The environmental problems caused by CO₂ emission are getting grave and the efficient application of plant oil is closely watched for one of the solution of those problems.
We screened genes that could increase the seed oil content by introducing chimera repressor transcription factor (TF)-fused genes based on CRES-T method into Arabidopsis thaliana. We had estimated that CRES-T method, known to be effective as creating dominant factors suppressing the expression of specific target genes with resultant dominant loss-of-function phenotypes, could improve the phenotype of oil content that is a quantitative trait.
We nondestructively measured the oil contents of recombinant T2 seeds into which TF-fused genes with 35S promoter were introduced by ¹H pulse NMR. For the ¹H pulse NMR, at least 3 mg seed was sufficient for quantification. As a result, we could get seeds containing more oil than that of the host plant.

View full abstract

It is generally considered that plant seed oil is a potent resource for bio-diesel fuel and bio-plastics. However, there have been a few examples of molecular breeding that successfully increase the oil content of plant seeds, mainly because the mechanism governing oil storage in plant seeds have been poorly understood.
In this study, to find out genes that could regulate seed oil storage, we prepared 200 of the chimeric gene constructs for transcription factor to which the plant specific repression domain was fused, and were introduced into Arabidopsis thaliana individually. The contents of T2 seeds oil were nondestructively measured in 10 stocks for each gene transformant by ¹H-pulse NMR. Some strains were obtained whose oil contents were increased about 20 % in comparison with those of wild type. In these strains, AP2/ERF and MYB family were found to be included.

View full abstract

Phosphatidate phosphatase (PAP) is the enzyme which produces diacylglycerol (DAG) by dephosphorylating phosphatidic acid (PA). PAPs play an important role in glycerolipid metabolism since DAG is a common precursor of biosynthesis for glycolipid,phospholipid and neutral lipid (triacylglycerol).
We recently reported lipid phosphate phosphatases (LPP) which are localized to plastids in Arabidopsis (Nakamura et al. (2007) J Biol Chem, 282: 29013-29021). Here, we identified a novel type of PAP (PAH1, PAH2). Analysis for their mutant lines showed that pah1 and pah2 single mutants had no change in lipid metabolism, whereas in a pah1pah2 double mutant accumlation of PA, alteration of the lipid composition and morphological change in leaves were observed. Role of PAPs in lipid metabolism under Pi-sufficient and Pi-depleted conditions will be discussed in this presentation.

View full abstract

The sudden loss of turgor pressure from motor cells of pulvini is thought to cause the rapid seismonastic movement of Mimosa pudica. The change in turgor pressure occurs along with an efflux of ions and water from symplast into apoplast. In the present study, we measured the osmotic water permeability (Pf) of motor cell protoplast. An obvious decrease in Pf values was observed by adding mercury chloride, showing that aquaporin contributes to the water transport through the membrane of motor cell. We have reported that the water transport activity of aquaporin, which is heterologously expressed in Xenopus oocytes, is regulated by PKA-mediated phosphorylation. In the presence of PKA activators, Pf value of protoplast was increased, indicating that PKA-mediated phosphorylation play a key role in the water transport regulation. In this report, we will show how this PKA-mediated phosphorylation regulates the water permeability of motor cell protoplast.

View full abstract

Petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from reddish-purple to blue during flower-opening period. The color change is caused by an unusual vacuolar pH increase from 6.6 to 7.7 in the epidermal colored cells. We have already clarified the involvement of ItNHX1 in the pHv increase. To clarify the mechanism and function of ItNHX1 in morning glory petals we studied cellular differences between the red and blue parts in chimera petals in which red parts appeared in the blue colored petal. Anthocyanin components in both blue and red parts were the same HBA. The pHv of red cells measured by a pH-sensitive microelectrode was 6.9 being significantly different from that of blue cells. K⁺ content in red cells was lower than that in blue cells. The function of ItNHX1 in petal color change and cell enlargement will be discussed.

View full abstract

Plasma membrane aquaporin, PIP2 subfamily is considered to be regulated by protein phosphorylation. Putative phosphorylation residues, Ser115 and Ser280, in pear PcPIP2;2 were mutated to Ala (this mimics non-phosphorylated state) or Asp (this mimics phosphorylated state) and water transport activity of these mutants were measured in Xenopus oocytes. Water transport of PcPIP2;2s having only one mutation from Ser to Ala was almost the same as that of WT PcPIP2;2. By contrast, PcPIP2;2 mutant, whose Ser115 and Ser280 were mutated to Ala, showed lower water transport activity. There is no report telling Ser115 phophorylatinon in planta, although Ser280 phophorylatinon have been reported. This means that Ser115 is not phophorylated in planta or it^,s phophorylation level is quite low, suggesting the importance of Ser280 phosphorylated state. Therefore we prepared phosphor-specific antibody against Ser280. Western blot showed that phosphorylated state of PcPIP2;2 accorded with diurnal growth of pear fruits.

View full abstract

Inorganic ions absorbed from soil move through the xylem vessel to leaves and are taken up by leaf cells. Both apoplastic and symplastic pathways mediate its route, but the detail is not clear. In the present study, we analyzed the mechanism of Pi transport in the leaves. First, we established the isolation method of living mesophyll cells and vascular tissues from the rosette leaves of Arabidopsis thaliana. Then, we measured the Pi uptake activity of the isolated mesophyll cells. The mesophyll cells isolated only from the leaves developed in Pi-deficient condition have Pi uptake activity. The expression patterns of genes encoding Pi transporters in the mesophyll cells and the vascular tissues are analyzed by real-time PCR. Some transporter genes are highly expressed in the vascular tissues. In the mesophyll cells from the Pi-deficient plants, their expression levels increased.

View full abstract

Vacuole is the largest organelle in plant cells, and contributes to increase in cell volume and intercellular homeostasis. Many kinds of substances are stored in the vacuole and working for cellular functions. Since various membrane proteins must be involving in vacuolar membrane transports, we carried out proteomic analysis of the vacuolar membrane proteins, and found many uncharacterized proteins. Although, V-type H⁺-ATPase and H⁺-PPase, which energize the vacuolar membrane, have been well-studied at molecular level, their functional statuses in situ are not fully analyzed, yet.
In this study, we analyzed the subcellular localization of those vacuolar membrane proteins using immunofluorescent staining. The V-type H⁺-ATPase and H⁺-PPase showed dot-like localization in the cytoplasm. In order to investigate their distribution on the vacuolar membrane, we isolated intact vacuoles from suspension culture cells or mesophyll cells. We discuss the distribution of transporters on these isolated vacuoles, too.

View full abstract

Boron (B) is essential in higher plants and B deficiency hampers crop yields in over 80 countries. Transgenic Arabidopsis overexprssing BOR1, a borate exporter for xylem loading, exhibited improved fertility under B limitation (Miwa et al, 2006). In this study, we aimed to further improve B-deficiency tolerance through overexpression of NIP5;1, a boric acid channel for B uptake in roots.
An NIP5;1 activation tag line showed improved root elongation under B limitation. We introduced a construct that mimics the NIP5;1 locus of this activation line into transgenic Arabidopsis overexpressing BOR1. Several independent transgenic lines showed improved root elongation under B deficiency and one of them exhibited elevated short- and long-term B uptake and improved fertility under B limitation. This study demonstrated that it is possible to generate nutrient-deficiency tolerant plants through overexpression of a channel for an essential mineral nutrient.

View full abstract

ugar transporters play indispensable roles in sugar transport within and between cells and also in long distance carbon distribution. Sugar transporters of plants are classified in eight families and two homologues of SFP family (SlSFP1 and SlSFP2) were found in a tomato EST library generated by KAZUSA DNA Res. Inst. SlSFP1 and SlSFP2 encode proteins of 486 and 490 amino acids, respectively, and both have 12 putative trans-membrane domains and typical sugar transporter motifs. Real-time PCR analysis revealed that SlSFP1 expression is especially high in early and late stages of tomato fruit development, while SlSFP2 expression is almost constant during fruit development and in other organs. Both SlSFP1 and SlSFP2 were induced by NaCl, cold stress and dehydration. GFP fusion proteins of SlSFP1 and SlSFP2 localized in vacuolar membrane of Arabidopsis cells, suggesting their contribution to sugar accumulation in tomato fruits.

View full abstract

Plants are exposed to many kinds of environmental stresses. To overcome these stresses, plants respond with biochemical and physiological changes, for example, expressing genes involved in metabolism and transport. Especially, sugars accumulating during environmental stresses are thought to play important roles in stress tolerance. In this study, we analyzed a stress-inducible monosaccharide transporter gene ERD6A and its homolog ERD6B. ERD6 was originally isolated from a cDNA library from Arabidopsis plants that were exposed to dehydration stress for one hour. At first, we analyzed expression of ERD6A and ERD6B genes in response to dehydration, cold and high salinity stresses by RNA gel blot analysis. The expression levels of these genes were increased by those stresses. We also investigated tissue-specific expression patterns, subcellular localization and phenotypes of ERD6A and ERD6B knock out mutants.

View full abstract

Plants respond to abiotic stresses by altering the transport activity of various metabolites and ions. Microarray analyses identified various osmotic-stress inducible genes for membrane-proteins including a potassium transporter, KUP6, in Arabidopsis. Expression of KUP6 was induced by mannitol and drought treatments in plants. The GUS activity was detected in root tips and vascular cells, and increased in young seedling roots by mannitol and salt treatments of the transgenic plants expressing the KUP6 promoter-GUS gene. The KUP6-GFP protein was localized to the plasma membrane in root tips of the transgenic Arabidopsis. A T-DNA insertion mutant of KUP6 showed enlarged leaf sizes under normal growth condition. The root growth of kup6 showed decreased ABA sensitivity and increased auxin sensitivity compared with that of wild-type plants. Furthermore, drought stress tolerance of kup6 was decreased significantly. These results suggest that KUP6 plays an important role in drought stress tolerance in Arabidopsis.

View full abstract

To investigate the mechanism of mechanosensing in plants, we have isolated genes of Ca²⁺-permeable mechanosensitive channel candidates from Arabidopsis thaliana, MCA1 and MCA2 (Nakagawa et al. , PNAS 104:3639-3644, 2007). Mca1 and Mca2 have 73 % identity in amino acid sequence and can complement the lethality of the yeast mid1 mutant, although sequence similarity between Mca1/Mca2 and Mid1 is low. Mca1 and Mca2 possess a regulatory domain of rice putative protein kinases, an EF-hand-like motif, and a coiled-coil motif in the N-half and transmembrane segments and a Cys-rich domain in the C-half. We have found that Mca1 is involved in Ca²⁺ influx and touch sensing in roots. Here, we performed molecular characterization of Mca1/Mca2 using yeast. We found that Mca1/Mca2 could form a dimer and a tetramer and that the C-half of Mca1 expressed in yeast did not retain Ca²⁺ uptake activity and the N-half was lethal to yeast.

View full abstract

We have identified a gene encoding an iron-phytosiderophore transporter (HvYS1) in barley. HvYS1 is a specific transporter for Fe(III)-phytosiderophores, and involved in primary iron acquisition from soils in barley roots. In contrast, ZmYS1 in maize possesses broad substrate specificity, despite a high homology with HvYS1. The distinct difference in the substrate specificities between ZmYS1 and HvYS1 motivated us to investigate the mechanism by which the transporters distinguish their substrates. In this study we revealed, by assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, that the outer membrane loop between the 6th and 7th transmembrane regions is essential for the substrate specificity. Circular dichroism spectra indicated that a synthetic peptide corresponding to the loop of HvYS1 forms an α-helix in solution, whereas that of ZmYS1 is flexible. We propose that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter.

View full abstract

ALMT1 confers aluminum (Al)-tolerance by secretion of malate from root apices, which excludes Al ions. In Japanese varieties, Al tolerance was correlated with malate efflux, but not with the ALMT1 transcription level, suggesting a possible post-transcriptional regulation for functional expression of ALMT1 (Sasaki et al., 2006). To reveal the possibility, we compared a pair of Japanese lines which were derived from the same cultivar but differed in Al tolerance. The Al-tolerant line showed less accumulation of Al and higher malate efflux. However, both lines showed the same levels of ALMT1 transcript and ALMT1 protein. We obtained F₂-progenies by the cross of these lines, and Al sensitivity of them was segregated to the 1 (sensitive) : 3 (tolerant) ratio. These results suggest that at least one gene is involved in the post-translational modification of ALMT1 which is necessary for its functional expression.

View full abstract

The ionic transport systems have important roles on the ionic homeostasis in the cells under the salt stress. We hardly know about intracellular behavior of chloride ions, a counter ion for sodium ions, under the high salt condition. It was reported that AtCLC-a and -c play a role in controlling the intracellular nitrate status in Arabidopsis. However, other functions were not known. We investigated about the two chloride channel genes, OsCLC-1 and OsCLC-2in rice. To assess the function of rice chloride channel genes OsCLC-1 and -2, we isolated deletion mutants from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. clc-1 and clc-2 had slightly reduces root length compared with wild type. Therefore, we investigate the morphology of roots from clc-1, clc-2, and the double mutant of them in detail.

View full abstract

Selenite (SeO₃^2-) was uptaken by the coccolithophorid, Emiliania huxleyi, as a source of selenium (Se), an analogous element of sulfur, to synthesize essential selenoproteins. In this research, we characterized kinetics of the selenite uptake systems under various conditions.
When the cells were grown under selenite-depleted conditions, Km values for the high-affinity transport system were decreased less than one-sixth in 3 days, whilst values of Vmax were almost constant. When 10 nM selenite was supplemented into the selenite deficient cells, the uptake activity was decreased in half in 24 h. These results suggested that high-affinity selenite transporter might be induced under the selenium deficient conditions.
Although sulfate did not affect the selenite uptake activity, the Km values of selenite uptake were increased to 3-fold of the control activity by the presence of sulfite or selenate. These results indicated that sulfite and selenate competitively inhibited the active transporter of selenite.

View full abstract

The quantitative trait loci (QTL) for the control of amino acid contents in rice (Oryza sative cv) grain were analyzed in order to investigate the genetic factor responsible for regulating primary metabolite levels in albumen tissues. Following the extraction of metabolites from the albumen of 85 back-crossed inbred lines (BILs) of rice derived from Sasanishi (japonica) and Habataki (indica), the concentration of 18 amino acids were determined by using LC-ESI-MS. The QTL analysis by the interval mapping method identified 25 QTLs for controlling the amino acid contents.

View full abstract

Gamma-Glutamyl Hydorolase (GGH) cleaves the polyglutamate chain of folates. We have previously reported that GGH1 is expressed in procambial cells or meristematic tissues of mature plants, and that over-expression of any of three GGH genes or suppression of all GGH genes caused developmental defects. To assess whether GGH-mediated regulation of glutamate chain lengths of folates is involved in regulating meristematic activity, we analyzed expression pattern of GGH1 as well as effect of folate supplementation during callus induction and shoot regeneration from hypocotyl explants. The expression of GGH1 was suppressed in callus on callus inducing medium, while up-regulated in shoot inducing medium. Furthermore, pteroyl monoglutamate, but not pteroyl pentaglutamate, had shoot inducing activity. These results suggest significance of regulating glutamate chain lengths of folates in shoot development and involvement of GGHs in the process.

View full abstract

In higher plants, the shoot apical meristem generates most of aerial parts. In Arabidopsis, the NAC transcription factors CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 play important roles in the formation of the shoot apical meristem and shoot organ boundaries. We have previously selected 24 candidate downstream genes of CUC by a microarray-based screening. Here we further characterized these candidates by using transgenic plants expressing a fusion protein of CUC1 and the glucocorticoid receptor (CUC1-GR). Three hours of dexamethasone (DEX) treatment, which activated CUC1 function, resulted in upregulation of three genes among the 24 candidates. Furthermore, simultaneous application of DEX and the protein synthesis inhibitor cycloheximide resulted in the upregulation of ten candidates including the above three genes, suggesting that these ten genes were direct targets of transcriptional regulation by CUC1. Now we are trying to identify target regulatory sequences of CUC1 in candidate gene promoters by using transient assays.

View full abstract

The above ground part of a plant is composed of a unit called phytomer, which is derived from a population of indeterminate cells located at the shoot apical meristem (SAM). The balance between indeterminate and determinate cells and the differentiation of cells, which undergo organogenesis are regulated at SAM so that the entire plant structure is organized. We have analyzed the course of the first step of lateral organ formation at SAM in rice. It is known that the indeterminate cells at SAM express OSH1 gene, a member of KNOTTED type homeobox gene family in rice, whereas it is down-regulated at the determinate cells which will differentiate into lateral organs. Using anti-OSH1 antibody, we conducted immunohistochemical analysis to detect the domain where determinate cells exist in SAM during the course of a plastochron. In this presentation, we will discuss the mechanism of differentiation of determinate cells visualized by OSH1 down-regulation.

View full abstract

Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1/abc2 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1/abc2 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. CYO1/ABC2 protein localizes to the thylakoid membrane in chloroplasts, and CYO1/ABC2 protein copurified with the PSI/LHCI and PSII/LHCII complexes. CYO1/ABC2 has a C4-type zinc finger domain similar to that of Escherichia coli DnaJ. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1/ABC2 has protein disulfide isomerase activity. These results suggest that CYO1/ABC2 has a chaperone-like activity required for thylakoid biogenesis in cotyledons.

View full abstract

The shoot apical meristem is characterized by the expression of KNOX1 genes. KNOX1 genes have been implicated in the acquisition and/or maintenance of meristematic fate. One of the earliest indicators of a switch from indeterminate meristem to determinate leaf primordium is the down-regulation of KNOX1 genes orthologous to SHOOT MERISTEMLESS (STM) in the initiating primordia. In simple-leafed plants, this down-regulation persists during leaf formation. In compound-leafed plants, however, the expression is later re-established in the developing primordia, creating an indeterminate environment for leaflet formation.
Here we identify two conserved sequences within the 5' upstream region of STM genes in both simple- and compound-leafed species across monocots and dicots. We show that one of them is involved in the regulation of the persistent repression and/or the re-establishment of STM expression in the developing leaves but not the initial down-regulation. We also show that this regulation is significant for leaf develpment.

View full abstract

Postembryonic development in plants depends on the activity of the shoot (SAM) and root (RAM) apical meristems. It is well known that the size of SAM is regulated by CLAVATA (CLV) signaling pathway in Arabidopsis, in which CLAVATA3 (CLV3) acts as a signaling molecule to negatively restrict the stem cell population in both the vegetative and reproductive SAM. Here we show that two proteins related to CLV3, FLORAL ORGAN NUMBER2 (FON2) and FON2-LIKE CLE PROTEIN1 (FCP1), have functionally diversified in rice: FCP1 is critical for the regulation of SAM and RAM in the vegetative phase, whereas FON2 is responsible for regulating the aerial meristems in the reproductive phase. FON1, a putative receptor of FON2, may not necessarily be required for FCP1 action. In addition, we identify the critical amino acid that differentiates the action of FCP1 and FON2.

View full abstract

Postembryonic development in plants is dependent on the activity of shoot and root meristems. CLAVATA3 (CLV3), a putative peptide ligand of Arabidopsis thaliana, regulates the stem cell population in the shoot apical meristem (SAM). Previously, we have reported that functional CLV3 encodes dodecapeptides with two hydroxyproline residues, and chemically synthesized CLV3 peptide also functions in in vitro bioassay systems, resulting in reduced SAM size (Kondo et al.2006). In order to confirm that the synthetic peptide acts through endogenous CLV pathway, we analyzed SAM defective phenotype of clv mutants in the presence of CLV3 peptide. As a result, clv1 and clv2 plants, but not clv3 plants, showed resistance to SAM defect caused by CLV3 peptide. This suggests that synthetic CLV3 peptide acts through endogenous CLV1/CLV2 receptor system.

View full abstract

Legumes possess systemic feedback mechanism to control the nodulation. HAR1, which was identified from Lotus japonicus as a shoot factor of this mechanism, encodes a receptor-like kinase (RLK). Among all Arabidopsis RLKs, HAR1 showed the highest similarity with CLAVATA1 (CLV1). CLV1 is estimated to recognize CLV3 peptide. This raises the possibility that HAR1 interact with CLV3-like (CLE) peptide. So, we identified 32 putative LjCLE sequences from Lotus genome. Expression analysis showed three LjCLE genes were significantly upregulated in the root by rhizobial infection. Among them, over-expressed LjCLE1 and LjCLE2 in hairy root system inhibited nodulation strongly. This suppression was cancelled in har1 mutant. Further analysis showed that LjCLE1 and LjCLE2 are upregulated immediately after rhizobium inoculation. Furthermore, Nod Factor (NF) and some components of NF signaling pathway are required for the induction of LjCLE1 and LjCLE2. These results suggest that LjCLE1 and LjCLE2 function in systemic regulation of nodulation.

View full abstract

Rautenberg and Kuhn observed in 1864 that nitrogen sources inhibit nodulation. Since then, it has been shown that leguminous hypernodulating mutants (har1 etc.) exhibit a nitrate-tolerant phenotype. However little is known about the molecular mechanism.
In L. japonicus HAR1 is involved in systemic regulation of nodulation. Okamoto in our laboratory found that LjCLE1 and LjCLE2 are significantly up-regulated in response to Mesorhizobium loti and have the strong ability to suppress nodulation via HAR1. So we noticed that har1 mutant exhibits a nitrate-tolerant phenotype in nodulation and searched for LjCLE induced by nitrate treatment.
Expression analysis revealed that LjCLE2 among 32 LjCLE genes was specifically and significantly up-regulated by 10 mM nitrate. The increase of transcript level induced by rhizobial infection was cancelled by pretreatment of 10 mM nitrate. Based on these results, we propose a model in which LjCLE2 induced by nitrate suppresses nodulation via HAR1-mediated signaling.

View full abstract

Asparagus spp. have a unique organ, phylloclade, which is thought to be homologous to a stem, the morphology of phylloclade is leaf-like and true leaves are reduced. In Asparagus, it is known that phylloclade has ability to photosynthesize, so that phylloclade seems to be an intermediate organ between stem and leaf, but nothing has been studied about the molecular mechanisms. In seed plants, genetic regulatory systems that distinguish stem and leaf identities remain unclear. From this point of view, phylloclade in Asparagus is an interesting model to study shoot/leaf differentiation.
We are studying developmental mechanisms of phylloclade using Asparagus asparagoides (L.) W. Wight. In addition, we are studying expression patterns of molecular marker genes involved in leaf and shoot development in phylloclade. In this report, we will show the identity of phylloclade in terms of the marker-gene expressions, and discuss the developmental mechanisms of phylloclade.

View full abstract

Unifacial leaf, in which leaf surface consists only of the abaxial side, has been evolved in a number of divergent species in monocots. The unifacial leaves provide very unique opportunities for the developmental studies of the leaf axes formation in monocots. In addition, the mechanism of the parallel evolution of such drastic changes in leaf polarities is of interest from an evolutionary viewpoint.
We are studying molecular genetic mechanisms of the unifacial leaf development and their evolution, mainly using Juncus as a model system, which is very suitable for molecular genetic studies. In addition, Juncus are known for a wide variety of leaf forms. We carried out developmental and gene expression analyses in Juncus, and combined their results with phylogeny of Juncus species. As a result, we revealed the evolutionary patterns of unifacial leaves in Juncus, and illustrated key regulatory changes of gene expressions for the unifacial leaf development.

View full abstract

Developmental program that stops cell proliferation plays an important role in the determination of leaf size. To investigate this regulation genetically, we have isolated three lines of grandifolia-D mutants of Arabidopsis thaliana that produce larger leaves than wild-type plants do by extending the duration of cell proliferation. Unexpectedly, our genetic mapping revealed that each gra-D loci has genetic linkages with two different chromosomal regions: depending on the lines used, the gra-D loci are mapped on the top part of either chromosome 2 or 4, and all three lines shows a second linkage at the lower part of chromosome 4. Curiously, this region contains the AINTEGUMENTA (ANT) gene that is known to positively control the duration of cell proliferation in leaf primordia. RT-PCR analysis revealed that ANT is overepressed in the gra-D mutants backgrounds. We are now characterizing the relationship between ANT overexpression and gra-D phenotype in more details.

View full abstract

We are interested in the underlying mechanisms how gain-of-function mutant rotundifolia4-1D has shorter leaves. In order to get some insights about it, we are studying ROT4 molecular function. ROT4 encodes small protein of 6.2kDa. Previously, we roughly showed that functional domain resides in conserved C terminal region of the molecule, but the precise region of it has not been characterized. Furthermore, whether this domain is processed out of the other part of the molecule has remained unknown. In the present study, we first identified the domain more precisely by overexpressing deletion series of the coding region. Secondly, we constructed two types of fusion protein (ROT4:GFP and GFP:ROT4) and analyzed the phenotype of the overexpressors. Subsequently, we detected the protein by immunoblotting to clarify if processing event occurs physiologically and if GFP-fusion protein is functional. Based on these and other experiments, we will discuss molecular function of ROT4.

View full abstract

We isolated four arabidopsis mutants that showed increased cell number and decreased cell size in leaves. Three of them showed accelerated heteroblasty. In Arabidopsis thaliana, adult leaves have more and smaller cells than juvenile leaves. Therefore, leaves of these three mutants might have characteristics of adult leaves of wild type (Usami et al., 2007 Annual meeting of JSPP).
In the present study, we focused on the role of miR156 mediated regulation of SPL transcription factors in heteroblastic changes of cell number and size. One of the three accelerated heteroblasty mutants we isolated had a mutation in miR156 target site of SPL15 gene and showed elevated SPL15 mRNA level. And in miR156 constitutively expressing plants, number and size of leaf cells did not differ significantly between higher and lower nodes. These results indicate that SPL transcription factors might be involved in cell number and size regulation in leaves.

View full abstract

Leaf develops along proximal-distal, medial-lateral and adaxial-abaxial axes. Although leaf is composed of leaf blade and petiole, the organogenesis along proximal-distal axis have received less attention. To understand mechanisms of junction formation between leaf blade and petiole, we carried out developmental analysis using wild-type Arabidopsis thaliana as well as a CYCB1;2:GUS transgenic line, which visualizes mitotic cells. As a result, it was found that leaf blade/petiole junction began to be visible at a relatively early stage of development, where mitotic cells distributed uniformly. Notably, although mitotic cells were known to distribute basipetally in developing leaf blade, this trend was reversed in the developing petiole. Similar analyses are underway using 35S:LEP and bop1bop2 that exhibit abnormal organogenesis along proximal-distal axis. Furthermore, to identify genes involved in the junction formation, we searched for enhancer trap lines specific to leaf blade/petiole junction.

View full abstract

ANGUSTIFOLIA (AN) is a plant homologue of animal CtBP (transcription corepressor)/ BARS (a protein involved in Golgi maintenance) and thought to control polar cell expansion in the leaf-width direction. Although AN has been expected to function as CtBP, the molecular function of AN is not yet elucidated. In this study, swapping experiment was carried out between the AN gene and Drosophila melanogaster dCtBP. As a result, it appeared that AN did not function as CtBP in drosophila. In addition, analysis of intracellular-localization-site-directed mutagenesis of AN suggested that the function of AN differs from that of CtBP. The observation of the intracellular localization revealed that AN localizes in novel dot-like structures closely associated with the Golgi apparatus. This localization is also unexpected from the hypothesis that AN might be a plant BARS. Further analysis is in place to uncover the relationship between such particular localization and the polar cell expansion.

View full abstract

Tobacco EIN3 like (TEIL) is a homolog of the Arabidopsis EIN3, which encodes a transcriptional factor in ethylene signaling. TEIL has been reported to binds the promoter sequence of acidic tobacco PR1 gene (Kosugi and Ohashi, 2000), and was presumed to regulate basic PR gene expression and flower formation (Hibi et. al, 2007). We have indicated that ethylene emission in wounded leaves was altered in TEIL-suppressed tobacco plants. Here, we report that altered expression of NtERFs in TEIL-suppressed plants especially the decrease of ACC-induced ERF2 expression. In the study to analyze the function of TEIL on disease resistance, a time-course observation showed the tendency that the resistance to a fungal pathogen R. solani was increased in TEIL-overexpressors and decreased in TEIL-suppressed tobacco plants. These results indicate that TEIL contribute on ethylene signaling via expression of NtERF genes and on disease resistance to a kind of pathogens.

View full abstract

A plant hormone ethylene inhibits Agrobacterium-mediated gene transfer, but the inhibitory effect has not been clear. The gene transfer mechanism includes the bacterial growth, induction of virulence gene expression in the bacteria, transfer of T-DNA into the plant cells, and finally integration of the T-DNA into the host genome. Since ethylene inhibits T-DNA transfer, plant ethylene response would affect one or more of these steps during the transfer process. We investigated an effect of the plant ethylene response on the induction of virulence gene expression in A. tumefaciens. The host plants produce some phenolic compounds that are required for induction of virulence gene expression in the bacteria. It seems that plant produced virulent gene inducing compounds were regulated by ethylene response.

View full abstract

To clarify the processes involved in plant immunity, we have isolated and characterized Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response. Inoculation of cad1 mutant plants with virulence pathogen shows that the cad1 mutation results in the restriction of bacterial growth (Plant Cell Physiol. 2005, 46: 902-912). The mutant enhances gene expression of the plant defensin (PDF1.2) which is related to the plant defense pathway regulated by ethylene and jasmonic acid (JA). All the results suggest that resistance to the pathogen in the mutant is associated to the genes of ERF protein which controls ethylene-responsive genes. Microarray analysis using the cad1 mutant revealed some gene for the ERF genes which are likely involved in plant defense. Characters of the ERF gene will be reported

View full abstract

The Arabidopsis mutant cad1(constitutively activated cell death 1)shows a phenotype that mimics hyper-sensitive response (HR)-like cell death. The mutant was observed the significant increase of salicylic acid (SA) levels and activated defense mechanism during pathogen infection. We therefore concluded that the CAD1 negatively controls the SA-mediated pathway of programmed cell death in plant immunity. (Plant Cell Physiol. 2005, 46: 902-912)
Induction of systemic acquired resistance (SAR) results in accumulation of SA in both local cells and systemic organs in coincidence with induction of pathogenesis-related (PR) genes as molecular marker for the SAR. To evaluate the relationship between the SAR and CAD1 function, we used DEX-induced RNAi for the CAD1 knockdown in this study. The results will be reported.

View full abstract

The interactions between plants and incompatible pathogens often lead to hypersensitive response, in association with rapid and localized cell death, hypersensitive cell death, at the infected sites of host tissues. However, little is known about molecular mechanism that operates hypersensitive cell death in plants. We previously reported that a vacuolar processing enzyme is essential for the execution process of hypersensitive cell death through disrupting the vacuole. To investigate the involvement of the vacuolar system in hypersensitive cell death, we inoculated avirulent bacteria into Arabidopsis mutant plants that exhibited abnormal vacuolar system. In several mutants, the hypersensitive cell death was delayed than in wild-type plants. We discuss the involvement of vacuolar system in plant defense systems.

View full abstract

Plant hypersensitive cell death (HR cell death) is a form of plant programmed cell death and plays a major role in plant immunity. We previously demonstrated that OsNAC4, a plant specific transcription factor, is involved in induction of HR cell death in rice. To elucidate mechanism of HR cell death induced by OsNAC4, localization of OsNAC4 was investigated. OsNAC4 was translocated into nucleus during HR cell death and its translocation was regulated by phosphorylation. Next, we compared global gene expressions in OsNAC4 RNAi line using oligonucleotide microarray. OsHSP90 were identified as upregulated gene by OsNAC4. When OsHSP90 was overexpressed in rice cells, loss of plasma membrane integrity was observed.

View full abstract

Plants respond to pathogen infection using an innate immune system with at least two distinct recognition mechanisms. One mechanism recognizes microbe-associated molecular patterns (MAMPs). The other is resistance (R) gene-based surveillance system. In spite of importance of salicylic acid (SA) -mediated responses in R gene-mediated defense, it has been unclear if MAMP-triggered defense requires SA-mediated signaling mechanisms. Here we report intimate interactions between MAMP-triggered and SA-mediated signaling. We found that SA accumulated after treatments with a MAMP, flg22. Expression profile analysis revealed the importance of SA signaling in the MAMP-triggered gene expression change. Effects of flg22 pretreatment on resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (PstDC3000), are partially dependent on SA signaling. These results indicate that the SA production triggered by flg22 is a component of the flg22-triggered signaling mechanism leading to resistance. We are currently studying signaling mechanisms involved in SA accumulation induced by flg22.

View full abstract

In order to robustly execute defense responses against a multitude of pathogens with various defense suppressors, plants have developed a complex and highly interconnected signaling network. To dissect the Arabidopsis defense signaling network, we have employed a reverse genetic approach combined with gene expression profiling. We assayed pathogen-inducible gene expression in a collection of twenty-five Arabidopsis mutants which were predicted to perturb various points in the signaling network. Plant mutants were infected with the bacterial pathogen Pseudomonas syringae, and gene expression profiles were analyzed using a non-linear dimensionality reduction. By identifying the similarities in expression profiles between mutants, we developed a signaling network model that infers interactions between mutated genes. Using this model, we have developed and tested hypotheses about signal flow through this network including the mode of signal amplification in a subnetwork of salicylate-mediated signaling and mutual inhibition between subnetworks.

View full abstract

Molecular mechanisms of plant cell death after the interaction between a disease resistance gene (R) and an avirulence gene are little known. We produced rice cell lines expressing chimeric genes (CRXa), which consist of the extracellular region of CEBiP, a rice receptor gene for chitin elicitor (GN7), and the intracellular region of Xa21, an R gene to rice bacterial blight. GN7 treatment resulted in the enhanced cell death and oxidative burst in CRXa callus, but not in callus expressing the truncated forms. Responses to lipopolysaccharide elicitor among callus lines were the same. These results indicated that GN7 signal was converted to cell death signaling through CRXA. We next analyzed the generation of reactive nitrogen species (NO and ONOO). GN7 treatment caused a great increase of both species in CRXa callus. Treatment with NO but not ONOO scavenger suppressed the cell death, suggesting that NO is involved in Xa21-dependent cell death.

View full abstract

Recent studies of symbiotic mutants of Lotus japonicus have led to isolation of a number of host genes, which are required for endosymbioses with both rhizobia and arbuscular mycorrhiza. These genes compose a common signaling pathway (CSP), in which symbiotic signals are converted into Ca signals (Ca-spiking). CCaMK, Ca/CaM-dependent protein kinase, also belongs to CSP and may act as a Ca decoder. Ca binding to CCaMK leads to activation of downstream pathways responsible for endosymbioses. Introduction of gain-of-function (gof) CCaMK leads to formation of spontaneous nodule. This fact indicates a pivotal role of CCaMK for nodule organogenesis.
During nodule formation, nodule organogenesis is coupled with bacterial infection. To clarify the involvement of several symbiotic genes in nodule organogenesis and/or bacterial infection, we introduced gof-CCaMK into nod- mutants and analyzed symbiotic phenotypes of them. Based on the results, we propose a model of early signaling pathway of nodulation.

View full abstract

Legume plants have an ability to fix atmospheric nitrogen via symbiosis with soil microbes. The transport of various metabolites between legume plants and rhizobia plays central roles in the establishment and physiological function of the nodules. ATP-binding cassette proteins constitute one of the largest protein families in plants, and have various functions in planta. cDNA array analysis revealed several ABC transporter genes are strongly induced during nodulation, suggesting the involvement of ABC proteins in nodulation processes. We have carried out a genome-wide analysis of ABC proteins in Lotus japonicus, and identified 91 putative ABC proteins. In this study, we focused one gene, named LjPDR1 (LjABCG1), which was specifically up-regulated in nodule formation. The expression of LjPDR1 was strongly induced by methyl jasmonate, and LjPDR1 was shown to be localized at the plasma membrane. We have created transgenic RNAi plants and are currently evaluating the phenotypes of these plants.

View full abstract

In Lotus japonicus, inoculation of M. loti induced transient nitric oxide (NO) production in roots accompanying with the expression of LjHb1 (class 1 Hb of L. japonicus). When nodAC deficient mutant was inoculated, transient NO production was induced in roots, whereas LjHb1 was not induced. Inoculation of exo mutants induced neither NO production nor the expression of LjHb1. Inoculation of pathogen induced continuous NO production in roots. We had shown that application of crude lipopolysaccharide (LPS) of M. loti induced NO production and the expression of LjHb1. When purified LPS of M. loti were applied, NO production was induced in the roots. We focused on NO production and expression of LjHb1in L. japonicus and the rhizobial components that induce NO production and LjHb1 expression in host plant root will be discussed.

View full abstract

Nitric oxide (NO) and reactive oxygen species (ROS), that are signal molecules in plant defense system, are produced in host plant cells by inoculation of rhizobium. The mutant of AtNOA1, which relates in nitric oxide synthesis in Arabidopsis thaliana, exhibit lower nitric oxide level than that of wild type and declined defense response against pathogens. We cloned and analyzed expression profiles of LjNOA1, the homolog of AtNOA1 of Lotus japonicus. When the expression of LjNOA1 was compared with, the expression of plants under symbiotic condition is higher than that of the plants with nitrogen fertilizer in all the tissues of plants. The higher expression of LjNOA1 may enhance the defense system of host plants L. japonicus.

View full abstract

Mergaert et al. (PNAS, 2006) suggest that possible factors responsible for differentiation of rhizobia in galegoid legumes have plant origin. It was found that the bacteroid differentiation in indeterminate nodule formed in galegoid plants is distinguished than that in the other legumes and was characterized with lack of bacterial cell division ability, formation of enlarged and polyploid bacteroids. Moreover, were found genes expressed only in indeterminate but not in determinate nodules. These genes were called ncr (nodule-specific cystein-rich) and the NCR peptides have structure resembles the plant defensins with potential antimicrobial activity. It was suggested that the NCRs could be the searched plant factor responsible for bacteroid differentiation. Further, by hairy root transformation of Lotus japonicus we tried to confirm or reject this hypothesis. We found changes in morphology and characteristics of bacteroids from NCR expressed Lotus plants.

View full abstract

Leguminous plants form root nodules for symbiotic nitrogen fixation with soil bacteria Rhizobia. To utilize symbiotic nitrogen fixation, it is indispensable to understand the molecular mechanism of nodulation process. Fix^- mutants, which form root nodules but have defect in nitrogen fixation activity, are useful materials to clarify the molecular mechanism which nitrogen fixation by endosymbiotic bacteria is controlled by host plant genes. A novel Fix^- mutant Ljsym89 was isolated from L. japonicus Gifu plants by EMS mutagenesis. Preliminary observations of the mutant nodules at 14 dpi by optical and electron microscopy revealed that the Ljsym89 nodules show abnormal structures of bacteroids, together with typical symptoms of premature senescence, such as enlargement of symbiosomes and disintegration of the infected cells. Here, we report further characterization of a novel Fix^- mutant Ljsym89 and positional cloning of its causal gene.

View full abstract

Actinomycetes Frankia have symbiotic ability to form nitrogen-fixing nodules on roots of actinorhizal plants. To screen Frankia genes involved in symbiosis, root exudate of its host actinorhizal plant was used.
Cultures of Frankia sp. strain CcI3 were treated by root exudate of its symbiotic host plant, Casuarina glauca and a non-host plant, Alnus glutinosa respectively. Suppression subtractive hybridization (SSH) is a powerful technique for screening differentially expressed cDNAs. Although SSH was originally designed for eukaryotic mRNA, we achieved the application of SSH to prokaryote Frankia preparing eukaryote-like mRNA. CcI3 total RNA was removed 16S and 23S ribosomal RNA and add poly(A) tail by RNA poly(A) polymerase. Using this technique, we are trying to screen CcI3 genes specifically induced by root exudate of the host plants.

View full abstract