that V₁ protein highly accumulated in the immature leaves at P4 stage, but barely accumulated in mature leaves that correspond to P5 and P6 stages. In v₁ mutant leaves, normal expression of chloroplast transcription/translation apparatus are disturbed and accumulation of plastid-encoded chloroplast proteins were severely suppressed. These results suggest that V₁ has important roles in the construction of the chloroplast transcription/translation apparatus that occurs exclusively at the P4 stage.

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The Obg subfamily of monomeric GTPases has been identified in most organisms including bacteria, mammals and plants. Bacterial Obg proteins seem to play a specialized role in ribosome function. However, the molecular function of eukaryotic Obg proteins is largely unknown. Prokaryotes have one Obg protein, whereas eukaryotes posses several homologues, suggesting that each eukaryotic Obg protein may function in different organelles. We identified two Obg homologues (AtObg1 and AtObg2) in Arabidopsis. AtObg1 carries a potential chloroplast transit peptide at N termini. To confirm its subcellular localization, stable transgenic plants expressing AtObg1-GFP fusion proteins were examined with confocal microscopy. The AtObg1-GFP fusion proteins are localized in the chloroplast in all tissues, including leaves and roots. Knockout of AtOBG1 gene resulted in a defect in embryo development. On the other hand, AtObg1 over-expression lines showed variegated phenotypes. These results suggest that AtObg1 may play a crucial role in ribosomal function in chloroplasts.

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Plastid DNA exists as a large DNA-protein complex that is called a plastid nucleoid. The nucleoids contain various DNA-binding proteins, the majority of which remains to be identified. In this work, we showed that the small proteins containing the SWIB domain were related to plastid nucleoids. The SWIB domain is a conserved region of BAP60/BAF60, which are subunits of the SWI/SNF class chromatin remodeling complexes. Several bioinformatics tools reliably predicted the Arabidopsis hypothetical proteins containing SWIB domain proteins (PSWIB1, 2) to be targeted to plastids. We transiently expressed the PSWIBs fused with GFP in tobacco leaves or Arabidopsis protoplasts, and found that they localized in chloroplast nucleoids. In stable transformants, PSWIB-GFP fusion proteins were also localized in the nucleoids of various plastids. In addition, transgenic lines overexpressing the PSWIB-GFP exhibited pale green or albino phenotype. Further functional analysis of the PSWIBs is currently being undertaken.

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Plastid DNA forms a nucleoid with a number of proteins. In pea and soybean, sulfite reductase (SiR) was identified as a main constituent in isolated plastid nucleoids, and was reported to repress transcription in vitro. SiR catalyzes reduction of sulfite to sulfide using ferredoxin as an electron donor and has been believed to exist in plastid stroma. Immunofluorescent staining of isolated pea chloroplasts with anti-SiR antibody showed localization of SiR to nucleoids. DNA-binding properties of SiR were analyzed by gel mobility shift assay using recombinant pea SiR. Stepwise band shift was observed, suggesting that different number of SiR molecules were bound. Apparent dissociation constants for 40mer and 20mer double-stranded DNAs were ~25 nM and ~225 nM, respectively. Single-stranded DNA was also shifted. Available evidence suggests that the DNA-binding was not sequence-specific. These results suggest that SiR binds to entire plastid genome, involved in the transcriptional repression.

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Recently, close relation between the segregation of nucleoids and the cell division has been clarified in Escherichia coli. However, the relationship between nucleoid segregation and plastid division is poorly understood in plants. We developed a new fluorescent probe for live visualization of plastid nucleoids. The PEND protein contained a DNA-binding domain called cbZIP at its N-terminus. In transformants of Arabidopsis thaliana, cbZIP-GFP fusion proteins were localized in the nucleoids of plastids. In resting chloroplasts, nucleoids existed as small particles. During the division of plastid, nucleoids formed a fibroid structure, which suggests an active segregation of nucleoids. This also point to a mechanism of coupling of nucleoid partition and plastid division, as in the case of the chromosome distribution and segregation in eukaryotic cell division. Moreover, during the division of plastid in Cyanidioschyzon merolae, nucleoids were connected across the plastids. Therefore nucleoid segregation machinery may be present widely in plants.

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We have isolated two genes encoding bacterial DNA polymerase I homologues from BY-2 cultured tobacco cells, and named them NtpolI-like 1 and NtpolI-like 2. The two genes were highly similar to each other (97.2% identical at amino acid sequence level), suggesting that they were orthologous genes each derived from respective progenitor species of Nicotiana tabacum, an allotetraploid plant. Although plastid localization was predicted for the both gene products, western blotting analyses, as well as experiments with NtpolI-like1/2-GFP fusion proteins, demonstrated dual-targeting of the gene products to plastids and mitochondria, suggesting that the DNA replication in the plastids and mitochondria might be conducted by the same DNA polymerase. Further biochemical analyses to examine the relationships between (i) NtpolI-like1/2 gene products and organelle DNA polymerases and (ii) DNA polymerases present in the plastids and mitochondria are now on progress, using plastid- and mitochondrial nuclei isolated from BY-2 cultured tobacco cells.

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Plant has three genomes in nucleus, mitochondria, and plastids, respectively. Gene expressions from these genomes are seemed to coordinated with each other through an undefined crosstalk mechanism. They are proceeded by T7 phage-type RNA polymerase (NEP) in mitochondria, whereas by NEP and eubacteria-type RNA polymerase (PEP) in chloroplasts. Arabidopsis has three different NEPs, namely, mitochondria-localized RpoT;1(RpoTm), chloroplast-localized RpoT;3(RpoTp), and possible dual-localized RpoT;2(RpoTmp). In this study, we addressed if the RpoT;2 had critical roles in transcription of mitochondria and/or chloroplast genome(s), through the analysis of a RpoT;2 knockout mutant. Although Baba et al. has already reported about an allele of the RpoT;2 mutant in 2004, the definite function of RpoT;2 is still unclear. We performed northern analysis, QPCR analysis, microarray analysis, measurement of photosynthesis activity and so on. We will show the data and suggest critical roles of RpoT;2 in some gene expression.

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To elucidate the cellulose synthesis mechanism, we isolated a mutant of Arabidopsis (css1) that showed changed sensitivity to cellulose biosynthesis inhibitor. The analysis of phenotypes indicated that the css1 mutation influenced various fundamental metabolisms including amino acid metabolism, triacylglycerol degradation and polysaccharide synthesis (cellulose and starch). Unexpectedly the map-based cloning of responsible gene for the mutation identified a protein that was assumed a splicing factor of mitochondrial group II intron. In accordance with this result, this mutant exhibited improper splicing of NAD4 transcript. We seem that defect in the function of mitochondria influences various aspects of basic metabolism including cellulose synthesis. It is also possible that At-nMat1 gene product influences nuclear gene expression utilizing its RNA-related activity. Our results suggested that sucrose synthase (SuSy), an enzyme involved in the biosynthesis of cellulose, plays key roles in the connection between mitochondria and cellulose synthesis.

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Although many researches have been made since the RNA editing of plant mitochondria was discovered in 1989, the cis recognition elements in RNA editing, guide DNA or RNA, a trans factor (protein), and deaminase have not been decided until now.
We determined 519 RNA editing sites of 36 kinds of protein genes in tobacco mtDNA by RT-PCR method. Multiple alignments of the DNA sequences was carried out for 29 mitochondrial genes of tobacco, Arabidopsis, rapeseed and rice, and 780 RNA editing sites were marked.Consequently, the sites common to four plants had only 170 sites (22%) and 303 sites (39%) were unique to one species. From these results it was presumed that
(1) Land plant was diversified to acquire and fix the RNA editing site for each lineage.
(2) A trans-factor would recognize many RNA editing sites.
(3) A trans-factor also evolved uniquely for each lineage.

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FtsH, also called AAA protease, is a membrne-bound ATP-dependent metalloprotease and present in chloroplasts and mitochondria in plants. In mitochondria, several homologues have been identified in inner membranes with its catalytic domain faced either to matrix (m-AAA) or to intermembrane space (i-AAA). We have shown that in Arabidopsis, twelve potential FtsHs are present and that three are targeted into mitochondria. Moreover, a recent study indicated that one of the homologues FtsH11 is dual-targeted into chloroplasts and mitochondria. The overall results demonstrated that four FtsHs, two m-AAAs (FtsH3 and 10) and two i-AAAs (FtsH4 and 11) are present in Arabidopsis mitochondria. We isolated T-DNA insertion mutants for these four FtsH genes. Homozygous mutants were successfully isolated for all genes, suggesting that loss of one of the mitochondrial homologues is dispensable. We are currently generating double mutants, and initial characterization of the double mutants will be presented.

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We successfully transformed respiratory deficient mitochondria of Chlamydomonas reinhardtii with DNA-constructs containing compensating genes that restored the deficiency. PCR and sequencing analysis showed that homologous recombination occurred between the mitochondrial genome and the artificial DNA-constructs, and the frequency was roughly proportional to the length of sequence homology. Through this investigation, we found that respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those that could not be transformed in our experiments. All of the transformed mitochondria have deletions (0.7 to 1.5-kb) extending from the left terminus to somewhere in the cob gene of their linear 15.8-kb mitochondrial genome. In comparison, mitochondria of all the non-transformable strains have both of their genome termini in intact. This led us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have higher potential for recombination.

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The role of plant purple acid phosphatases (PAPs) has ever been proposed for the degradation of organophosphates, when plants are under phosphate starvation. However, not only wall regeneration on the surface of tobacco protoplasts was accelerated by the addition of wall-bound tobacco PAP, but also cellulose deposition was enhanced in the transgenic tobacco cells overexpressing the PAP (NtPAP12). Since PAPs are members of a group of nonspecific phoshomonoesterase, containing protein phosphatases, calcineurin (type 2B) and protein phosphatases type 1, the tobacco PAP could probably function as the type of protein phosphatase. We suggest the potential substrates for NtPAP12 in suspension-cultured tobacco cells by screening proteins phosphorylated.

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UDP-D-galacturonic acid (UDP-GalUA) is a donor of galacturonic acid in pectin. This compound is not commercially available, and its preparative method with high efficiency is needed. UDP-GalUA pyrophosphorylase involved UDP-GalUA synthesis has been neither purified nor cloned. The UDP-sugar pyrophosphorylase from Pisum sativum possesses a broad substrate specificity. Here, we investigated UDP-GalUA pyrophosphorylase activity of this enzyme and applied it to the preparation of UDP-GalUA.
The UDP-sugar pyrophosphorylase catalyzed the formation of UDP-GalUA and pyrophosphate from GalUA 1-phosphate and UTP, showing that this enzyme has UDP-GalUA pyrophosphorylase activity. The enzyme had optimum pH of 6 and the K_m values for GalUA 1-phosphate, UTP, UDP-GalUA, and pyrophosphate were 2.3 mM, 1.2 mM, 0.70 mM, and 1.3 mM, respectively. In the presence of inorganic pyrophosphatase, the enzyme produced UDP-GalUA in 91 % yield on a preparative scale. Thus this UDP-sugar pyrophosphorylase is useful for the highly efficient production of UDP-GalUA.

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Nucleotide sugars are synthesized through both de novo and salvage pathway, and serve as glycosyl donors for the synthesis of polysaccharides in higher plants. UDP-sugar pyrophosphorylase catalyzes the conversion of various monosaccharide 1-phosphates to UDP-sugars. Using genomic database, a putative gene of Arabidopsis has been identified as UDP-sugar pyrophosphorylase. The gene, designated AtUSP, encodes a protein with 614 amino acids, and shares high similarity with a UDP-sugar pyrophosphorylase from pea. AtUSP was expressed in young seedlings, rosette leaves, and flowers of Arabidopsis, and relatively strong expression was detected in the vascular tissue of leaves and the pollens. Recombinant AtUSP expressed in Escherichia coli exhibited broad specificity toward monosaccharide 1-phosphates forming various UDP-sugars such as UDP-Glc, UDP-Gal, UDP-GlcA, UDP-Xyl, and UDP-L-Ara. A mutation in AtUSP< gene caused by T-DNA insertion completely abolished male fertility, indicating that AtUSP plays an essential role in the salvage pathway in pollen development or germination.

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Pectin is one of the major cell wall polysaccharide found in dicotyledonous plants. We have investigated the properties of galactosyltransferase (GalT) participating in the synthesis of β-1,4-galactan side chains of pectin, and partially purified through several chromatographic procedures. Here, we report the chain elongation of galactan by a partially-purified GalT.
A crude enzyme was prepared from etiolated soybean hypocotyls. GalT was solubilized by treatment with a detergent and partially purified through ion-exchange chromatography. The chain elongation reaction was performed with 2-aminobenzamide (AB)-derivatized β-1,4-Gal₇ as an acceptor substrate. The crude enzyme attached less than four Gal residues onto the acceptor, whereas the partially-purified enzyme transferred successively more than 25 Gal residues. Enzyme activity toward varying lengths of acceptor galactooligomers was examined. Activity increased with increasing degree of polymerization (DP) of the galactooligomers from 4 to 7, while the acceptors with DP 8-11 showed less acceptor efficiency than that of Gal₇-AB.

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Arabinogalactan proteins (AGPs) were the extracellular proteoglycans associated with the plasma membrane, and their functions have been implicated in diverse developmental roles including differentiation, cell-cell recognition, and embryogenesis. AGPs are characterized by large amounts of carbohydrate components rich in galactose and protein components containing hydroxyproline. In spite of the importance of AGPs, study of enzymes which attack to carbohydrate moiety of AGPs is limited. Recently, we succeeded in the cloning a beta-1,3-galactanase gene from Phanerochaete chrysosporium for the first time. In the present study, we searched the database of the Arabidopsis thaliana genome based on the sequence, and found two homologous regions. The amino acid sequences deduced from the nucleotide sequences suggest that the genes encode membrane enzymes belonging to glycoside hydrolase family 43. The genes were successfully expressed in the methylotrophic yeast Pichia pastoris, and both recombinant proteins hydrolyzed beta-1,3-galactan. The result indicates that the genes encode to beta-1,3-galactanase.

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Arabinose is a major component of numerous plant polysaccharides, and it exists predominantly in the α-L-arabinofuranosyl (α-L-Araf) form. However, small amounts of arabinopyranosyl (Arap) residues were detected. In in vitro studies of the biosynthesis of arabinose-containing polysaccharides UDP-arabinopyranose (UDP-Arap) has been used as the glycosyl donor. However, the enzymatically formed products and acceptor substrates have not been well characterized. We studied arabinosyltransferase (AraT) activity and the structure of the products formed when UDP-Arap and UDP-arabinofuranose (UDP-Araf) are reacted with fluorescent-labeled arabinofurano-oligosaccharides in the presence of mung bean microsomal membranes. Our data show that the membranes contain AraT activity that transfers a single arabinopyranosyl residue to O-3 of the non-reducing terminal Araf of the oligosaccharides when UDP-Arap is the glycosyl donor. A small amount of Araf residues are incorporated to O-5 of the non-reducing terminal Araf of the oligosaccharides, in addition to incorporation of arabinopyranosyl residues when UDP-Araf is used.

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Rhamnogalacturonan I (RG-I) is a pectic polysaccharide in primary cell walls. RG-I has a backbone consisting of rhamnose and galacturonic acid residues. The rhamnose residues are substituted with arabinan, galactan, and arabinogalactan side chains. Arabinosyl residues of arabinan and arabinogalactan are mainly arabinofuranosyl (Araf) form.
UDP-Arabinopyranose (UDP-Arap) has been used for the biosynthesis of arabinan. Nunan and Scheller and we reported that a single [¹⁴C]Arap residue was incorporated into the exogenous acceptor when UDP-[¹⁴C]Arap was used as a donor. To study the biosynthesis of arabinan, we developed an assay to measure (1-5)-α-arabinan synthase activity in Golgi membranes from mung bean using UDP-Araf instead of UDP-Arap as a donor substrate and fluorescent-labeled arabino-oligosaccharides as acceptors. As the results, (1-5)-linked arabinofuranosyl oligosaccharides were formed.

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Xyloglucan endotransglucosylases (XETs) catalyze the transfer of a portion from a donor xyloglucan to another xyloglucan molecule, and are considered to contribute to the loosening or rigidity of the cell wall, because xyloglucans cross-link adjacent cellulose microfibrils. To identify and define the reaction mechanism of XETs, we purified XET isoenzymes from growing pea stems. Five XETs were obtained by sequential lectin affinity chromatography, gel permeation chromatography, cation exchange chromatography, and chromatofocusing. The pI values (with molecular masses) of purified five enzymes were 7.8 (31 kDa), 7.1 (29 kDa), 7.0 (31 kDa), 6.8 (29 kDa) and 6.5 (29 kDa), respectively. All the enzymes showed a transferase activity but no hydrolase activity for xyloglucans. These isoenzymes have different pH optima (range 5.5-7.0). They were highly specific for xyloglucans, and showed no transglucosylation activity for other glucans and xylans.

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Xyloglucan endotransglucosylase (XET) is a cell wall enzyme capable of cleaving and reconnecting xyloglucan molecules. Integration of whole xyloglucan and its oligosaccharides into pea cell wall by XET causes suppression and acceleration of cell elongation, respectively. The immunohistochemical observation showed that XET could coexist with xyloglucan in Golgi before the integration of xyloglucan-XET complex to the cell wall in the elongating region of pea stem. In pulse-chase experiment the molecular mass of xyloglucan increased from 8.8 kDa to 110 kDa by endotransglucosylation when 230 kDa xyloglucan was supplied. We propose that the XET binds to non reducing end of xyloglucan in trans Golgi to form enzyme-acceptor complex.

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Xyloglucan endotransglucosylases/hydrolases (XTHs) are a class of enzymes capable of catalyzing the molecular grafting between xyloglucans and/or the endo-type hydrolysis of a xyloglucan molecule. They are encoded by 33 genes in Arabidopsis, and are divided into three subfamilies. Members of Group III subfamily are implicated in hydrolysis of xyloglucans, but their physiological roles remain elusive. We analyzed expression and function of all members of this subfamily (AtXTH27,-28,-29,-30,-31,-32,-33). Morphology of T-DNA-insertion lines for these XTH genes was indistinguishable from that of the wild type, except for the xth27 mutant, indicating that these XTHs are functionally redundant. To investigate their roles in more detail, we generated 20 double mutants. Promoter GUS expression analyses have shown that some of these genes are co-expressed temporally in specific plant tissues. Double mutation of XTH27 and XTH28, which are both expressed in anther, conferred a short-silipue phenotype.

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A synthetic auxin, 2,4-D stimulates the activity of rice EGase with the broad substrate specificity with respect to sugar backbones in β-1,4-glycans (glucose and xylose) at the early stage of lateral root formation. The nascent protein encoded by the auxin-responsive cDNA corresponding to the purified EGase (51 kDa) has a greater molecular mass (68 kDa) due to the presence of an extra peptide of 125 amino acids at C-terminus. Northern-blot analysis revealed the presence of a single transcript, but three distinct polypeptides (51, 68 and 110 kDa) were detected in immunogel-blot analysis of EGases extracted from primary root segments. Two larger polypeptides appeared in the microsome while the 51 kDa polypeptide was mainly detected in the buffer soluble fraction. Two polypeptides were auxin-responsive while one polypeptide (110 kDa) was constitutive. C-terminal truncation of 68 kDa protein may be involved in the generation of multiple forms of this auxin responsive EGase.

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Tension wood is a reaction wood of angiosperm woody plants. Reaction wood causes the stem to bend by its mechanical action. Tension wood generates much higher tensile growth stress than normal wood. However, the mechanisms how the tissue switches normal to tension wood, or how it generates tensile stress in tension wood is still unknown. Buffer insoluble proteins from tension wood and normal wood were compared on 2-D PAGE. The signals of which sizes were increased in tension side were cut off from the gel, digested by trypsin, then analyzed by LC/MS/MS. The proteins were searched on database with the mass spectrum patterns that obtained by LC/MS/MS. Many enzymes were annotated but nothing was found as a significant protein which was able to explain neither differentiation of tension wood nor generation of tensile stress.

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We have reported the adapted mechanism for boron deficiency by using poplar cells (1/20-B) grown in low boron concentration medium (5 μM). In these cells, pectin methylesterase (PME) activity was higher than that of non-resistant cells (1/1-B). This result indicates that the increase of activity plays a part of the mechanism in tolerance to boron deficiency.
In this study, I report the expression of PME gene in both cell lines during a culture period. The expression of PME gene in 1/1-B was observed only in 1-9 day after subculture. However, that of 1/20-B was recorded through the 14-days of culture period. Because the boron deficiency affects in cell elongation growth rather than cell division, and the later half of culture period was cell elongation phase in this cell line, it was indicated that the expression of PME gene in cell elongation phase took part in the tolerance to boron deficiency.

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A temperature sensitive mutant of Arabidopsis, lignecens (lig), which accumulates lignin aberrantly at higher temperatures, was analyzed for elucidation of regulatory mechanisms of lignin biosynthesis. Whereas the lig seedlings grew normally at 18C, their growth was inhibited severely at 28C. Lignin was accumulated in the root sub-apical region of the lig seedling 2 days after temperature shift to 28C from 18C. We investigated effects of the lig mutation on expression patterns of several genes related to lignin formation, including AtPAL1, AtCCRs, AtCAD-D, and AtMYBs. Exposure of the lig seedlings to 28C increased the expression of AtPAL1, AtCCR2, and AtCAD-D. Ethylene synthesis inhibitor AVG suppressed aberrant lignification in the lig seedlings at 28C. A map-based approach identified a missense mutation in a putative serine/threonine kinase gene of the lig genome. Based on these results, a possible function of the LIG gene will be discussed.

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The shoot-apical-meristem (SAM) of Arabidopsis is maintained primarily through two independent pathways by WUS and STM. However, mechanisms for transcriptional regulation of these regulators remain largely unknown. We isolated a T-DNA insertion mutant, sha1 (shoot-apical-meristem arrest 1) in Arabidopsis, in which the T-DNA is inserted in the 5^, promoter region of the gene encoding a novel ring-finger protein SHA1. The vegetative SAM of sha1 appears normal during juvenile stage (JV), but becomes fasciated after entering adult stage (AV), and disappears completely at the stage when wildtype begins to bolting. Consequently, no primary inflorescence and floral primordia are formed in sha1. Gene expression analysis reveals that in sha1, CLV1, CLV2 and STM are not significantly altered during both JV and AV, while WUS and CLV3 remain unchanged during JV but are extremely reduced during AV. These results suggest that SHA1 is required for promoting WUS transcription in postembryonic SAM maintenance.

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It is well known that the size of meristem is regulated by CLAVATA (CLV) signaling pathway in Arabidopsis. On the other hand, the genetic mechanisms of such regulation are poorly understood in other plants. We have been studying the function of FLORAL ORGAN NUMBER1 (FON1) and FON2 genes that regulate the size of floral meristem in rice. We have already reported that FON1 encodes a CLV1-like receptor-like kinase (Suzaki et al. Development 131: 5649-5657). We have revealed that FON2 encodes a small secreted peptide similar to CLV3.
Here, we report phenotypic analyses of transgenic plants that constitutively express FON2 in rice and Arabidopsis. The results obtained here suggested that the genetic network that regulates meristem maintenance is conserved in rice, and FON2 has the function similar to that of CLV3. Furthermore, our results suggested that additional or modified pathways may be involved in the regulation of meristem maintenance in rice.

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The shoot organs such as leaves and flowers arise from the shoot apical meristem (SAM). Organ primordia emerge in regions where auxin is highly concentrated in the peripheral region of the SAM. The Ser/Thr kinase PINOID (PID) is involved in establishment of auxin distribution through regulating cellular localization of PIN-FORMED1 (PIN1), an auxin efflux facilitator. To identify additional genes involved in this process, we carried out a screen for pid enhancers, named macchi-bou (mab). We identified four loci, MAB1~4. mab3 was a novel pin1 allele. The MAB1 and MAB4 genes were cloned by map-based approach. These genes encode a mitochondrial pyruvate dehydrogenase E1 beta subunit and a novel protein that belongs to the NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) family, respectively. A possible role of the MAB genes in organogenesis will be discussed.

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To explore basic components of plant morphogenesis, we have isolated and investigated a number of temperature-sensitive mutants of Arabidopsis thaliana that are defective in in-vitro organogenesis. Of these, rid3 (root initiation defective 3), rpd2 (root primordium defective 2), and rgd3 (root growth defective 3) were characterized by being temperature-sensitive for shoot and root redifferentiation while being almost insensitive for dedifferentiation and callus cell proliferation. Analysis of expression patterns of SAM-related genes during shoot redifferentiation revealed that the expression levels of CUC1 and CUC2 are suppressed in the rgd3 explants cultured at restrictive temperature, which might be partially responsible for the defect of shoot redifferentiation in this mutant. Phenotypic analysis of mutant seedlings indicated that the rgd3 mutation also affects the structural maintenance of both SAM and RAM.
Map-based cloning of the responsible genes of these mutants are in progress, and RID3 was found to encode a novel WD40-repeat protein.

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