In Arabidopsis, stromal and thylakoid membrane-bound ascorbate peroxidase isoenzymes (sAPX and tAPX, respectively) are encoded by two different loci. The aim of this study is to clarify the regulatory mechanism of each chloroplastic APX gene in Arabidopsis. Arabidopsis suspension cells were grown under low light intensity (50 μmol/m²/s), then transferred to mild light intensity (ML; 420 μmol/m²/s), which is the condition to show the maximum O₂ evolution. Both the activity and protein levels of tAPX significantly increased in about 24 h of ML illumination, but those of sAPX remained unchanged. Northern hybridization revealed a constant level of two chlroplastic APX transcripts during the experiment period. These results suggest that the light induction of tAPX is post-transcriptionally regulated. Expression pattern of these two chloroplastic APX genes in Arabidopsis plant is now being examined.

View full abstract

We evaluated the scavenging activity of H2O2 in WWC of illuminated intact chloroplasts from wild tobacco leaves. An illumination caused intact chloroplasts to uptake O2 and to accumulate H2O2. With the O2-uptake both APX and GAPDH lost their activities. The loss of APX activity lowered the activity of WWC, as judged from the decrease in ψ(PSII)xPFD. For the verification of these implications, we made a transplastomic tobacco plant where APX originated from red alga, Galdieria partita, was overproduced in chloroplasts. In illuminated intact chloroplasts from transplastomic tobacco, neither O2-uptake nor H2O2-accumulation was observed. Furthermore, both activities of WWC and GAPDH were maintained. The present work is the first report that APX was inactivated by H2O2 intrinsically photoproduced in intact chloroplasts from wild plants. This would be the reason why plants die when plants are exposed to abiotic stress which suppresses photosynthesis.

View full abstract

During culture, Brassica napus leaf protoplasts become swollen and finally degrade. Both morphological and biochemical approaches revealed that the process through which the death occurred were subjected to apoptotic-like manner. The Protease activity remained lower in leaves, but gradually increased during the culture. Several protease inhibitors added to the enzyme solution for protoplast isolation had no apparent effect on protoplast survival. The previous results showed that the amount of extracellular hydrogen peroxide and superoxide present in the medium during the isolation of leaf protoplasts may not be involved in triggering apoptosis from B. napus. DCFH-DA staining visualizes intracellular active oxygen species (AOS), especially hydrogen peroxide. Our data showed that greater levels of AOS were accumulated in B. napus chloroplasts. However, addition of radical scavengers to both the isolation and the culture medium had no effect on the cell death of B. napus protoplasts.

View full abstract

GPX-like protein from the halotolerant Chlamydomonas W80 (C. W80) shows activity towards unsaturated fatty acid hydroperoxides (PUFAOOH), but towards neither H₂O₂ nor phospholipid hydroperoxide. Here, we generated transgenic tobacco plants expressing a GPX-like protein in the cytosol (TcGPX) or chloroplasts (TpGPX). The activities towards α-linolenic acid hydroperoxide in TpGPX and TcGPX were 32.7-42.1 and 47.5-75.3 nmol/min/mg protein, respectively, while no activity was detected in the wild-type plants (WT). The transgenic plants showed increased tolerance to oxidative stress caused by application of methylviologen (50 μM), chilling (4°C), or salinity (250 mM NaCl). Under these stress conditions, the lipid hydroperoxidation in TcGPX and TpGPX was clearly suppressed compared with that of WT. Interestingly, TpGPX lines seemed to be more tolerant compared with TcGPX lines.These results clearly indicate that a high level of GPX-like protein functions to remove PUFAOOH generated in cellular membranes under stress conditions.

View full abstract

Molecular mechanisms of axillary bud dormancy resulting from apical dominance are still unknown. To analyze the mechanisms controlling dormancy in axillary bud of Arabidopsis, we compared gene expression profile before and after release from bud dormancy. Of the 22,746 genes represented on the Affymetrix ATH1 GenomeArray, expression of the 1,678 genes and 1,188 genes was down- and up-regulated by decapitation, respectively. We identified sugar-repressive element (SRE), which was enriched in the down-regulated genes, and two elements, Up1- and Up2-box, involving in the up-regulation. Both in silico and in vivo analyses demonstrated these elements are the functional components defining gene expression profiles. SRE was involved in the suppression of gene expression by sugar, and mainly regulated the expression of metabolism-related genes in dormant buds. In contrast, Up-boxes were principally related to protein synthesis after release from bud dormancy. We propose mechanisms of growth regulation that govern in axillary bud dormancy.

View full abstract

Recent evidence provide a reliable concept that the gibberellin-signaling causes derepression of the repressible function of the DELLA protein. Working of the DELLA protein in the germinated cereal aleurones has been identified, but little is known as to how this protein contributes to the germination process. To delineate between the GA-signaling and nuclear functions, the effects of the DELLA protein on the expression of the GA-primary response genes, such as GAMyb and OsDof3, have been evaluated. Transient expression of reporter genes controlled by the upstream portion from the primary response genes showed the GA-dependency in transfected aleurone cells. 5'-deletions of the promoter regions gradually decreased the reporter activities, while keeping a constant responsibility to GA. Swapping of the 5'-upstrem regions with a general promoter CaMV35S further sustained the GA-responses. These findings suggest that the expression of the primary response genes should rest on regulatory elements apart from the 5'-upstream regions.

View full abstract

Rice α-amylases were known to be encoded by multigene, and 11 isoforms were identified and characterized in the germinating seeds and tissue culture.α-Amylase I-1 (RAmy1A) was a glycoprotein bearing N-linked carbohydrate chain, that was actively synthesized and secreted from the secretory tissues. Here, we analyzed the transgenic rice seeds transformed with p35SΩ-α-amylase I-1 cDNA. The obtained transgenic rice lines had 2-5 copy of the exogenous gene. Most of them exhibited the co-suppression of α-amylase I-1. The co-suppression lines showed a marked delay of germination and seeding growth compared with the wild-type seed, however, supplementation of sucrose did overcome its abilities. Interestingly, it was observed that starch markedly accumulated in the bottom part of second leaf of transgenic seedlings, suggesting that α-amylase I-1 is involved in the accumulation and degradation of starch in the second leaf of germinating rice seeds.

View full abstract

The thermoinhibition of lettuce seed germination at 28 C was prevented by an ABA biosynthesis inhibitor, fluridone. At 33 C, however, the combined application of fluridone and GA₃ was necessary to overcome the thermoinhibition. The exogenous GA₃ lowered endogenous ABA contents in the seeds, enhancing catabolism of ABA. Fluridone also decreased the ABA contents. Nevertheless, neither fluridone nor GA₃ alone induced the germination at 33 C, because sensitivity of the seeds to ABA was further increased at the elevated temperature. Consequently, the combined application of fluridone and GA₃ decreased the ABA contents to a sufficiently low level allowing the seeds to germinate at 33 C. We conclude that ABA regulates thermoinhibition of lettuce seed germination and that GA affects temperature responsiveness of the seeds through ABA metabolism.

View full abstract

To investigate the regulatory mechanism of dormancy breaking in seeds by cold treatment, we examined the dormancy of several ecotypes of Arabidopsis, generous gifts from ARBC. The dormancy of an ecotype Abd-0 was broken by cold exposure (4°C) and also by exogenous supply of gibberellins (GA) without cold treatment, suggesting that GA may be involved in the dormancy breaking. Interestingly, Abd-0 seeds exposed to 4°C were found to be more responsive to GA administered exogenously to break dormancy than non-treated control. Quantitative PCR analysis showed that accumulation of RGL2 mRNA, a negative regulator of GA signaling, significantly decreased 48h after imbibition at 22°C in Col-0 seeds, whereas it remained at a high level for at least 7 days in Abd-0 seeds. The Abd-0 phenotype was transmitted through a dominant gene(s) and further molecular studies are in progress to elucidate genes responsible for dormancy breaking in the seeds of Arabidopsis.

View full abstract

In higher plants, somatic embryogenesis is thought to a direct evidence of 'totipotency', but little is known about the mechanism of acquisition of embryogenic competence in somatic cells. In carrot, somatic embryos can be induced by the stress treatment. In this system, expression of many embryo-specific genes were found during the stress treatment without any morphological change. It was expected to identify the genes involving in the acquisition of embryogenic competence by expression analysis during the stress treatment. From the analyses using Arabidopsis microarray, there were some up-regulated genes by the stress treatment. One of these up-regulated genes was No Apical Meristem-like 1 (NAM-like 1) and expressed in floral tissue and calli, but not in somatic embryos. Overexpressors of this gene formed numerous shoots on the basal part of inflorescence as compared to wild type. These results indicate that NAM-like 1 gene might involve in stimulation of organogenesis.

View full abstract

Somatic embryos of Japanese larch (Larix leptolepis GORDON) consist of cytoplasmic dense embryo-proper and vacuolated suspensor. Somatic embryogenesis was strongly inhibited in high-cell-density cultures. The inhibition was caused by factor(s) that were released by the cells into the culture medium. In this study, we purified and identified one of the inhibitory factors found in the medium of high-cell-density cultures of larch cells. The inhibitory factor showing the strongest inhibitory activity was purified by dialysis, fractionation with ethylacetate, chromatography on an octadecylsilyl silica gel-column and HPLC. The inhibitory factor was identified as vanilline benzyl ether (VBE) by mass spectrometry and 1H- and 13C-NMR spectroscopy. VBE synthesized from vanilline and benzylchloride by Williamson synthesis. The authentic VBE inhibited somatic embryo formation in Japanese larch, especially the development of suspensor. These results suggest that VBE accumulates in high-cell-density cultures and inhibits somatic embryogenesis in Japanese larch.

View full abstract

In Arabidosis, LEAFY COTYLEDON1 (LEC1) is thought to play important roles in embryogenesis and shares significant sequence identity with HAP3 subunit of the CCAAT-box binding factor (CBF). We had already isolated C-LEC1, a carrot homolog of LEC1, and found that the expression of C-LEC1 was limited in embryonic tissues, especially in the early stage of somatic and zygotic embryos. Based on the published reports on CBF in other organisms, it is considered that C-LEC1 may form a complex with carrot-HAP2 and carrot-HAP5 and bind to the CCAAT sequence in the promoter of embryo-specific genes.
By the yeast two-hybrid screening and PCR using degenerate primers, we had cloned four factors that might interact with C-LEC1. C-HAP2A and C-HAP2B showed high similarity to HAP2. C-HAP5A and C-HAP5B showed high similarity to HAP5. C-HAP5A and C-HAP5B could interact with C-LEC1 in vitro. Moreover, C-HAP2B/C-LEC1/C-HAP5A or C-HAP5B could form complex showing CCAAT-box binding activity.

View full abstract

Arabidopsis ABI3 gene is known as a transcriptional factor which expresses from very early stage of embryogenesis. In our previous work, we had already identified an embryo-specific promoter cis-element, Carrot Embryonic Element 1 (CEE1), in the C-ABI3 (carrot-ABI3) promoter. In this study, we analyzed the Arabidopsis ABI3 promoter region and identified the region inducing the ABI3 gene expression in embryo. At first, promoter activity of 4 ABI3-promoter-deletion:GUS constructs in zygotic and somatic embryos were analyzed. Next, binding activity with CEE1-binding-factors in carrot was checked on some CEE1-like sequences which exist in ABI3 promoter. From the results of both experiments, it was indicated that 100 bp region (Atd3-1L1) including two reverse-CEE1-like sequences is related to the ABI3 gene expression in embryo. Then, the Atd3-1L1 fragment was fused to CaMV35S minimum promoter and the gain-of-function experiments were conduced. The Atd3-1L1 region could induce gene expression in zygotic embryos from its early stage.

View full abstract

Many replication initiation sites are synchronously activated in the early stage of carrot (Daucus carota L.) somatic embryogenesis. Using this experimental system, we tried to label replication initiation sites with bromodeoxyuridine (BrdU). We performed pulse labeling of DNA with BrdU during development of globular stage embryos. After 15 min. of labeling, cell nuclei were isolated, and the genomic DNA fiber was spread on slides. Replication initiation sites were visualized by immunolabeling with FITC-conjugated antibody. The replicon size in the early glubular stage was singnificantly shorter than that in other stages. This result was consistent with the observation by Fujimura et al (1981 in this meeting). We successfully isolated labeled regions of DNA fiber by laser microdissectioning.

View full abstract

Carrot somatic embryos are directly formed when hypocotyls are briefly treated with 2,4-D and then cultured in hormone-free medium. We are interested in morphological events of cell cluster to globular embryo in embryo-forming process. In serial observation of somatic embryogenesis, we found that a part of cell cluster developed into embryo, whereas another was degenerated. Furthermore, we isolated a gene that may be probably involved in a partial degeneration of cell cluster. This gene, named 'nucellin-like protein' has similarity to barley nucellin (Chen et al., 1997) that encodes aspartic protease and may be involved in death of nucellar cell surrounding the entire embryo sac after pollination. Northern analysis revealed that nucellin-like protein transcripts were highly detected in cell clusters and slightly in globular embryos, but hardly in heart- and torpedo-shaped embryos and plantlets.

View full abstract

Many transposable elements exist in all living things from prokaryote to eukaryote. By dissecting the En/Spm functional components it was demonstrated in tobacco plants that two elemnt-encoded proteins TNPA and TNPD are both required and sufficient to mobilize En/Spm, and it was shown that that TNPA binds specifically to a subset of subterminal repeats (STR) sequences. The general structure of En/Spm is typical for many transposable elements, and Tdc is one of them.
In carrot genome, more than 20 copies of En/Spm-like transposable elements, Tdc, were found, and three subfamilies of Tdc were characterized.
The ends of Tdc also contain the 10 bp of STR sequences. We identified Tdc cDNA, which was defective, from cDNA library of carrot as the factor of binding to STR by yeast one-hybrid system. Furthermore, to identify of core cDNA region, we made deletion cDNA of transposase and estimated efficiency of binding to the sequence.

View full abstract

To control the metabolism of industrially useful materials in plants, we are analyzing metabolism-related genes by high-through-put approaches. We prepare resources of transgenic suspension-cultured Arabidopsis T87 cell lines carrying Arabidopsis and Lotus japonicus genes expressed under strong promoters. Unless developmental phenomena are concerned, suspension cultured cells are ideal for functional analyses. However, continuous maintenance of many cultured lines is laborious, constraining high-through-put analyses. Here we established an easy and reliable method of cryopreservation of suspension-cultured Arabidopsis cells. Cultured cells suspended in sodium alginate solution were dispensed into calcium chloride solution for beads formation. The cells in beads were appropriately dehydrated on silica gels, and then immersed in liquid nitrogen for storage. The stored cells were warmed to room temperature and grown on agar medium. Under optimal conditions, the recovery of viable cells was almost 100%. We are examining the effects of cryopreservation on metabolite and gene expression profiles.

View full abstract

Our laboratory has initiated a functional genomics program to study the model plant, a crucifer Arabidopsis thaliana at the transcriptome and metabolome levels. We chose a cultured cell line of A. thaliana T87 which were able to control of biological variation for metabolome analysis. Metabolic profiling of A. thaliana T87 cell culture was performed using multidimensional analysis technique based on the sensitivity and selectivity of mass spectrometry (MS). These analytical systems include reverse-phase HPLC coupled to UV photodiode array detection and electrospray ion-trap mass spectrometer, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometer, and gas chromatography (GC) coupled to time-of-flight (TOF) mass spectrometry. Profiling of the phenolic compounds in A. thaliana T87 cell culture using HPLC/MS detected kaempferol-type flavonoid metabolites accumulating in leaf of wild-type A. thaliana. Another metabolite profiling using GC/MS and CE/MS will be presented.

View full abstract

Integration of transcriptomics and metabolomics may provide precise information on regulatory networks of metabolic pathways. To identify key genes which control productivity of certain metabolites in Arabidopsis thaliana, we analyze gene expression profiles by DNA microarray and metabolite profiles by combination of mass spectrometry. The suspension-cultured A. thaliana cells, T87, is suitable for this analysis because of its homogeneity. First of all, we investigate the transcriptome of T87 in normal growth conditions. Transcripts involved in chloroplast function were maintained at high levels, suggesting that T87 is applicable for analyzing metabolic pathways in chloroplast. By methyl jasmonate treatment, transcripts of jasmonate biosynthesis genes increased within 1 hr and gradually decreased. Similar pattern was observed for genes encoding monoterpene synthase, 4-coumarate-CoA ligases, a few kind of transcription factors, P450s and glycosyltransferases. Functional analysis of these genes through a comparison with metabolome analysis is now in progress.

View full abstract

In order to improve productivity of metabolites from industrially useful plants, we are currently working on bioinformatics for correlating genes with metabolic pathways. Integration of both transcriptome and metabolome data on pathway maps will provide insight into gene function. In this study, we develop a web-based plant metabolic pathway viewer, Kazusa Pathway Viewer (KPV). KPV stores both metabolic and transcriptional profiling data to display simultaneously these profiles on metabolic pathways. For construction of pathway maps, we intend to integrated related pathways on single maps for intelligent viewing of complex paths, and adopted the scalable vector graphic (SVG) format that allows us displaying data dynamically. In the initial version, we set up the viewer for the model plant Arabidopsis thaliana. We plan to add pathways of the legume model Lotus japonicus, industrially useful plants, and crops.

View full abstract

We have reported previously in 2002, a starch synthase I (SSI)-deficient mutant that has 1/6 SSI activity. In this study, we isolated new SSI mutant lines that had different SSI activity levels by PCR method from about 40,000 knockout rice population and analyzed the function of SSI. Four mutant lines have 1/4, 1/5, 1/6 and zero SSI activity compare to the wild-type. The proportion of α-polyglucans with DP of 8-12 in amylopectin was reduced in all lines. The degree of change in amylopectin structure was negatively correlated with the SSI activity. Leaf amylopectin in the null mutant also showed the same change. These results suggest that SSI plays a distinct role in the synthesis of α-1,4 polyglucans of DP8-12 in starch metabolism. Because the seed weight of the null mutant was unchanged, it appears that other SS isozymes complement the α-1,4 polyglucans synthesis in the endosperm.

View full abstract

The specific vesicle fusion during the vesicular transport is mediated by membrane-associated proteins called SNAREs. Recently, the SNARE proteins have been classified into five classes based on the similarities of the SNARE motif for Qa-SNAREs/Syntaxins, Qb-SNAREs, Qc -SNAREs, R-SNAREs/VAMPs and SNAP-25. We identified 54 SNARE genes (18 Qa-SNAREs/Syntaxins, 11 Qb-SNAREs, 8 Qc-SNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis genome. RT-PCR analyses revealed that all SNARE genes were differentially expressed among tissues. In order to characterize the subcellular localization of the SNARE proteins involved in various vesicular transport pathways, we performed a series of transient expression assay by using the GFP (green fluorescent proteins) fused proteins and found 6 ER-localized SNAREs, 9 Golgi apparatus-localized SNAREs, 7 TGN-localized SNAREs, 2 endosome-localized SNAREs, 21 PM-localized SNAREs and 9 vacuole-localized SNAREs. These results predict the possible combination of SNARE molecules in each transport pathway. We discussed the complex post-Golgi transport pathways.

View full abstract

We have been studying mechanism, dynamics and physiological roles of endocytosis in Arabidopsis by focusing our attention on endocytic Rab/Ypt GTPases. We previously reported that Ara6, a plant unique Rab GTPase with the highest similarity to mammalian Rab5, and Ara7 and Rha1, conventional type Rab5 homologs, are all on the subpopulation of endosomes. Using fluorescent proteins and CSLM, we examined whether these proteins are on the same endosomes or on the different population of endosomes. We also compared the subcellular localization between Rab5 homologs and some putative endosomal SNAREs, which revealed that Ara6 is localized on the later compartment in the endocytic pathway. On the other hand, Ara7 and Rha1 are on the different population of endosomes from Ara6, where GNOM-dependent recycling should occur. We also show some data suggesting transition of these endosomes from one to another.

View full abstract

We have isolated an Arabidopsis mutant, eye (embryo yellow) that has a defect in the cell shape and forms many adventitious leaves in the shoot apex. The EYE gene was predicted to encode a protein homologous to a subunit of a Golgi-localized complex that is required for normal Golgi morphology and function. This result suggests that phenotypes of the eye mutant might be caused by defects in the function of the Golgi. We examined whether the eye mutant has defects in morphology and function of the Golgi by using a Golgi marker, AtERD2-GFP. In the eye mutant, the fluorescence of AtERD2-GFP was observed in the ER and small dots on the ER. This result indicates that AtERD2-GFP was not transport from the ER to the Golgi. The role of EYE will be discussed from this result.

View full abstract

To elucidate the molecular mechanism of the shoot gravitropism, we have isolated many shoot gravitropism (sgr) mutants in Arabidopsis. Previously we have described zig/sgr-4 mutant exhibited abnormal shoot gravitropism and morphological defects in stems and leaves. In zig-1, defective vacuoles were formed in the gravity-sensing endodermal cells, while the fragmented vacuoles or abnormal vesicles were observed in other tissues. ZIG encodes AtVTI11, a Qb-SNARE that is likely to function in the trans Golgi network-vacuole vesicle transport pathway. Thus, the vesicle transport mediated by ZIG may have pleiotropic functions in planta. To dissect ZIG function genetically, we performed a screening for the second mutation that can suppress defects of zig-1. The suppressor lines were isolated from zig-1 seeds mutagenized by EMS. The zig suppressor 3 (zip3) partially suppressed both abnormal gravitropism and morphology of zig-1. We have cloned the ZIP3 gene and its functional analysis is in progress.

View full abstract

BY-2 cells transformed with a cytochrome B5 and RFP fusion construct (CytB5-RFP). This protein distributed in the log-phase cells as punctate pattern. Upon reaching to the stationary-phase, some of the RFP fluorescence was detectable in the lumen of the vacuole. To address what is the trigger of such the relocation of CytB5-RFP at the stationary-grown cells, we incubated the cells with different medium and found that nutrient starvation caused the relocation of RFP fluorescence into the vacuole. To examine the involvement of autophagy for the relocation, we expressed the fusion protein of YFP and Apg8 homolog of BY-2. The YFP fluorescence was detectable in the cytoplasm with few punctate spots. The punctate spots increased under starvation conditions, and some of them appeared to be colocalized with some CytB5-RFP aggregates. These findings suggest that BY-2 cells upon starvation use the autophagic process for degradation of the aggregates.

View full abstract

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. In yeast, we previously found that the C-terminus of Atg8 protein is modified with a lipid molecule by ubiquitination-like reaction after cleavage of its C-terminus by Atg4 protease. These Atg8 lipidation reactions are essential for yeast autophagy. Arabidopsis homologues of ATG8 and ATG4 (AtATG8s, AtATG4s) are supposed to function in a similar way in plant autophagy. Yeast Atg8 is known to be delivered to the vacuole as a consequence of autophagic process. This time, transgenic Arabidopsis expressing GFP-AtATG8 were observed by fluorescence microscopy. In wild-type plants, GFP-AtATG8s, which are localized on some ring structures in the cytoplasm, were delivered to the lumens of vacuole under nitrogen-starvation condition. However, in T-DNA insertion double mutant of AtATG4, GFP-AtATG8s were not delivered to the vacuole under nitrogen-starvation condition. This will provide a monitoring system assessing autophagic process in a whole plant.

View full abstract

Plant cells highly develop complicated and sophisticated endomembrane systems including endoplasmic reticulum (ER), Golgi complex and vacuole. However, little is known how such structures of endomembrane systems are organized in the plant cells. Recently, we generated a transgenic Arabidopsis plants expressing a vacuole-targeted GFP to visualize the endomembrane systems. We mutagenized this transgenic Arabidopsis plants and screened for mutants, which exhibited abnormal structures of endomembrane systems. We isolated a mutant, katamari1, in which endomembrane formed an aggregation. Fluorescent markers and ultrastructural analysis showed that most of ER, Golgi, endosomes, peroxisomes and vacuoles were aggregated in the mutant cells. Interestingly, some of actin filaments were also aggregated. Positional cloning revealed KATAMARI1 gene encoding a type II membrane protein. We will discuss a function of the KATAMARI1 protein responsible for a proper organization of endomembrane.

View full abstract

Seed storage proteins are synthesized on rER as larger precursors, are transported to protein storage vacuoles (PSVs) by vesicle-mediated machinery, and then are processed into mature forms. Recently, we have identified AtVSR1 as a sorting receptor for seed storage proteins in Arabidopsis.1) Storage protein precursors are accumulated in the extracellular space in the seeds of AtVSR1-deficient mutant. We report here A06 mutant that accumulates the precursors in addition to the mature forms of the storage proteins. Electron microscopic analysis revealed that A06 mutant abnormally secreted storage proteins from cells as the AtVSR1-deficient mutant. A06 seed cells possessed smaller and much more PSVs in the cytoplasm than those of wild-type cells. These phenotypes of A06 mutant are different from those of AtVSR1-deficient mutant. Immunoblot analysis revealed that AtVSR1 was present in the seeds of A06 mutant.
1) T. Shimada et al. Proc. Natl. Acad. Sci. USA, 100, 16095 (2003)

View full abstract

To enhance our understanding of gibberellin (GA) metabolism in rice (Oryza sativa L.), we identified 29 candidate genes encoding the following GA metabolic enzymes: ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox). In contrast to Arabidopsis, multiple CPS-like, KS-like, and KO-like genes were identified in rice, most of which are contiguously arranged. We demonstrated that the enzymes catalyzing the early steps (i.e. CPS, KS, KO, and KAO) are encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by gene families. The remaining CPS-like, KS-like, and KO-like genes were likely to be involved in biosynthesis of diterpene phytoalexins rather than GAs, because the expression of some genes were up-regulated by UV irradiation and elicitor treatment.

View full abstract

Seed dormancy and germination are controlled by multiple environmental and endogenous factors. We have previously shown that both red light and cold treatment upregulate the gibberellin (GA) biosynthesis gene AtGA3ox1. Besides the synthesis of bioactive GAs, their deactivation by GA 2-oxidases is another possible mechanism for controlling the level of this hormone. Our QRT-PCR analysis indicated that AtGA2ox2, which encodes a GA 2-oxidase, was highly upregulated when seeds were imbibed in the dark at 22°C following a far-red (FR) light pulse that inhibits germination. In a loss-of-function AtGA2ox2 mutant, the inhibitory effect of FR light and dark incubation on germination was reduced, suggesting a role of AtGA2ox2 in maintaining a low level of bioactive GAs. These results suggest that GA deactivation plays a role in modulating the level of bioactive GAs in response to environmental signals during Arabidopsis seed germination.

View full abstract

GAMYB is known to be a transcription factor that activates a gibberellin-inducible α-amylase gene expression at aleurone layer in germinating cereal seed. Recent studies for two long-day plants, Lolium and Arabidopsis, showed that GAMYB mRNA is expressed at inflorescence meristem and tapetum in premature anther after flower initiation . We found that OsGAMyb is also expressed at developing inflorescence in rice, and observed (1) delay of germination, (2) suppression of internode elongation, and (3) morphological changes of inflorescence (decrease of primary branch and secondary branch) in GAMyb mutant which showed the reduction of the OsGAMyb transcripts. Furthermore we analyzed that transgenic rice carrying with Ubi::OsGAMYB RNAi gene, and consequently found that the Ubi::OsGAMYB RNAi rice showed the similar phenotype to GAMyb mutant .We discuss about role for OsGAMyb in germination and formation of inflorescence.

View full abstract

GAMYB was first isolated as a positive transcription regulator of gibberellin (GA)-dependent α-amylase expression in aleurone. However, the role of GAMYB outside of aleurone is not well understood. To investigate the function of GAMYB outside of aleurone, we isolated loss-of-function mutants from a panel of rice mutants by the Tos17 insertion. Through PCR screening, we isolated three independent mutant alleles. No α-amylase expression in the endosperm was induced in these mutants, indicating that the Tos17 insertion had knocked-out OsGAMYB function. We found no significant defects in the growth and development of the mutants before and at the flowering time. After the pahse transition to reproductive stage, however, defects in floral organ development, especially a defect in pollen development, were observed. These results demonstrate that, in addition to its role in the induction of α-amylase in aleurone, OsGAMYB is also important for the floral organ development and essential for pollen development.

View full abstract

RSG is a bZIP transcriptional activator, which regulates shoot growth by controlling GA level. Suppression of RSG resulted in severe dwarfism. We have demonstrated 1) RSG interacts with 14-3-3 proteins, which are broadly conserved regulatory factors, through phosphorylated serine-114 in RSG; 2) Binding with 14-3-3 inhibits the transport of RSG to nucleus; 3) RSG is not statically sequestered in the cytoplasm by 14-3-3, but dynamically shuttling between nucleus and cytoplasm; 4) endogenous level of GA regulates localization of RSG.
It is known that disappearance of DELLA protein, such as RGA, from the nucleus by protein degradation upon GA stimuli triggers GA responses in the cell. We examine whether the similar mechanism works in the case of RSG. After GA stimuli, protein levels of RSG and mutant RSG which can not bind to 14-3-3 are not changed. Consequently, GA regulates RSG mainly by controlling intercellular localization of RSG through binding with 14-3-3.

View full abstract

We isolated a rice brassinosteroid-related mutant, which displayed dwarfism, erect leaves and stunted leaf sheath. Application of brassinolide rescued the mutant phenotype, indicating that the mutant was defective in BR biosynthesis. Mutated gene was located on the Chr. 10 and this locus contains a homologous gene to Arabidopsis DIMINUTO. The putative amino acid sequence of this gene (OsDIM) shared 80% identity with AtDIM. Sequencing analysis revealed that the mutant produced a premature stop codon in the OsDIM gene. We could not found any OsDIM homologous genes in the rice genome. Although the phenotype of the Osdim was typical for BR-deficient mutants, the severeness of its phenotype was much milder than that of the brd1-1 plants, which is defected in BR-6 oxidase. This suggests a possibility that there is an alternative BR biosynthesis pathway not involved DIM in contrast to Arabidopsis, in which defect in DIM induces a severe phenotype.

View full abstract

We have investigated the mechanism of regulation of seed length in rice. The rice d11 fruits small grain seeds and shows dwarfism with extremely short of second-internode. We isolated the D11 gene by map-based cloning method. The D11 gene seemed to encode a novel cytochrome P450. By BLAST searches, the deduced amino acid sequence of D11 gene showed high similarity to Arabidopsis DWF4 (43% of identity and 62% similarity). The accumulation of D11 mRNA was much more in the d11 mutant than in wild-type plants. We also studied the effect of BL on the expression of D11 mRNA, because the transcription of cytochrome P450s involved in BR biosynthesis are reported to be regulated in a feedback manner by the end product of BR-biosynthetic pathway, BL. The expression of D11 mRNA in the mutant was dramatically reduced with exogenously supplied BL.

View full abstract

We have already isolated brd1 dwarf mutant of rice and identified the causative gene, OsBR6ox, a BR biosynthesis gene¹⁾. In this research, we tried to find genes up-regulated or down-regulated by BL using brd1 mutant and 22K rice microarray. Gene expression pattern in the brd1 mutant in the presence of BL was compared to that in the absence of BL. As a result, 21 genes were up-regulated and 20 genes were down-regulated 3hr after the addition of BL compared to the control. While 24hr after the addition, 95 genes were up-regulated and 17 genes were down-regulated. About 90% of the expression patterns of the 43 genes analyzed by real-time PCR were coincided with those of microarray. Further, about 50% of the expression patterns of the analyzed genes by real-time PCR were biologically repeated.
¹⁾ Mori et al., Plant Physiol. 130:1152-1161. (2002)

View full abstract

Brz (brassinazole) was synthesized as the first specific inhibitor of brassinosteroid biosynthesis by Asami et al. Target of Brz220 is the cytochrome P450 enzyme encoded by [I]DWF4[/I]. In order to analyze in detail the mechanisms of brassinosteroid signal transduction, we screened for mutants that showed longer hypocotyls than wild type when grown with Brz220 in the dark, and designated [I]bil[/I] mutants ([I]Brz-insensitive-long hypocotyl[/I]). We identified a recessive mutant, [I]bil5[/I], from fast neutron-treated lines. Hypocotyl elongation of these plants on Brz medium was less than that of [I]bil1-D[/I], but still at least twice that of the wild type. Interestingly, adult [I]bil5[/I] plants showed pale green, thin stems, thin leaves and shortened stem length. The pale-green leaves of [I]bil5[/I] were in contrast to the dark-green leaves of brassinosteroid-deficient mutants, and chloroplast gene expression was lower in [I]bil5[/I] than in the wild type.

View full abstract

The early auxin-inducible genes, IAA3, 5, 19 and SAUR-AC1 as well as a synthetic auxin-response element, DR5, were induced quickly in response to brassinosteroid (BR) treatment. Either of BR- and auxin-induction kinetics was similar between the early auxin-inducible genes and the DR5. We identified BR and auxin-inducible genes comprehensively using GeneChip. The frequencies of the auxin-response element, TGTCTC, was not enriched in auxin-specific genes, but was enriched in genes regulated by both BR and auxin. AXR1 , an homolog of the ubiquitin-activating enzyme E1, functions as an positive regulator of auxin-signaling. The axr1 mutant was insensitive to BR-induced hypocotyl elongation and SAUR-AC1 gene expression. YkB, an inhibitor of auxin action, inhibited BR-induced gene expression, whereas PCIB, an antagonist of auxin, did not inhibited BR-induced gene expression. The results suggested that the signaling pathway of BR interacts with that of auxin at upstream of ubiquitin-proteasome-mediated protein degradation system.

View full abstract

Here, we analyzed axr2/iaa7 and axr3/iaa17 mutants mainly in BR-mediated gene expression. Either of BR- and auxin-induction of IAA5 and IAA19 gene expression was inhibited in both axr2 and axr3 mutants. The inhibition was observed in 7-day-old seedlings, but not in 14-day-old seedlings. In the axr2, auxin induction of the SAUR-AC1 gene expression was inhibited, whereas BR induction of that was not inhibited. Histochemical staining of pSAUR-AC1-GUS was lost in hypocotyl of the axr2. Consistently with this finding, BR induction of hypocotyl elongation was lost in the axr2. The expressions of BR-biosynthetic genes were higher in the axr2 than in WT in 7-day-old seedlings, but some of them were normal in 20-day-old seedlings of the axr2. We also identified genes regulated by axr2 using GeneChip analysis. These results suggested that the IAA proteins regulate expression of each BR-regulated gene in an organ- and a growth-stage-specific manner.

View full abstract

We isolated and characterized a rice dwarf and abnormal leaf morphology 1 (dal1) mutant, which forms the round leaves at the tip. For cloning the DAL1 gene, we produced 4,000 F₂ plants by crossing the Japonica dal1 plants and an Indica rice, Kasalath. The DAL1 locus was narrow into ~40kb between two molecular markers. In this region, there were five possible genes and we focused on one gene, which encodes a katanin-like protein, because Arabidopsis katanin-like protein, FRA2, is involved in microtubule disassembly for normal growth and development of leaf and stem. We performed sequence analysis of this candidate gene in four alleles (dal1-1~4) and found that all dal1 mutants had base substitutions or deletions in DAL1. This strongly indicates that DAL1 encodes a katanin-like protein, and also suggests that the defect of the katanin-like gene causes the aberrant cortical microtubules resulting in stunted leaf and stem with abnormal morphology.

View full abstract

Individual microtubules dynamically alternate between two states, growth by polymerization and shrinkage by depolymerization, the property termed "dynamic instability." In plant interphase cells microtubules organize into cortical arrays that oriented perpendicular to the direction of cell elongation in rapidly elongating cells. To study how microtubule dynamic instability affects cortical microtubule orientations during cell elongation ( and thereby the direction of cell elongation ), the transgenic Arabidopsis plants in which β-tubulin fused with GFP were expressed to visualize individual microtubules were analized in vivo by confocal laser scanning microscopy and microtubule dynamic instability parameters were quantified. Next the dynamic instability parameters were compared between plants grown on agar media containing the microtubule-interacting drugs and control plants. At the drug concentrations used, seedling roots showed left-handed helical growth. We also plan to analyze the dynamics parameters of helical growth mutants.

View full abstract

In plant cells, cortical microtubule (MT) arrays generally regulate the direction of elongation. Analysis of right-handed and left-handed twisting Arabidpsis in our lab mutants provided supporting evidence.
When we tagged α-tubulin (TUA) at the N-terminus and expressed under the CaMV35S promoter in transgenic plants, it was incorporated into MT polymers and caused right-handed helical growth in petioles and petals. In contrast, TUA tagged at the C-terminus and β-tubulin tagged at the N-terminus did not produce helical phenotypes, although they were incorporated into MTs. We speculated that TUA with an extra sequence at the N-terminus interferes its GTPase activating function, leading to longer GTP cap and more stable microtubules, resulting in right-handed helical growth. To test this hypothesis, we examined the effect of TUA mutations which are predicted to be involved in this GTPase activation. Plants expressing the mutant tubulins caused right-handed helical growth, and had increased trichome branching.

View full abstract

Microtubule(MT) structure is highly conserved in eukaryotes. Stable heterodimers of α-tubulin and β-tubulin are stacked longitudinally to form a tubulin protofilaments and 13 protofilaments are associated laterally to form a hollow MT. Previously, we showed that dominant negative left-handed helical growth mutants lefty1 and lefty2 have a missense mutation in two α-tubulins at the α-β interacting region of intradimer surface and form unstable MTs.
Here, we screened a large collection of mutants that are hypersensitive to MT destabilizing drugs and investigated relationships between tubulin mutations and twisting phenotypes. Three left-handed mutants had mutations at intradimer regions, 3 left-handed mutants and 4 right-handed mutants carried mutation at interdimer regions, 10 right-handed mutants and 1 left-handed mutant were tubulin mutated at lateral regions, and two right-handed mutants were found to be mutated at GTPase activating regions of α-tubulin. Cortical microtubule arrays and dynamics in the mutants are being analyzed.

View full abstract

The propyzamide hypersensitive1-1 (phs1-1), semi-dominant Arabidopsis mutant, roots show left-handed helical growth phenotype when grown on hard agar plates. In the presence of 3μM propyzamide, a microtubule (MT) destabilizing drug, the root epidermal cells swell radially and are hyper-sensitive to this and other MT destabilizing drugs. The cortical MTs of phs1-1 root epidermal cells are shorter, and less well organized (with a tendency to form right-handed helices) than wild-type MTs. We identified the PHS1 gene by a map-based cloning approach. The C-terminus of PHS1 protein has a dual-specificity protein tyrosine phosphatase (DsPTP) catalytic domain. PHS1 was fused with glutathione-S-transferase, overexpresed in E. coli, and purified. PHS1 fusion protein dephosphorylated at a artificial substrate. The phs1-2 allele in which PHS1 gene was disrupted by T-DNA was recessive embryo-lethal. Analysis of transgenic plants expressing PHS1 genome regions indicated that phs1-1 R64C mutation is dominant-negative.

View full abstract

Cell division in basal land plants reveales a surprising variety of microtubule organizing centers (MTOCs) that is thought to represent milestones in the evolution of the typical diffuse acentrosomal plant MTOCs. In monoplastidic meiosis of the bryophytes, γ-tubulin is known to localize on the plastid surface in prophase and on the nuclear surface in telophase. As the membrane-based MTOC is not common in animal and seed plants, we further characterized these MTOCs in meiotic cells of the liverwort Dumortiera hirsuta using a drug study and in vitro experiments. γ-Tubulin localization patterns on plastid and nuclear surfaces are not affected by destruction of microtubules with oryzalin. γ-Tubulin is still detectable on the surface of isolated plastids and nuclei suggesting the tight association with nuclear and plastid envelope. In isolated platids, we could repolymerize microtubules from the plastids surface showing MTOC activities on the plastid surface.

View full abstract

We have reported that myosin XI purified from tobacco cultured BY-2 cells can walk processively on an actin filament with 35nm steps (EMBO J. 2003 vol. 22 pp. 1263-1272). In the present study, we studied the mechanism of inhibitory regulation by Ca²⁺. When myosin was treated with Ca²⁺ at concentrations higher than pCa5.5, light chain (calmodulin) detached and affinity between myosin and actin filament decreased. Ca²⁺ did not affect the actin activated ATPase. In in vitro motility assay, sliding movement of actin filament was observed even in the presence of Ca²⁺, when myosin density was increased. We estimated the elasticity of myosin molecule by measuring the rotation of short actin filament attached to a single myosin molecule in the absence of ATP. Ca²⁺ treatment decreased the Brownian movement of actin filament, indicating that the long neck of myosin folded and became shorter and rigid by removal of calmodulin.

View full abstract

Myosin XI is a molecular motor responsible for the cytoplasmic streaming in plant cells. In cultured tobacco BY-2 cells, two subclasses of myosin XI, 170-kDa and 175-kDa myosins, have been identified biochemically and immunocytologically. It has been revealed that the 175-kDa myosin was accumulated in phragmoplasts in mitotic cells. In the present study, we identified the organelle transported by the 175-kDa myosin. In transgenic BY-2 cells expressing GFP-labeled endplasmic reticulum (GFP-ER), the intracellular localization of 175-kDa myosin and ER was studied. Especially in mitotic cells, both were accumulated in the phragmoplasts. This phenomenon was suppressed by laturanculin B, an actin-depolymerizing drug. These results suggested that the 175-kDa myosin is concerned with translocation of ER in the cytoplasmic streaming and in phragmoplast formation.

View full abstract

Chloroplasts are centripetally or centrifugally arranged in bundle sheath cells (BSC) of C₄ plants. The intracellular arrangement of BSC chloroplasts is acquired during cell maturation and can be re-established after disturbance by centrifugation. Therefore, it is expected that BSC chloroplasts possess molecular mechanisms to recognize their intracellular positions and move to certain positions. In this study, we investigated the effects of various inhibitors to re-establishment of centripetally arrangement of BSC chloroplasts in finger millet (Eleusine coracana), an NAD-ME-type C₄ plant, after centrifugal treatment. Although microtuble polymerization inhibitor, colchicine, was not effective, actin polymerization inhibitors, cytochalasin B and latrunculin B, inhibited the re-establishment. Myosin ATPase inhibitor BDM and cycloheximide also showed inhibitory effects. These results suggested that de novo protein synthesis and myosin-actin system have important roles in intracellular arrangement of BSC chloroplasts.

View full abstract

In green plant cells, microtubules run along the plasma membrane to form an interphase cortical array. To address mechanism of cortical microtubule formation, we analyzed assembly of microtubules on isolated plasma membrane ghosts in vitro. When cytoplasmic extract with rhodamine-tubulin was added onto the ghosts isolated from protoplasts of tobacco BY-2 cells, newly assembled microtubules were detected on ghosts. Upon the addition of cytoplasmic extract, the number of γ-tubulin spots on the cortical microtubules greatly increased and microtubules elongated from the spots. Antibodies against γ-tubulin inhibited the microtubule formation. The formation was also inhibited by removal of cortical microtubules before addition of the extract. We hypothesize that cytoplasmic γ-tubulin binds onto cortical microtubules, and new cortical microtubules initiate from γ-tubulin on the microtubules in vivo. To examine the hypothesis, analyses of microtubule organization in γ-tubulin silenced Nicotiana benthamiana are in progress.

View full abstract