Beta vulgaris accumulates large amounts of sucrose, betaine, and raffinose in tap roots. Regulation of betaine synthesis is important, but its mechanisms are poorly understood. This is partly due to the difficulty of transformation of betaine-accumulating plants. Here, we examined the changes of accumulations of osmoprotectants and cations. One month old Beta vulgaris accumulates betaine 2 - 5 μmol/gFW even under the control conditions. Upon salt stress, the levels of CMO and betaine increased significantly in all the leaves, stems, and roots. The levels of Na⁺ also increased upon the salt stress whereas the levels of K⁺ did not change so much. The suppress expressison of CMO has been examined. The changes of accumulation of sucrose and proline under these conditions will be also presented.

View full abstract

Most of higher plants can not grow under high salt condition. However, some plants (e.g. mangrove) can tolerate high salt condition. Salt accumulation in the vacuoles is one of the mechanisms for salt tolerance. During salt accumulation, a rapid increase of vacuolar volume without increasing cell-size was reported in barely roots and mangrove suspension cultured cells.
In this study, we analyzed behaviors of membrane vesicles and vacuoles in Arabidopsis under high salt condition. High salt condition induced a rapid increase in the vacuolar volume and activated dynamic state of the acid vesicles in Arabidopsis root cells.The same phenomenon of the vacuole was observed in Arabidopsis suspension-cultured cells. Using fluorescence Na⁺ indicator, we observed Na⁺ accumulation in the vacuole and dot-like fluorescence around the vacuole under high salt condition. We further analyze the dynamics of vesicles and the ion transporter proteins under high salt condition.

View full abstract

Boron (B) cross-links pectic polysaccharides at rhamnogalacturonan II regions and thereby contributes to build the cell wall structure. However, it remains unknown how B deficiency causes various metabolic disorders and brings about cell death. To understand this mechanism, we have analyzed the response of cultured tobacco BY-2 cells to B deprivation.
When 3-day-old cells were transferred to a B-free medium, dead cells were evident as early as 12 h after the treatment. We previously showed that the genes for antioxidative enzymes are upregulated in low B-acclimated cells, which suggests that oxidative damages are involved in low B stress. Here we found that lipid peroxides and reactive oxygen species were accumulated in -B cells. In addition, an antioxidant butylated hydroxyanisole reduced cell death. These results indicate that oxidative damage does occur under B deficiency as the direct cause of cell death.

View full abstract

To remediate the worldwide-spreading acid-sulphate soil area and utilize it for human beings, acid tolerant leguminous plants are expected to be useful as crops and pioneer plants. We report the isolation of acid-tolerant genes by functional cloning of a soybean cDNA library and their characterization in planta. E. coli cells were transformed with a cDNA library of soybean constructed with the pDEST14 expression vector and cultured on a LB medium adjusted to pH 4 or pH 5. A total of 27 unique resistant clones were isolated. Some of the corresponding genes in soybean seedlings were constitutively expressed, while others were induced by the aluminium stress. Selected six clones were introduced into A. thaliana using the pGWB2 vector. All the clones tested were able to confer acid tolerance to A. thaliana, that is, the overexpressors grew better than the wild type under the acid and aluminium stresses.

View full abstract

Low-temperature is an important factor determinig germination of rice, but the underlying genetic mechanism of germination in low-temperature has not been well elucidated. We constructed a genetic map using recombinant inbred lines derived from two crosses Kakehashi x Dunghanshali and Iwatekko x Arroz da Terra in order to locate quantitative trait loci (QTLs) conferring low-temperature germination. Six QTLs were mapped. Three of six QTLs for low-temperature germination were contributed by Dunghanshali,and four of six were contributed by Arroz da Terra and two were shared between Dunghanhasli and Arroz da Terra. We mapped two of the most effective QTLs on chromosome 2 and chromosome 3, and are tying to isolate the causal genes by using F3 generation and BC2F2 of near isogenic lines. As a parallel effort, we performed screening of low-temperature germination mutants from EMS-treated rice mutant lines.

View full abstract

Tolerance to abiotic stress is an important agronomic trait in crop and is a complex trait controlled by many genes, which is called quantitative trait loci (QTLs). Identification of these QTLs would archive a stable crop production in the world. Previously, we mapped three QTLs controlling low temperature germinability using back crossed inbred lines (BILs) derived from a cross between a vigourous low temperature germinability temperate japonica variety, Italica Livorno, and a normal temperate japonica variety, Hayamasari. A major QTL, qLTG-3-1, low temperature germinability on chromosome 3, explained more than 30 % of total phenotypic variation in the mapping population. To understand the molecular basis of the most effective QTL, qLTG-3-1, we identified it by positional cloning method and found that it encoded unknown function protein.

View full abstract

Seed morphogenesis is one of the most important developmental steps in the plant body plan. To clarify the molecular mechanism of the seed formation of Arabidopsis, we applied forward genetic approach with the transcriptional factor mutant library of CRES-T (Chimeric REpressor Silencing Technology). We identified a seed-shape defective mutant. The responsible gene was a factor (CR245) that belongs to the plant-specific NAC transcriptional factor superfamily. CR245 has the most closely related homologue CR248 that exhibits a similar transcriptional profile to CR245 in the array dataset of AtGenExpress. Although each T-DNA insertion mutant of CR245 and CR248 had the normal seed shape, cr245cr248 double mutant had the abnormal shapes of seeds. Each of CR245 and CR248 was localized in the nucleus, and had a functional transactivation domain in the C-terminal region, respectively. Our results suggest that both CR245 and CR248 regulate seed morphogenesis redundantly.

View full abstract

Although plastid development is dramatically changed through embryogenesis to seed germination, its biological relevance is not clearly understood. Here, we present evidences that plastid retrograde signaling pathways are involved in genetic programs of embryogenesis and seed germination. We have applied chemical genetics approach to Arabidopsis seed germination and identified E compounds from non-annotated chemical library. E compounds inhibit greening and expansion of cotyledons through ABA and GA signaling components. Genetic analysis revealed genome uncoupled loci, which are well characterized plastid retrograde signaling components, were also involved in the response to E compounds. Based on marker gene expressions for plastid retrograde signaling, E compounds were found to have both positive and negative action at different time points after germination. This can be explained by sequential activation (or repression) of two plastid retrograde pathways perturbed by E compounds during seed germination.

View full abstract

Seed dormancy and germination is controlled by two phytohormones, abscisic acid (ABA) and gibberellins (GAs). We have previously identified and characterized Arabidopsis mutants that show ABA-insensitive seed germination. One of the mutants, named chotto1 (cho1), carries a mutation in a gene encoding a seed-specific APETALA2 transcription factor. We found that the transcript levels of genes for gibberellin 3-oxidase, GA3ox1 and GA3ox2, were up-regulated by after-ripening treatment during imbibition of the wild-type seed (Col-0). In contrast, transcript levels of GA3ox1 and GA3ox2 were similarly high regardless of after-ripening treatment in cho1 seeds. These results indicated that GA3ox gene expression is de-repressed in cho1 independently of after-ripening treatment, and also that CHO1 negatively regulates the GA3ox gene expression. We will show recent results of simultaneous ABA/GA analysis in the presentation.

View full abstract

We carried out gene expression analysis on early stages of seed imbibition. Non-dormant Arabidopsis seeds imbibed at 15-, 30-, 60- and 180-min were analyzed using Affymetrix ATH1 Arrays. No up-regulated genes were found until 30 min, and Zat12 (At5g59820) was the only gene significantly up-regulated at 1 hour. After 3 hours, expression of about 2,200 genes was induced. These included AtNCED9, CYP707A2 and ABI4, and several genes associating with biogenesis of reactive oxygen species. Moreover, we found about 1,900 down-regulated genes at 3 hours. ABRE (CACGTG) was overrepresented in their promoters. LC-MS/MS analysis revealed that endogenous ABA level was drastically decreased after 3 hours. QRT-PCR analysis showed that CYP707A2 expression was induced 3 hours after imbibition. Interestingly, expression levels of ABI3, ABI4, and ABI5 were changed after 3 hours. These results suggested that the reduction in the ABA levels affects gene expression at 3 hours of imbibition.

View full abstract

Arabidopsis L1L is a B subunit of the hetero trimeric CCAAT binding factor NF-Y, most closely related to LEC1. L1L is specifically expressed during seed development and requires the key regulators of seed development, LEC1 and FUS3 for its expression. L1L can activate the promoter of a seed storage protein CRC gene in T87 cells when co-expressed with an NF-YC subunit, HAP5B and ABRE-binding factors, bZIP12/bZIP67. We showed here evidence for complex formation between L1L-HAP5B and bZIP67. We also demonstrated that the expression of a seed specific sucrose synthase gene, SUS2 was reduced in a l1l mutant and that L1L-HAP5B and bZIP67 could activate its promoter like the CRC promoter. The transcriptional activation by these factors required ABREs but not CCAAT motifs and could not be enhanced by NF-YA subunits. These observations suggested that the L1L-HAP5B hetero dimer activates transcription by acting on ABREs through the interaction with bZIP12/bZIP67.

View full abstract

The flo2 mutant shows decreased expression of most genes involved in starch synthesis and storage proteins. Flo2 gene may encode a multi-functional regulatory factor for biosynthesis of storage substances in the rice grain. In order to isolate the Flo2 gene, we generated the F2 lines crossed between the flo2 mutant and Kasalath. Using the recessive F2 lines, we mapped the Flo2 locus in detail and determined to be in the 37 kb region. The nucleotide sequence analysis of the corresponding region revealed a point mutation in an ORF, which generated a translation stop codon. The corresponding mutation was found in all F2 lines showing the flo2 phenotype. We also analyzed the corresponding ORF in other flo2 mutants which were independently established, and detected that similar mutations occurred in this ORF on these mutants. These results strongly suggest that this ORF is the Flo2 gene.

View full abstract

A bZIP transcription factor ABI5 is involved in ABA signaling during seed maturation and germination. Amino-acid substitution of putative target sites for Ser/Thr protein kinases resulted in suppression of ABA-dependent transactivation. The ABI5 polypeptide was phosphorylated by ABA-activated SnRK2 protein kinases, SRK2D, SRK2E, and SRK2I. These genes were expressed differentially during seed maturation and germination. A srk2d/srk2e/srk2i triple mutant showed stronger in ABA insensitive phenotypes in seed germination than srk2d/srk2i double mutant, but not srk2d or srk2i single mutants. Changes in seed dormancy consistent with ABA insensitivity were observed. Viviparous phenotype was also observed in the triple mutant under high humidity conditions. Microarray experiments indicate that many genes down-regulated in the abi5 seeds were suppressed in the srk2d/srk2e/srk2i seeds. These results demonstrate that three redundant SnRK2 protein kinases, SRK2D, SRK2E, and SRK2I, have important roles in ABA signaling during seed maturation and germination of Arabidopsis.

View full abstract

In plant sphingolipids, C4 positions of the LCBs are mostly hydroxylated by dihydrosphingosine C4 hydroxylase (DSH). We found five DSH genes (OsDSH1 to OsDSH5) in the rice genome. Among them, OsDSH1 and OsDSH4 restored the yeast DSH mutation, suggesting that both OsDSH1 and OsDSH4 functioned as DSH in yeast. The expression pattern of OsDSH1 was determined using the GUS reporter assay system. Since the strong expression of the GUS gene was observed in SAM, RAM, vascular bundle and stigma, it was suggested that OsDSH1 was specifically expressed in these organs. In the transformants with the OsDSH1 RNAi, serve reduction of their fertility was observed. These result strongly suggested that OsDSH1 was involved in the fertility.

View full abstract

Multiprotein bridging factor 1 (MBF1) is a transcriptional co-activator that mediates transcriptional activation in yeast and animals. Although Arabidopsis thaliana has three MBF1 genes, their functions remain unclear. In this study, AtMBF1 was fused to a strong transcriptional repression domain SRDX (AtMBF1-SRDX), and we tried to make transgenic Arabidopsis overexpressing this fusion protein. Since it is considered that AtMBF1-SRDX can exert dominant negative effect to endogenous AtMBF1s and prevent functions of AtMBF1-related transcription factors, it is expected to appear phenotypes based on functions of AtMBF1-related factors. Transgenic Arabidopsis overexpressing AtMBF1-SRDX (AtMBF1-SRDXOE) showed dwarf leaves phenotype, and its leaf cells were also tiny compared to those of wild type. We found ploidy level of leaves of AtMBF1-SRDXOE was reduced, and expression level of some negative regulators of endoreduplication was also increased in AtMBF1-SRDXOE. These results indicate that AtMBF1-SRDX suppresses endoreduplication in leaf cells through promoting expression of such negative regulators.

View full abstract

The amyloplasts sedimentation in the endodermal cells is important for gravity perception in Arabidopsis shoot. It has been reported that amyloplasts are surrounded by actin microfilaments (MFs), and that treatment with actin polymerization inhibitor enhances gravitropic organ curvature. However, not only the molecular link between amyolplasts and MFs, but also regulatory role of MFs in gravitropic response is still unclear.
Our previous study suggests that SGR5(SHOOT GRAVITROPISM5) and SGR9 are synergistically involved in regulation of amyloplast movement, and shows that sgr5 sgr9 double mutant completely loses gravitropic response. Here we found that treatment with actin polymerization inhibitor restored gravitropic response of sgr5 sgr9 double mutant stems. The result suggests that abnormal amyloplasts movement in the double mutant could result from inhibition of MFs depolymerization, leading to abnormal gravitropism. We are investigating whether SGR5 and SGR9 are involved in amyloplasts movement by regulating actin remodeling in gravity perceptive cells.

View full abstract

NAC domain proteins, Vascular-Related NAC Domain Protein6 (VND6) and VND7, are master transcription factors that control xylem vessel element differentiation (Kubo et al, 2005, Genes Dev). Here we succeeded in the induction of xylem vessel element differentiation by activating the master transcription factors. We generated transgenic Arabidopsis plants, tobacco BY-2 cells, and Arabidopsis T87 cells, in which VND6 or VND7 fused to the VP16 transactivation domain and the glucocorticoid receptor (GR) is overexpressed under the control CaMV35S constitutive promoter. In the transgenic Arabidopsis plants, various types of cells including root hairs and trichomes transdifferentiated efficiently into vessel elements after the DEX-treatment. Activation of VND7 in BY2 and T87 also effectively induced the transdifferentiation. The detailed morphological observation of the transdifferentiated cells and analysis of cell wall composition during the transdifferentiation are now underway.

View full abstract

Temperature is a primary environmental cue for seed germination of many weeds and vegetables. To investigate the mechanism of germination regulation by temperature, we have selected five high temperature (thermoinhibition) resistant germination mutants of Arabidopsis. The seeds of trg1 shows slightly reduced sensitivity to ABA at 22 ^oC, and the insensitivity is apparent at higher temperature conditions. In addition, trg1 plants have shorter siliques and slightly shorter stems than the wild type. The mutation was mapped between molecular markers TJ-5 and FJ-4 at the bottom arm of chromosome 1. We sequenced all the seven predicted genes in this region, and found 14 bp deletion in At1g68560 which codes for α-xylosidase. The mutation introduces pre-mature stop codon in pro-peptide region of the precursor protein. The thermoinhibition resistant germination of trg1 seeds may be caused by disorganization of cell wall metabolism since α-xylosidase is thought to hydrolyse xyloglucan oligosaccharaides in cell wall.

View full abstract

Poplar cellulase (PaPopCel1) was overexpressed in a tropical leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium method. Expression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than those of the wild type. The pairs of leaves in the transgenic plants closed slower than those in the wild-type plants, showing a delay in the closing movements of leaves during sunset. Based on the carbohydrate analyses of cell walls, the leaves in the transgenic plants contained less wall-bound xyloglucan than those in the wild-type plants. The expression supports the concept that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase to loosen xyloglucan intercalation followed by an irreversible wall modification, which could promote plant growth and disturb the closing movements of leaves within their biological clock.

View full abstract

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. The wood fiber of tension wood has a special secondary cell wall named G-layer which contains no lignin and high amount of cellulose. This layer is thought to be the main part to generate the growth stress of tension wood. On the other hand, tension wood in the transgenic poplar overexpressing xyloglucanase generates much less growth stress than that in the wild type. We compared the ultrastructure of the G-layers from the transgenic and wild poplars. Many cracks were observed in the transgenic plants with clear microfibril structures.

View full abstract

L-Arabinofuranosyl (Araf) residues are an important constituent found in plant cell walls. UDP-L-arabinopyranose (UDP-Arap) is synthesized by several enzymes and then enzymatically converted to UDP-L-Arabinofuranose (UDP-Araf), which is a real sugar donor for Araf-containing cell wall components. We showed that rice seedlings had UDP-Ara interconversion activity and purified the enzyme, UDP-arabinopyranose mutase (UAM), to homogeneity. The reaction is reversible and the formation of UDP-Arap is favored over the formation of UDP-Araf (90:10). Amino acid sequence of the purified UAM revealed that rice has three corresponding genes, UAM1~3, and that these proteins are identical to those previously reported as "reversibly glycosylated polypeptide" (RGP). The recombinant UAMs were obtained using baculovirus/insect cell system and characterized. Recombinant UAM1 and UAM3 had mutase activity with similar enzymatic properties but no activity was observed for UAM2. RGPs are found in the whole plant kingdom, indicating that UAM function is necessary for normal plant growth.

View full abstract

Interconversion between UDP-xylose (UDP-Xyl) and UDP-L-arabinose (UDP-L-Ara) is catalyzed by a membrane-anchored UDP-Xyl 4-epimerase in the Golgi apparatus and essential for the syntheisis of L-Ara-containing cell wall polysaccharides in higher plans. In this study, a soluble UDP-Xyl 4-epimerase distinct from the enzyme in the Golgi apparatus was isolated from pea (Pisum sativum L.) sprouts and characterized. The N-terminal amino acid sequence revealed that the enzyme is encoded by a UDP-glucose (UDP-Glc) 4-epimerase gene, PsGalE. Recombinant PsGalE (rPsGalE) expressed in Escherichia coli showed both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities, confirming that PsGalE is a UDP-Glc 4-epimerase with broad substrate specificity toward UDP-sugars. The apparent K_m values of rPsGalE for UDP-Glc UDP-galactose, UDP-Xyl, and UDP-L-Ara were 0.21 mM, 0.21 mM, 0.17 mM, and 0.14 mM, respectively. The results suggest that the interconversion between UDP-Xyl and UDP-L-Ara in cytoplasm is catalyzed by UDP-Glc 4-epimerase isoforms homologous to PsGalE in higher plants.

View full abstract

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. AGPs are implicated in several aspects of cell wall biology including the signaling of cell context and cell expansion, yet remain elusive in terms of their mechanistic involvement in cell processes. They are characterized by large amounts of carbohydrate components rich in galactose and arabinose, and protein components rich in hydroxyproline. The study of glycoside hydrolases that cleave AGPs is very important because the enzymes specific to particular sugar residues and type of glycosidic linkage will provide useful tools for the structural analysis of the sugar moieties of AGPs. Recently, we demonstrated that Streptomyces avermitilis possess the both exo-1,3-galactanase and endo-1,6-galactanase, suggesting S. avermitilis possess more AGPs degrading enzymes. By using the information of genome sequence of S. avermitilis, we cloned some possible AGPs degrading enzymes. We will present how these enzymes act on AGPs.

View full abstract

We have investigated the properties of β-galactosyltransferase (GalT) involved in the synthesis of the sugar portion of arabinogalactan-proteins whose basic structure comprises a β-(1->3)-galactan, branched with consecutive (1->6)-linked β-Gal residues, to which α-L-Ara and uronosyl residues are attached.
The activity of GalT was assayed by a reaction mixture containing a microsomal fraction prepared from etiolated soybean hypocotyls, UDP-Gal, and various galactooligosaccharides. Resulting Gal transfer products were derivatized with p-aminobenzoic acid ethyl ester (ABEE) and analyzed on HPLC.
The GalT catalyzed incorporation of Gal onto β-(1->3)-galactotriose as an acceptor through β-(1->6)-linkages, producing a branched tetrasaccharide, β-Gal-(1->3)[β-Gal-(1->6)]-β-Gal-(1->3)-Gal. The GalT appeared to prefer the formation of single β-(1->6)-linked Gal branches on the linear β-(1->3)-galactan chains. The GalT also transferred Gal onto β-(1->6)-galactotriose, producing β-(1->6)-galactotetraose. These results indicate that the enzyme involves in the synthesis of β-(1->3)(1->6)-galactan framework in AGPs.

View full abstract

Cell walls of higher plants are actively metabolized during cell growth. We had reported that the degradation of galactan occurred during cell elongation growth by using cell suspension cultures of poplar cells. In this study, I purified a galactan degrading enzyme from the medium of cultured Populus cells and some properties of the enzyme were revealed. The galactan degrading activity in the medium increased rapidly in late stage of elongation phase (10-14 days after subculture). The enzyme protein was purified from the medium on the 14-day to about 100-fold by anion-exchange, hydrophobic, cation-exchange, and gel permeation chromatography. The purified enzyme showed about major 70 and minor 66 kDa bands on SDS-PAGE. The molecular weight of major one agreed with one of the galactan degrading enzymes purified from cell walls. The optimum temperature and pH were 40˚C and 5.5 respectively. Other some properties will be reported in the meeting.

View full abstract

Yieldin (YLD) is the wall protein, isolated from cowpea cell wall (Okamoto-Nakazato et al. 2000), regulating the wall yield threshold (y). Since the molecular cloning study of YLD exhibited that a carbohydrate-binding site was conserved within the YLD molecule, we have assumed YLD to have a carbohydrate-binding activity. To confirm the assumption, we analyzed the carbohydrate-binding activity of YLD by a hemagglutination test using rabbit red blood cells.
As the results, YLD was shown to have a hemagglutinating activity (HA activity) towards the trypsinated rabbit red blood cells and suggested to bind to the carbohydrate(s) on the surface of the red blood cells. The pH-dependency of the HA activity of YLD was similar to that of the YLD-regulated y in the cell wall. These results suggest that YLD is a kind of lectin and the carbohydrate-binding activity of YLD is related to the y-regulation in the cell walls.

View full abstract

Wall-yielding properties have been described with Lockhart's wall parameters (extensibility and yield tension) determined by fitting the stress-extension rate curves with two linear regression lines. These two parameters are regulated pH-dependently in the elongation growth. It has been reported that yieldin from cowpea (Vigna unguiculata L.) hypocotyls does not regulates extensibility but yield tension. On the other hand, we have recently demonstrated that cucumber expansin (CsEXPA1) is capable of regulating both wall parameters.
To clarify the roles of yieldin and expansin on the wall extension, we obtained both yieldin (VuYLD1) and expansin (VuEXPA1) from the cell wall of cowpea hypocotyls and examined the function of VuYLD1 and VuEXPA1 using the isolated wall preparation of cowpea hypocotyl. We show that VuEXPA1 also regulates both extensibility and yield tension by reconstitution assays. The function of VuYLD1 will also be shown.

View full abstract

Plant wall polysaccharide, which is mainly composed of cellulose, hemicellulose and pectin, determines the shape of the cells. During cell enlargement, wall loosening occurs by cleavage of xyloglucan tethers of cross-linkage between cellulose microfibrils and, hydrogen-bonds,in which endo-1,4-b-glucanase, xyloglucan endotransglycosylase/hydrolase and expansin could be involved. To realize the mechanism of wall loosening induced by these apoplastic proteins, we analyzed property of the proteins heterologously expressed in Nicotiana Benthamiana using a virus vector. I present the expression, characteristic and localization of the proteins.

View full abstract

In higher plants, programmed cell death is highly regulated by various factors. Bax Inhibitor-1 (BI-1) is a widely conserved cell death suppressor. Arabidopsis BI-1 (AtBI-1) is approximately 26kDa protein with 7 transmembrane domains and localizes in ER. Overexpression of AtBI-1 suppresses H₂O₂-. SA- and elicitor-induced cell death in plant cells. Moreover, AtBI-1 possesses coiled-coil structure in the C-terminus, which is essential in AtBI-1 function and interacts with calmodulin. However, the mechanism of AtBI-1-mediated cell death suppression has not yet been elucidated.
Recently, we revealed that AtBI-1 interacted with AtFAH (Arabidopsis fatty acid hydroxylase) via cytochrome b₅ (Cb5). Cb5 is an electron transfer protein, which is related to fatty acid synthesis, and AtFAH is a hydroxylase of sphingolipid fatty acid localizing in ER. Lately, the relationships between sphingolipids and cell death have been discussed vigorously. AtBI-1 may suppress cell death through sphingolipid regulation.

View full abstract

Rice EL5 is a membrane-localized RING-type ubiquitin ligase (E3). Transgenic rice plants overexpressing mutant EL5 proteins (mEL5) that are deficient in E3 activity showed a rootless phenotype accompanied by cell death in root primordia. Thus, we concluded that EL5 functions as an anti-cell death E3 in root (Koiwai et al, 2007). We report here that treatment with higher concentration of NO₃^-, NO₂^- or Ca²⁺ specifically caused necrosis of root tip in mEL5-expressing plants. Treatment with SNP, nitric oxide (NO) donor, also enhanced cell death in mEL5-expressing callus. These results suggest that EL5 is involved in NO stress tolerance in rice. We next searched EL5-interacting proteins. Three isoforms of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC1, GapC2, and GapC3) were pulled-down with the His-tagged C-terminal region of EL5 (A195-N325) from the extract of suspension-cultured rice cells. Analysis using the bacterial two-hybrid system strongly suggested that GapC2 interacts with the P249-S276 region of EL5.

View full abstract

AAL-toxin is a pathogenicity factor of Alternaria alternate f. sp. lycopersici. Little is known about the signaling pathway of AAL-toxin leading to programmed cell death. To investigate candidate genes involved in AAL-toxin-induced cell death, we used virus-induced gene silencing (VIGS) and Nicotiana umbratica that is sensitive to the AAL-toxin. VIGS analyses indicated that AAL-toxin-induced cell death requires for a mitogen-activated protein kinase kinase, MEK2 that is involved in the ethylene synthesis, and the ethylene signaling components, EIN2 and EIN3, suggesting that ethylene biosynthesis and the downstream signaling are required for AAL-toxin-induced cell death. However, EIN2 and EIN3 were not required for HR cell death induced by INF1 elicitor or MEK2^DD. We identified an ERF gene involved in AAL-toxin induced cell death. Overexpression of the ERF did not induce the cell death, suggesting that ethylene signaling is necessary, but not sufficient for AAL-toxin-induced cell death.

View full abstract

Programmed cell death plays an essential role to the development and maintenance of bodies. Dysfunction of DAD1 (Defender against Apoptotic cell Death) has been linked to programmed cell death in animals and plants. One type of programmed cell death in animals is triggered by cytochrome c release from mitochondria via pores formed by BAX proteins. This type of programmed cell death is prevented by expression of BAX inhibitor 1 (BI-1). To study the regulation of Arabidopsis AtDAD1 and AtBI-1 in the plant tissue, we generated transgenic Arabidopsis plants expressing GUS driven by promoter fragments of AtDAD1 and AtBI-1. The promotor-GUS assay have shown that the genes of AtDAD1 and AtBI-1 express the root cap, vascular bundles of the root, abscission zones of the silique and septum of the maturity silique. These results suggest the involvement of the AtDAD1 and AtBI-1 in the developmental programmed cell death.

View full abstract

Arabidopsis LSD1 is a negative regulator of programmed cell death (PCD). We have demonstrated that LSD1 is a cytoplasmic retention protein, which inhibits the nuclear transport of nuclear proteins by interacting via its zinc finger motifs in cytoplasm. Because most of characterized LSD1-interacting proteins belong to transcriptional regulators (AtbZIP10, LIN1, IAA8), the function of LSD1 would be involved in transcriptional regulation participating in the promotion of PCD. To clarify the mechanism of physical interaction between LSD1 and these transcriptional regulators, we examined the target recognition sequence of LSD1 by phage display method. As a result, we found that "GxP" motif is a core sequence of target recognition by LSD1. Using yeast two-hybrid system, it was shown that LSD1 interacts with AtbZIP10, LIN1 and IAA8 via this "GxP" motif. The interaction between LSD1 and these proteins in plant cells has been recently investigated by GST-pull-down, co-immunoprecipitation and BiFC methods.

View full abstract

Our recent research suggested that LSD1 has a function to regulate indirectly the transcriptional control of genes involved in PCD by interacting with other proteins. To make the function of LSD1 clear more, we have focused on IAA8, which was previously identified as LSD1-interactor by yeast two-hybrid (Y2H) screening. Y2H analysis demonstrated that IAA8, one of Aux/IAA family proteins, interacts with LSD1 via same domain as with TIR1. Similarly, all Aux/IAAs with this domain we examined interact with LSD1, too. On the other hand, all LSD1-interactors should be localized in cytoplasm because LSD1 is localized exclusively in cytoplasm of plant cells. However, since only IAA8 is localized in cytoplasm of Arabidopsis mesophyll protoplasts, we suggest that LSD1 interacts only with IAA8 in vivo. By mutant analysis, IAA8 negatively regulated PCD induced in lsd1 mutant. This result strongly suggests that the function of IAA8 would participate in plant PCD inducing mechanism

View full abstract

The function of Arabidopsis bZIP transcription factor AtbZIP10 involved in programmed cell death is regulated by inhibition of its nuclear transport due to direct interaction with cytoplasmic retention protein LSD1. We also demonstrated that AtbZIP10 interacts with microtubule-plus-end-binding protein AtEB1. In this study, we tried to clarify the overview of functional control mechanism of AtbZIP10 by focusing on intracellular trafficking and selective degradation, which are important mechanism for precise transcriptional regulation. Using AtEB1 KO-mutant (ateb1a/b/c), we found that the function of AtEB1s does not participate in cytoplasmic location of AtbZIP10. In addition, because the phenotype of yeast bZIP transcription factor YAP1 mutant was partially complemented by AtbZIP10, the nucleocytoplasmic transport of AtbZIP10 may be regulated by intracellular redox state as YAP1. Furthermore, we supposed the presence of degradation mechanism for AtbZIP10 in nucleus and identified domain involved in degradation on AtbZIP10, which were examined based on the fluorescence of AtbZIP10-GFP.

View full abstract

Our previous research showed that the gene expression of Xylem Sap Lectin: XSP30 in root stele was circadian clock-controlled and the amplitude was up-regulated by the leaf-produced gibberellin in cucumber plant. In this research, GA-deficient mutant of Arabidopsis (ga3ox1/ga3ox2) was used to observe the effects of gibberellin in root area. Applying GA₄ on shoot area of the GA-deficient mutant showed obvious promotion in seminal root elongation and also root branching. In order to find out if gene expression in root area is affected by GA₄ application, the microarray analysis was performed on root samples of GA-treated or non-treated GA-deficient mutant. The results lead us to select several up and down-regulated genes which may be expressed in root and controlled by the shoot. The expressions of the selected genes were confirmed by real-time PCR. Some of them may be responsible in shoot-root interactions.

View full abstract

In the course of experiments to search for enzymes involved in phytohormone metabolism, we found a new cytochrome P450 monooxygenase (P450) that is involved in gibberellin (GA) metabolism in Arabidopsis. Using a yeast expression system, we found that this P450 enzyme catalyzed the conversion of ent-kaurenoic acid into steviol (ent-13-hydroxykaurenoic acid). When GA₁₂ was used as the substrate, 12α-hydroxy GA₁₂ was produced as a major metabolite and 13-hydroxy GA₁₂ (GA₅₃) was detected as a minor product. Transgenic Arabidopsis plants overexpressing this P450 gene showed semi-dwarfism like mild GA-deficient mutants. GA analysis indicated that the levels of non 13-hydroxy GAs including GA₄ were decreased, whereas those of 13-hydroxy GAs including GA₁ (which is less active in Arabidopsis) were increased in the transgenic plants. These results demonstrate that the ratio of GA₄ to GA₁ in plants can be modified using this gene.

View full abstract

Plant regulates its transpiration by controlling the size of the stomatal pore. In addition to such rapid responses, the number of stomata is also regulated by environmental factors. The phytohormone abscisic acid (ABA) is known to regulate stomatal closure in response to the changing water status. It is reported that ABA-induced stomatal closure is impaired in Arabidopsis ABA-insensitive mutants, abi1-1 and abi2-1. In this study, we found that the density and size of stomata were increased in the abi1-1 and abi2-1 mutants. We also found that ABA-deficient aba2-2 mutant also showed the similar phenotypes. These data indicate that ABA regulates stomatal differentiation.

View full abstract

Suppression of seed germination by environmental high temperature (thermoinhibition) during summer is crucial for Arabidopsis (Arabidopsis thaliana (L.) Heynh.) to establish vegetative growth in autumn. We have reported that high temperature enhances abscisic acid (ABA) level by up-regulation of ABA biosynthesis genes, and suppresses gibberellin (GA) action by down-regulation of its biosynthesis genes in conjunction with up-regulation of a GA negative regulator gene, SPINDLY (SPY). To observe the hormone interaction in thermoinhibition, we used ABA deficient mutant, aba2, and found that the expression of GA 3-oxydase genes are not suppressed and the expression of SPY gene is down-regulated even at high temperature. In addition, thermoinhibition resistant germination phenotype of aba2 seeds is suppressed by GA boisynthesis inhibitor, paclobutrazole. These results suggest that suppression of GA synthesis and signaling in thermoinhibited Arabidopsis seeds is mediated by ABA.

View full abstract

We have been utilizing LC-ESI-MS/MS for high-sensitive plant hormone analysis. Our goal is to establish a method for comprehensive analysis of all plant hormones and their precursors and metabolites. As reported in this meeting last year, we have established a method for bioactive hormones (GA₁, GA₄, ABA, IAA, JA, SA, cytokinins, brassinosteroids). To better understand how hormone levels are regulated in plants, the hormone precursors and metabolites should also be analyzed. We have previously reported the analysis methods for 14 GA related compounds and 7 ABA related compounds. In this presentation, we will report the analysis method for JA related compounds. Although JA plays important roles in various stress responses, the biosynthesis and metabolism pathways have not been fully clarified yet. The analytical method for OPDA, OPC:8, JA-leucine, JA-isoleucine will be discussed.

View full abstract

Jasmonic acid (JA), a plant hormone, has roles in regulating stress response and development. We identified transcription factors induced by jasmonates (JAs) and wounding using microarray analysis. In this study, we aimed to reveal the JA signaling by understanding physiological function of JA-responsive transcription factors.
One of the JA-response transcription factor INU1 is induced strongly by JAs and wounding at early time after treatment and this suggests INU1 has important roles in JA signaling. In addition, expression of INU1 depends on JA because INU1 expression was markedly reduced in JA biosynthesis mutant aos. We performed microarray analysis for methyl jasmonate dependent gene expression in inu1 mutant and found that genes related to flavonoid biosynthesis are up-regulated in inu1. By metabolome analysis, flavonoid contents were found to be changed in inu1. These results indicate that INU1 negatively regulates flavonoid biosysthesis.

View full abstract

It is well known that low nitrogen supply causes stimulation of root elongation in rice. Chromosome segment substitution lines (CSSLs) developed between Koshihikari and Kasalath were employed for mapping QTLs controlling root length under various NH₄⁺ concentrations. One of QTLs stimulated the root length by Kasalath allele was detected on long arm of chromosome 8 under 5 μM NH₄⁺. When Koshihikari and SL-225, a CSSL substituted Kasalath segment on this QTL region, were hydroponically grown with 5-1000 μM NH₄⁺, the root length of SL-225 was significantly stimulated under all concentration tested. Content of absorbed nitrogen in the SL-225 plants was significantly decreased under 50 μM NH₄⁺. To isolate a target gene from the QTL region, BC1F2 and F3 progenies derived from SL-225 were tested for the stimulation of root elongation grown with 5 μM NH₄⁺. As a result, target QTL was delimited at the 7 cM region.

View full abstract

Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1), i.e. OsGS1;1, OsGS1;2 and OsGS1;3. Both OsGS1;1 mRNA and OsGS1;2 mRNA were detected in roots, leaf sheaths, leaf blades, and spikelets. However, the OsGS1;3 mRNA was only detectable in spikelets. OsGS1;1 knockout mutants caused by insertion of endogenous retrotransposon Tos17 showed severe retardation in growth rate under NH₄⁺-containing hydroponic solution. Tos17-mediated mutants for OsGS1;2 and OsGS1;3 were recently identified. Their characteristics will be discussed.

View full abstract

In rice plants, glutamine (Gln) regulates expression of several key genes in nitrogen metabolism including NADH-glutamate synthetase 1 (NADH-GOGAT1) gene. However, little is known about molecular mechanisms for Gln-sensing and signaling systems in plants. To understand the Gln-sensing and signaling mechanisms, we have focused on rice ACT domain repeat proteins (OsACRs; encoded by OsACR1 to OsACR9) as a candidate for the Gln-sensor. We reported that cellular distribution of OsACR9 protein overlapped with NADH-GOGAT1 in various organs of rice. In the unexpanded leaf blades of tillers of RNAi-mediated OsACR9-knockdown rice transformants at T0 generation, there was a decrease in NADH-GOGAT1 mRNA content compared with that in the control. Involvement of OsACR9 in gene regulation will be discussed based on transcriptomic profile from the T1 plants.

View full abstract

Four distinct isoenzymes of glutamine synthetase (GS1) are known to share their functions in maintaining ammonium assimilation in Arabidopsis root. Among them, GLN1;2 showed unique characteristics favorable for an ammonium-rich environment in root: (i) transcripts of GLN1;2 were highly accumulated by ammonium supplementation; (ii) GLN1;2 showed low substrate affinity for ammonium (Km = 2.5 mM); (iii) GLN1;2 promoter activity was localized in root epidermis and root hairs in response to ammonium supplementation, thus, it is likely that GLN1;2 is one of the most essential components in primary ammonium assimilation in roots. We further investigated the GLN1;2 promoter region responsive to ammonium supplementation using GFP as a reporter. The ammonium-responsive region was narrowed down to a 21-bp sequence which was located 3.6-kb upstream of GLN1;2 coding region and was essentially required for driving the expression of GFP in root epidermis and root hairs under ammonium-rich conditions.

View full abstract

Some plants grew better in spite of low inorganic nitrogen concentration under the condition of organic matter application as nitrogen sources, compared with the chemical fertilizer application. This suggested the direct uptake of organic nitrogen by such a plants. PEON is considered to be the most promising nitrogen sources of organic nitrogen in soil. In this study, we tried to show the evidence of direct uptake of PEON using the immunohistochemical method.
We collected PEON from a soil and purified by dialysis. As nitrogen source, PEON was applied to sand medium on which chingensai, lettuce and syungiku were grown for 14 days. The growth of three differed seedlings applied with PEON were much better than those applied with NH4NO3. Roots of lettuce grown on sand-medium applied with PEON were subjected to immunohistochemical staining with anti-PEON antibody and Hrp-DAB system . Then, positive reactions with PEON in lettuce roots were obtained

View full abstract

Chloroplast NAD kinase enhances carbon and nitrogen metabolism
Chloroplast NAD kinase (NADK2) has been identified as an essential enzyme for chlorophyll synthesis, photosynthetic electron transport, and xanthophyll cycle. Metabolite analysis revealed that the contents of intermediates of the Calvin cycle were increased in transgenic plants overexpressing NADK2 gene (NADK2-OX). The activities of RubisCO and glyceraldehyde-3-phosphate dehydrogenase as well as CO₂ uptake were also increased, indicating that carbon fixation was enhanced in NADK2-OX. On the other hand, the accumulation of free amino acids, especially glutamine and glutamate were observed in NADK2-OX. Furthermore, the expression levels of chloroplast glutamine synthetase and NADH-dependent glutamate synthase genes were increased. The uptake and reduction process of nitrate were also increased. It therefore seemed that the activation of GS/GOGAT pathway and nitrate reduction contributed to amino acid overproduction. These results indicate that NADK2 overexpression enhances both carbon and nitrate metabolism.

View full abstract

We previously reported the enhancement of nitrogen assimilation in the transgenic Arabidopsis plants expressing the Dof1 transcriptional activator for expression of the genes encoding enzymes involved in carbon skeleton production. The major part of assimilated nitrogen is utilized for the biosynthesis of components involved in photosynthesis. Here, we show the effects of strengthened nitrogen assimilation on carbon metabolism under the high light condition (HL) in the transgenic plants. The amounts of carbon metabolites were enlarged in the transgenic plants under HL. The chlorophyll contents and carbon fixation rates were also increased in the transgenic plants under HL. Furthermore, the contents of protein and starch were increased in the transgenic plants under HL. These results indicate that genetic co-activation of nitrogen and carbon assimilations in the Dof1 transgenic Arabidopsis leads to the increase in substance production under HL.

View full abstract

Purine metabolism plays a central role in plant nitrogen metabolism. Unless salvaged to purine nucleotides, purines and their partial degradation products are converged to xanthine, the key starting compound of the catabolism which is consequently decomposed to inorganic nitrogen and carbon. To evaluate the physiological significance of purine catabolism, we constructed RNAi-silenced Arabidopsis in which the rate-limiting xanthine dehydrogenase (XDH) was downregulated. The RNAi plants displayed pleiotropic phenotype including retarded growth and overaccumulated xanthine. Excess xanthine didn't affect the growth of wildtype plants and the impaired growth of RNAi plants was reversed by a xanthine catabolite urate, suggesting that a block in purine catabolism possibly causes the growth defect of RNAi plants. Wildtype plants were able to use xanthine as sole nitrogen source whereas the growth of RNAi plants was severely impaired. Collectively, these data suggest the importance of purine catabolism and its catabolites in plant growth and physiology.

View full abstract

In the moss Physcomitrella patens, we previously identified eight nitrate transporter genes (PpNRT2;1-PpNRT2;8), the respective function of which has remained to be determined. In this study, we disrupted PpNRT2;1, PpNRT2;3 and PpNRT2;5, each of which has distinct structural characteristics and gene expression profile, by target mutagenesis to yield the mutants ΔNRT2;1, ΔNRT2;3 or ΔNRT2;5, respectively. These mutants grew normally under nitrate-replete conditions. Unlike ΔNRT2;1 and ΔNRT2;5, however, ΔNRT2;3 showed higher nitrate uptake activity than WT in the low substrate concentration range (0-100 μM). The expression levels of the other NRT2 genes, particularly PpNRT2;1 and PpNRT2;2, were higher in ΔNRT2;3 than in WT. These data suggest that PpNRT2;3 has relatively low affinity for nitrate. PpNRT2;1 and PpNRT2;2 are presumably high affinity nitrate transporters. In WT, high-level expression of PpNRT2;3 seems to indirectly control other NRT2 genes through the glutamine-mediated feedback repression mechanism.

View full abstract