Cuscuta japonica, a holoparasitic angiosperm, develops haustoria that are necessary to be parasitic on host plants. Previous cytological observations showed that haustorium formation is initiated by emergence of meristematic cells in the cortex. We hypothesized that the haustrium fomation is similar to apical meristem formation. In this study, we analyzed expression patterns of genes related to apical meristem formation during haustorium deveropment.
We isolated cDNAs of genes for apical meristem formation, e.g. homeobox genes (KNOX and WUSHEL-like), AS1 homologue and so on, from C. japonica. Their expression patterns during haustorium development were analyzed by RT-PCR. Our results indicated that some of the genes must be involved in haustrium development in Cuscuta. In addition, we also analyzed expression patterns of cell cycle related genes. We will discuss the role of their genes in haustrium development in Cuscuta.

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Several strains of Closterium were isolated from the field and aseptically cultured. When spotted on agar plates, cells of some strains spread widely over the plates during cultivation, while cells of the other strains stayed at the inoculation spot, forming a clump. The former type of cells secreted mucilage in a linear fashion. One of these strains, Closterium acerosum was used for studying the mechanism of directional mucilage secretion during phototaxis.
When a plate with actively growing cells was placed under illumination from only one direction, most cells exhibited positive phototaxis. Calcofluor staining showed abundant mucilage ejection from the tip of cells away from the light. To reveal the intracellular processes during this directional mucilage discharge, organelles such as Golgi bodies, ER, and vacuoles were stained and observed by fluorescent microscopy. Further details of the mucilage secretion process were visualized by TEM using the periodic acid-silver hexamine method.

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Aldrovanda vesiculosa once could be found in many places of the world except North and South America. The plants propagate by forming axillary buds, and produce winter resting buds in fall which sprout in late spring. Flowering is very rare. It is extremely difficult to maintain the plants. Aldrovanda vesiculosa was cultured and propagated in vitro, then grown outdoors, to prevent its extinction and to conduct morphological and physiological research throughout the year.
Apical segments of Aldrovanda vesiculosa were excised and sterilized in 0.5% NaClO₃ and cultured in 1/10 MS liquid medium. The plants proliferated well by forming axillary buds which were excised and subcultured once per month. The propagated plants were taken ex-vitro and kept in ca. 300 plastic containers. The plants in the containers outside kept growing and completed their life cycle, though their growth outdoors was often impeded by the influence of proliferating algae.

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Aldrovanda vesiculosa used in this study was grown ex-vitro after in vitro propagation. Fine structure was observed by LM, direct SEM observation and TEM of conventionally fixed or rapid-frozen specimens.
The shoot apex was found to be exposed, not covered by leaf primordia. Stamens and pistil were closely situated in the flower facilitating self-pollination. Endosperm in seeds contained mostly amyloplasts and protein bodies for storage, while the embryo contained lipid bodies and amyloplasts. A root structure was identified when the seeds germinated. Numerous well developed amyloplasts were observed in winter resting buds. Observation of young carnivorous leaves revealed characteristic structures of their glands and hairs: development of ER, tannin vacuoles, and Labyrinthin walls in digestive glands; abundant plasmodesmata and conspicuously developed ER in sensory hairs; and the proliferation of Golgi bodies in absorptive hairs. The localization of acid phosphatase in developing digestive glands was examined by cytochemistry using cerium.

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Arabidopsis thaliana BOR1, an efflux-type boron transporter, plays a major role in xylem loading of boron. For evaluating the physiological importance of pericycle specific expression of BOR1 in roots, transgenic Arabidopsis plants expressing BOR1 under control of cauliflower mosaic virus 35S RNA promoter were generated. When T2 plants were grown on kanamycin(Km) medium with 3μM boric acid, but not with 30μM, some of the plants resistant to Km showed growth inhibition similar to bor1-1 mutant. This was observed in several independent transgenic lines. Western analysis and microscopic observation confirmed overexpression of BOR1 in all types of cells in roots. We propose BOR1 proteins, expressed in outer layers of cells in roots, exported boron prior to xylem loading and inhibited transport of boron to shoots.

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The Arabidopsis genome contains 14 genes encoding sulfate transporters. Sulfate transporter, Sultr1;3 belongs to the family of high-affinity transporters, and was able to complement the sulfate uptake capacity of the yeast sulfate transporter mutant. Cell-type specificity of Sultr1;3 was studied in transgenic Arabidopsis expressing the fusion protein of Sultr1;3 and green fluorescent protein (GFP) under the control of Sultr1;3 promoter. Sultr1;3-GFP specifically localized in the phloem of cotyledons, hypocotyls and roots. Sultr1;3 mRNA accumulated under low-sulfur conditions. Inter-organ movement of ³⁵S sulfate was determined in the T-DNA insertion mutant of Sultr1;3. In this mutant, the efficiency of the movement of sulfate from cotyledons to the other organs was approximately 30 % of the wild type. These results suggest that Sultr1;3 participates in the loading of sulfate to the sieve element-companion cell complex and controls the inter-organ movement of sulfate in Arabidopsis.

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Sulfate transporter gene family of Arabidopsis is consisted of 14 isoforms classified into 5 groups. In this study, we demonstrated that Sultr3;5 is able to complement the sulfate uptake capacity of the yeast mutant lacking sulfate transporters. Transgenic Arabidopsis expressing the fusion gene construct of Sultr3;5 promoter and GFP showed the specific expression in the root pericycle and xylem parenchyma cells. The low-affinity sulfate transporter, Sultr2;1 is localized in the same tissue, therefore, we conducted functional analysis of both transporters using their knockout or antisensed plants. Measurement of translocation of ³⁵[S] sulfate from root to shoot revealed that movement of sulfate to shoot was accelerated in the Sultr2;1 antisense and Sultr3;5 knockout plants compared to their wild type. These results suggest that both Sultr2;1 and Sultr3;5 facilitate transport of sulfate into the pericycle and xylem parenchyma cells from apoplasic space in the stele, restricting translocation of sulfate to the shoot.

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In the nitrate assimilation pathway of plant, nitrite, produced after nitrate reduction by cytosolic nitrate reductase, enters into the chloroplast and is reduced to ammonium. Nitrate at a physiological concentration could be taken up to the chloroplast by an active transport. The transport of nitrite into chloroplast seems to be an important step in nitrogen metabolism in plants that use nitrate as major nitrogen source.
CsNitr1 cDNA was cloned and characterized as a plastid transporter for a nitrogenous compound. In this study, we analyzed the function of CsNitr1 by detecting quantitatively the translate using specific antibodies that were raised against hydrophilic region of CsNitr1. The expression of CsNitr1 was induced in green tissues by nitrate and nitrite only under the light depending on chloroplast integrity, suggesting that the function of CsNitr1 links with photosynthesis, especially with nitrite reduction in it.

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Plant utilizes nitrate and/or ammonia as nutrient. There are plant species having preference to nitrogen sources: rice likes ammonia whereas cucumber prefers to nitrate. We consider that such preference arises from the different rate of nitrite uptake by chloroplast. We cloned a cDNA, CsNitr1, which encodes a putative nitrite transporter of chloroplast envelope in cucumber leaves and introduced it into rice (Oryza sativa L. cv. Nipponbare) to modify the transport process of nitrite in the nitrate assimilation pathway.
Transgenic rice grew hydroponically with nitrate or nitrite as well as non-transformed rice with ammonia. However, the latter died after the replacement to new hydroponic medium that contained nitrate as a sole nitrogen source. Uptake of nitrate by the transgenic rice was higher than that by non-transformed ones indicating that the metabolic speed of rice for nitrate could be improved by the introduction of CsNitr1.

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Among root membrane proteins of tomato we found a metal binding protein and cloned its cDNA, LeGlp1. Two Cys residues at N-terminus may coordinate to metal ions. In this report, we constructed pBI121 vectors that express LeGlp1 mutants with the Cys residues mutated to Ser. LeGlp1 mutants were isolated from the leaves of transgenic tobacco and examined its metal binding by the affinity to Ni(II) column.
LeGlp1 mutant was synthesized as well as the native LeGlp1 but with rather unstable structure than the native one. Any of the mutants that lost one of the two Cys residues could not bind to the Ni(II) column indicating that the Cys is involved in the metal binding. Green-fluorescence protein fused with the N-terminal extension of LeGlp1 was localized at the Casparian band, which suggests the role of LeGlp1 in the uptake of metal ions.

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A Japanese indigenous Fern (Athyrium Yokoscense) is known as a heavy metal hyper-accumulator, which ability had been utilized as an index for searching mines at the old era. So, we aimed to clarify the genetic background of the tolerance and accumulation capacity. Here, we reported the condition for the callus formation, which would be useful to analyze the ability at the stable and homogenous conditions, and result from the analysis of the cadmium tolerance and accumulation capacity at the callus stage. Calli were formed from fronds of the fern when they were placed on the MS-medium supplemented with sucrose, agar and various combinations of Kinetin and 2,4-D. Then, these calli were grown in the same medium containing some concentrations of cadmium. The callus growth was comparable to that of the mature plant even in the higher concentration of cadmium, indicating that the cell itself would contain the ability.

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We traced the time course of ⁵²Fe translocation in graminaceous plants by a PETIS (Positron Emitting Tracer Imaging System) method. In all results, ⁵²Fe was first accumulated to 'discrimination center' (DC), which is the basal part of the leaf. I) Barley plants that had been cultured with and without Fe were supplied ⁵²Fe-deoxymugineic acid (DMA) to the root. Fe deficient barley accumulated ⁵²Fe to DC about 12 times as much as Fe sufficient barley. II) The maize ys1 mutant which is defective in Fe(III)-MAs transporter 'YS1' and wild type plants were cultured without Fe and supplied ⁵²Fe-DMA to the root. In the ys1 mutant, ⁵²Fe translocation at the DC was suppressed to about 20% of that of the wild type plant.

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The substances in apoplast have critical roles in plant growth and differentiation. A technique to obtain phloem contents by EDTA from cut leaves takes long time to collect apoplast fluid. We developed a method to obtain the apoplast fluid by pressurizing a plant in a pressure chamber. The contamination of apoplast fluid by symplast was examined using 7-10-day-old seedlings of Pharbitis nil. Symplast was collected by centrifugation from the used plant that had been frozen and thawed. The volume of apoplast was about 5% of symplast. The major sugar components of apoplast were Glc, Suc and Fru, in the ratio of 68, 25 and 7%, the ratio of symplast was 50, 20, and 30%. SDS-PAGE analysis showed that a band at 120 kDa was only for symplast and another at 20 kDa found only for apoplast, suggesting that the contamination of apoplast fluid by symplast was very little.

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Information of species of substances passing through the vascular bundle and their daily changes is limited. We developed a method to obtain the apoplast fluid by pressurizing plant in a pressure chamber. The apoplast fluid was obtained by pressurizing with 0 and 1 MPa at every 4h intervals from 7 to 10-day-old seedlings of Pharbitis nil cv. Violet grown under 16h LD photoperiodic condition. Components of sugar were determined by HPLC-PAD and GC. The concentration of sugar and protein at 1 MPa changed photoperiodically. The concentration of apoplast sugar in the light (4.5mg/ml) was 3 times higher than in the dark. In the light, the Glc, Suc and Fru ratio was 80:11:9, while in the dark were 45:33:22. GC analysis of hydrolyzed apoplast showed that Glc (64%) and Rha (33%) were major, and Xyl, Ara, Man and Gal were minor. A major component of symplast i.e. sugar was Glc (95%).

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It has been demonstrated that some pumpkin phloem proteins moved long distance in the organ-selective manner when introduced into rice sieve tube via insect stylet (Aoki et al., Plant Cell Physiol. 43: s192). Here we report the identification and primary structure analysis of pumpkin phloem sap proteins showing organ-selective long-distance movement. Biotinylated protein spots corresponding to long-distance movement signals were excised from 2D-electrophoresis of Q-sepharose bound fraction of the pumpkin phloem sap protein, and excised spots were subjected to in-gel digestion. Peptide fragments were then separated by micro HPLC and their primary structures were determined by internal peptide sequencing and peptide mass analysis. The protein moved specifically to the shoot was identical to the CmPP16-1. One of the two proteins which moved both to the shoot and root was identified as CmPP16-2, and the other was Cucurbita maxima ortholog of silver leaf whitefly inducible protein 1 (SLW1).

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A genomic clone of Wiv-1 gene for wound-inducible cell wall-bound acid invertase was isolated from tomato. The 5' upstream region (4kbp) of the gene contained some regulatory elements for phloem-specific expression. This region was fused to the beta-glucuronidase (GUS) gene, and then the chimeric gene was introduced to tobacco. In leaves of the transgenic tobacco, GUS activity continued to increase for 96 h after wounding. A strong GUS staining was observed in pollens and vascular tissues of stems close to petioles in transgenics grown in a greenhouse. These data suggest that the cell wall-bound enzyme encoded by Wiv-1 involves in the regulation of transport of sucrose in leaves and in the sink activity of pollens. In addition, results of some experiments for the induction of Wiv-1 gene expression by sugars will be reported as well.

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To identify the genes responsible for grain filling, we compared the transcription profilling of genes in superior (SS) and inferior (IS) spikelets in rice on 1, 7 and 13 days after flowering using a microarray system. Genes involved in carbohydrate metabolism were mainly analyzed and classified into 3 groups according to their expression patterns. Group1 gene expression decreased accompanying the spikelet development. It includes starch- and sucrose- synthases, and sugar transporters. Their expression in IS delayed than SS, which may delay the spikelet development in IS. Group2 gene expression increased accompanying the spikelet development. It contains branching enzymes, glutelins and prolamins. They expressed higher in SS than IS. It might bring better grain filling in SS. Group3 genes which expressed higher in IS than SS include alpha-amylases. They catalyze starch degradation and may cause poor grain filling in IS.

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Cryptogein is known as a proteinaceous elicitor, which induces HR in tobacco cell. We generated cryptogein mutants (K13V and N93A) and compared the biological activities of external alkalization and death progression. The quantitative results exhibited that N93A declined both the external alkalization and death induction activity. In the case of K13V, the death progression was inhibited to less than 10% of original activity, although its external alkalization retained normal.
The cryptogein-sterol complex is a key component of death induction. Using a fluorescent probe, dehydroergosterol, we examined the sterol binding activity of these mutants. From the results, we found that K13V lost its binding ability, and N93A retained a half of the original ability. These results suggest that cryptogein requires a sterol binding to induce an efficient death reaction on plant cell, although the external alkalization does not require the sterol binding inside of cryptogein molecule.

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N-acetylchitooligosaccharides elicit several defence responces in suspension-cultured rice cells (Oryza sativa cv Nipponbare). We have reported the isolation of two novel elicitor responsive genes, CIGR1 and CIGR2, of GRAS family of rice. It was previously suggested that both genes are the crossing point of the two distinct signaling pathways from elicitor and gibberellin. In this study, we investigated the behavior of both mRNAs in rice plant using in situ hybridization and northern blot analysis. In situ hybridization experiments indicated that both genes were rapidly activated by treatment with elicitor or gibberellin and that the activation of CIGR1 mRNA was localized in dumbbell-shaped silica cells at leaf epidermis and CIGR2 mRNA activated in the mesophyll cells.

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Tobacco BY-2 chitinase and glucanase activities were induced by the addition of the pathogenic fungus Alternaria alternata culture medium. The object of this study is isolation and analysis of the elicitor active compounds from culture medium.
The culture medium was autoclaved and filtrated to remove the fungus body. 99% methanol-insoluble fraction of the filtrated solution had elicitor activity. The activity fraction was separated by removing protein, sodium hydroxide extraction and 75% methanol extraction. Finally, the elicitor was isolated by a gel filtration chromatography. The elicitor activity was assayed by the BY-2 chitinases induction method.
The isolated elicitor was composed of carbohydrates with molecular weight of >4000. BY-2 chitinase was induced with the concentration of 200 μg ml^-1. Hydrolysates were converted the alditol acetates and analyzed with a gas chromatography. As the results, the carbohydrates elicitor from A. alternata was composed of mannose, glucose, and galactose.

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The tobacco BY-2 chitinases were induced by the addition of Alternaria alternata cultured medium. In this study, we focused on BY-2 β-1,3-glucanase, and investigated the effect of A. alternata and 12 chemical stresses on the β-1,3-glucanase induction.
BY-2 glucanase were induced by the addition of salicylic acid, laminarin and several chemical stresses, and especially, A. alternata culture medium induced glucanase activity markedly. On the other hand, methyl jasmonate and abscisic acid did not induce the glucanase. By addition of A. alternata, the glucanase activity increased after 12~24h, and reached the maximum at 60h and then decreased. The time course pattern of glucanase activity was different from the pattern of BY-2 chitinases. Now, we try to cDNA cloning of the β-1,3-glucanase.

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Flagellin, a main component of a bacterial flagellum, purified from rice-incompatible strain, N1141, of Acidovorax avenae specifically induces defense responses, such as hypersensitive cell death and defense-related genes. In this study, we analyzed the flagellin perception system in rice. Cultured rice cells did not respond to flg22, an elicitor-active synthetic peptide in Arabidopsis and tomato comprising the conserved 22 amino acids within the N-terminal part of flagellin, and a peptide corresponding to flg22 within flagellin of A. avenae. On the other hand, C-terminal recombinant flagellin fragment induced reactive oxygen species and defense-related gene in cultured rice cells. Furthermore, binding experiment for flagellin in rice microsomal membrane revealed the existence of a 45 kDa flagellin binding protein, which is apparently different from 115 kDa flg22 binding protein in tomato. These results suggest that rice has the different flagellin perception system from Arabidopsis and tomato.

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Resistance responses of cultured rice cells was specifically induced by a incompatible strain of Acidovorax (Pseudomonas) avenae and several pathogen-associated molecular patterns such as flagellin, hrpY, hrpW were involved in this induction of resistance responses. To investigate the transcript abundance and expression patterns during the resistance responses, cDNA microarray including 3353 cDNAs from libraries of the incompatible strain inoculated and non-inoculated cultured rice cells were constructed. Using the microarray, gene expression patterns during the infection in cultured rice cells were compared between the incompatible strain and the deficient mutants of several molecular patterns containing flagellin, hrpY, hrpW, and hrc. Total RNAs were extracted from cultured rice cells inoculated after 0.5, 1, 2, 3, 4, 5, 6 hours and labeled during reverse transcription, then hybridized to microarray. Among the genes induced by the incompatible strain, the gene expressions of 8% (Δfla), 4% (ΔhrpW), 2% (ΔhrpY) and 23% (Δhrc) were reduced, respectively.

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When challenged with the fungus Alternaria alternata Japanese pear pathotype, the Arabidopsis plants produced no lesion. The plants did not produce both reactive oxygen species and HR cell death after challenge with A. alternata, although they denoted the resistance to A. alternata. The spore germination was observed microscopically at the surfaces of Arabidopsis plants and the penetration attempts were found at the cell walls. However, the infection hyphae were not seen in the cytoplasm of epidermal cells. In Arabidopsis plants infected with A. alternata, papillae were formed at the sites of attempted fungal penetration. On the other hand, changes in the expression patterns of ca.7000 genes were examined by cDNA microarray analysis in Arabidopsis plants after inoculation with A. alternata. In the results, the abundance of 128 mRNAs was increased, whereas that of 86 mRNAs was reduced. Physiological, molecular and genetic characterization of several genes is in progress.

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In rice, the small GTP binding protein OsRac1 is thought to play a key role in defense signaling by regulating NADPH oxidase, which generates reactive oxygen species (ROS) during oxidative burst. The expression of a metallothionein, OsMT4, an ROS scavenger, was reduced in rice cultured cells that overexpress the constitutive active form of OsRac1. Transgenic plants that overexpress OsMT4 showed increased susceptibility to bacterial blight and blast fungus. Furthermore, OsMT4-overexpressing cells showed reduced elicitor-induced hydrogen peroxide (H₂O₂) production. In contrast, Tos17::OsMT4 insertional mutant and RNAi-OsMT4 transgenic cells, which were deficient in OsMT4 expression, showed increased elicitor-induced H₂O₂ production compared to the wild type cells. Furthermore, in vitro assay showed that recombinant OsMT4 protein possesses superoxide scavenging activity. OsMT4-GFP fusion protein was localized at the cytosol indicating, OsMT4 functions primarily in the cytosol. The results suggest that OsRac1 downregulates OsMT4 expression, thereby potentiating ROS accumulation and disease resistance.

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Plants respond to various biotic stresses by activating NADPH oxidase to generate reactive oxygen species (ROS) which are used to kill invading pathogens directly or to trigger defense signaling, or both. Although, rboh genes encoding NADPH oxidase have been cloned from various plants, the molecular regulatory mechanism of NADPH oxidase activation remains to be understood. In our previous studies, a small GTP-binding protein, OsRac1, was shown to regulate NADPH oxidase-dependent ROS generation in the rice cells during defense response. In the present study, we report that OsRac1 could interact with plant rbohs in yeast two-hybrid system. In addition, a sphingolipid elicitor, which induces OsRac1-dependent ROS production in rice cell cultures, was able to induce rboh mRNA expression. We will discuss the possibility of NADPH oxidase as a downstream target of OsRac in plant defense signaling.

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MAP kinase (MAPK) cascades are major components downstream of receptors or sensor that transduce extracellular stimuli into intracellular response. In plant, MAPK are activated by abiotic stress and by pathogen. A MAPK cascade functions downstream of the elicitor receptor FLS2 in Arabidopsis, and tobacco MAPKs are involved in reactive oxygen species production, cell death and defense gene activation. Rice MAPK genes are induced by rice blast (Magnaporthe grisea) and fungal elicitor, however little is known about MAPK cascade and disease resistance.
We are characterizing a new rice MAPK quickly activated by rice blast elicitor in cell culture and by incompatible rice blast infection in plant. This MAP kinase shows high homology to the plant NtSIPK and AtMPK6 involved in cell death and disease resistance. Therefore we are using multiple approaches (transgenic plants, transient assay) to further investigate the role of this MAPK in cell death and defense signaling in rice.

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The treatment of brassinolide (BL), the most active BR, induces resistance against a broad range of pathogens in rice, tobacco and Arabidopsis. The analysis of induction mechanism of brassinosteroid-mediated disease resistance (BDR) using Arabidopsis indicated that BDR needs NPR1 protein, a key component of systemic acquired resistance. However, BL treatment did not induce the expression of pathogenesis-related genes (PR-1, 2 and5) and the accumulation of salicylic acid. BDR was not induced in an ethylene insensitive mutant, ein2. Our data suggest that ethylene plays an important role in BDR.

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Several chemicals are identified to induce systemic acquired resistance (SAR) in plants. We analyzed the mode of action of a new pyrazole derivative for the induction of SAR. This compound induced disease resistance against a broad range of pathogens in rice, tobacco and Arabidopsis, and induced the expression of PR genes and SA accumulation in tissues. These results indicated that this compound stimulate upstream of SA in the SAR signaling pathway, as does probenazole (PBZ). The gene expression patterns in microarray analyses using Arabidopsis suggested that this pyrazole derivative induces SAR in a different manner from that of PBZ.

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Hypersensitive cell death plays a critical role in the plant defense system, constituting a major component of HR to restrict pathogen multiplication. Screening early responding genes during HR against TMV infection, we identified that encoding ornithine decarboxylase (ODC). Subsequent analyses showed not only ODC but also other genes involved in polyamine biosynthesis to be up-regulated, resulting in accumulation of polyamines in apoplast of TMV-infected leaves. Treatment of inhibitors for polyamine biosynthesis reduced the rate of HR and accumulation of polyamines. These results suggested polyamines to have critical role(s) in HR. This was experimentally confirmed, showing that H2O2 was generated in leaves infiltrated with polyamines. Further analyses revealed that accumulated polyamines were indeed degraded by polyamine oxidase detected in apoplast. It was concluded that, when HR is triggered, polyamines are synthesized, transported to apoplast, and degraded to induce H2O2, eventually resulting in hypersensitive cell death.

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Plastocyanin (PC) or cytochrome c₆ (Cyt.c₆) functions between cytochrome f and P700 in the photosynthetic electron transport system. In some green algae and cyanobacteria, PC accumulates in the cells grown with copper, whereas Cyt.c₆ accumulates under copper-deficient conditions. We have investigated the structure and expression of PC gene in the green alga Eudorina elegans Ehrenberg var. elegans NIES-456 that shows reciprocal formation of PC and Cyt.c₆. Partial sequence of the PC gene was determined by PCR and 3' RACE; the deduced amino acid sequence of Eudorina PC shows the highest homology with that for Chlamydomonas reinhardtii. Northern blot analysis using the PCR product as a probe showed that the amount of PC mRNA was reduced and the low molecular weight species of mRNA appeared under copper-deficient conditions, suggesting that the PC synthesis may be regulated at the level of mRNA stability.

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When Chlamydomonas cells are exposed to CO₂-limiting stress conditions, they sense the change of CO₂ levels and induce a set of genes including Cah1, which encodes a periplasmic carbonic anhydrase. Cah1 is not expressed under high- CO₂ conditions (5% [v/v] CO₂), whereas the transcription is activated within 1 hour after transfer to low- CO₂ conditions (0.04% [v/v] CO₂) in light. The CO₂ -dependent expression of Cah1 is controlled by its 5'-upstream regions (ref.1). A regulatory mutant with Cah1, I39 were isolated by DNA tagging mutagenesis.
In I39, Cah1 mRNA was accumulated under 5% [v/v] CO₂ but was not under 15% [v/v] CO₂. I39 has decreased sensitivity against the environmental CO₂. The inserted tags were present on a single chromosome and the mutated gene was tagged.
ref.1) Kucho et.al. Plant Physiol. (1999) 121: 1329-1337.

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Carbon concentrating mechanism (CCM) is induced by lowering CO₂ in light. To understand whether induction of CCM depends on light intensity or not, the photosynthetic rate and K_1/2(Ci) values were measured in various light intensities with different CO₂ concentration. As light intensities were stronger, the apparent affinities for Ci were higher in the presence of 1.2% CO₂. And by using cDNA macroarrays containing 10,368 ESTs, global gene expression was examined during acclimation to high-light and/or CO₂-limiting conditions. Chlamydomonas cells were cultured under 5% CO₂ with 120μEm^-2s^-1.For high-light treatment, cells were transferred to 1000μEm^-2s^-1, and for CO₂-limiting conditions, cells were transferred to 0.04 % CO₂. After treatments, cells were harvested after 0.3, 1, 2, and 6h. Expression levels of 391 genes were altered, 132 genes were high-light responsive genes and 317 genes were low-CO₂ responsive genes. We discuss the cross-talk between the light-signaling and the CO₂-signaling.

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Phosphoglycolate phosphatase (PGPase) is a key enzyme of photorespiration that catalyzes the conversion of phosphoglycolate into glycolate. PGPase-deficient mutant pgp1 requires elevated levels of CO₂ for growth in light. It is possible that carbon-concentration-mechanism (CCM) cannot operate in this mutant because of the accumulation of phosphoglycolate. In order to know how the pgp1 mutation affects the expression of CCM-related genes, we compared the overall expression levels between the mutant and the wild type C9 under low-CO₂ condition by using cDNA macroarray. Several genes including Ccp1 encoding the chloroplast envelope protein LIP-36 were not induced under low-CO₂ condition. And other genes (e.g. Mca encoding mitochondrial carbonic anhydrase) were identified whose expression levels were lower than that of the wild type under low-CO₂ condition. These affected genes were assumed to be regulated by unknown metabolic products, which accumulated in the pgp1 mutant.

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Diatoms may contribute about 25% the total primary production on the earth. Carbonic anhydrase(CA) is important factor for carbon assimilation. The regulation of expression of CA by CO₂ and light and its intracellular location were studied with P. tricornutum. Both under light and dark, CA expression increased at both mRNA and protein accumulation level upon transferring cells from 5%CO₂ to air. The expression level in the light was higher than that in the dark. By prolonged exposure to air for 5 days, the level of mRNA accumulation gradually increased and oscillated at an interval of about 24h. These results predicate that CA expression is triggered by changes in [CO₂] in the medium and light helps mRNA accumulation. Also CA expression seems to relate with cell cycles. Chimeric protein which possesses 46 N-terminal sequence of Β-CA precursor joined with GFP was expressed in P. tricorutum and found to locate in chloroplast.

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Microalgae are considered to play a key role in aquatic carbon fixation. Most of unicellular algae possess inorganic carbon concentrating mechanism which confers cells to do high-affinity photosynthesis for dissolved inorganic carbon. The expression of intracellular β carbonic anhydrase in the marine diatom Phaeodactylum tricornutum has been shown to be response to changes in pCO₂ in the culture media. In the present, the function of the promoter region of β-CA gene was analysed in P. tricornutum. The chimeric gene in which 5'-upstream region of CA gene was fused to a GUS reporter gene was constructed and introduced into the cell by microparticle bombardment. The results demonstrated that the GUS activity exhibited in air-grown transformants was 15-25 fold that in 5% CO₂-grown cells. The critical cis-elements in β-CA promoter which are required for CO₂ responsibility of β-CA are being investigated by loss- and gain- of function assay of this promoter region.

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Genes homologous to mammalian Na⁺/bile acid transporter (NBT) exist in Arabidopsis thaliana and rice genome sequences. Their functions are still unknown, but a gene encoding an NBT homologue is expressed higher in C4 species than in C3 species of Flaveria (Yamaguchi et al. 2002, Plant Cell Physiol. Suppl 43 3aB09). It suggests that the homologue may be related to C4 pathway.
We have isolated a full length cDNA of a putative NBT homologue (McNBT) from the leaf tissue of Mesembryanthemum crystallinum, a facultative Crassulacean acid metabolism (CAM) plant. McNBT encodes a protein which shows 78% homology to the Arabidopsis protein. Low level expression of this gene is detected in the leaf and root tissues of C3 mode of M. crystallinum by using semi-quantitative RT-PCR technique. During CAM induction by addition of 0.35 M NaCl to the rooting medium, mRNA level of the gene is increased only in the leaf tissue.

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The amount of Rubisco in leaves is largely affected by nitrogen nutrition during leaf expansion. The purpose of this study was to examine the relationship between nitrogen nutrition and gene expression of Rubisco in rice leaves. Rice plants (Oryza sativa L. cv. Notohikari) were grown with different levels of nitrogen supply (sufficient, control and deficient) from the emergence of the 8th leaves. The levels of mRNAs of rbcS and rbcL responded to the changes in the nitrogen levels quickly and reached the highest values one day after the treatments in the 8th leaves, irrespective of the treatments. The levels of rbcS and rbcL DNA, however, did not increase as the same extent as the mRNAs did, indicating that the regulation of the levels of mRNAs of rbcS and rbcL in expanding leaves of rice under different levels of nitrogen supply includes some important factors other than the template levels.

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It has been suggested that Rubisco might be degraded within or outside of intact chloroplasts because the amount of Rubisco decreases much faster than the decrease in chloroplast number. In this study, we investigated the possibility that Rubisco is degraded outside of chloroplasts.
By immunocytochemical analysis, besides the chloroplast stroma, we recently found that Rubisco was localized in small spherical bodies having 0.4-1.2 μm in diameter. These Rubisco-containing bodies (RCB) were also found in the specimens prepared by high-pressure freezing and freeze substitution methods. RCB seemed to be derived from chloroplasts because the protrusion of the chloroplast envelope enclosing the stromal portion was seen. RCB contained the other stromal protein, glutamine synthetase, but not thylakoidal proteins. The appearance of RCB was most remarkable when the amount of Rubisco started to decrease. These results suggest that RCB could take responsibility for the Rubisco degradation outside of the chloroplast during leaf senescence.

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Considerable studies have been done to investigate the effect of acid rain on plant growth and photosynthesis. However, little attention has been paid for the effect of 'normal' rain on leaf photosynthesis, nevertheless it sometimes inhibit plant photosynthesis considerably. We have investigated the effect of surface wetness on leaf photosynthesis in bean plants to clarify the mechanism responsible to this photosynthetic inhibition caused by mist exposure. Rubisco content was significantly declined in mist-treated bean leaves, suggesting that photosynthetic inhibition is caused by the decline in carboxylation enzymes. Rubisco degradation was enhanced in mist-treated leaves. These results suggest that the decline in photosynthesis by surface wetness would be caused by the inhibition of photosynthetic machinary, rather than by the stomatal responce to wetness.

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Decrease in photosynthetic activity of source-sink model plant of sweet potato under sink-limited conditions was mainly due to the binding of inhibitor to RuBPcase.The inhibitor was suggested to be sugar phosphate. To clarify the structure of inhibitor and its inhibition mechanism of RuBPcase, purifying the inhibitor in large quantities is needed. Steam girdling of stems of sweet potato grown in green house resulted in the decrease in photosynthetic rate and total activity of RuBPcase and the accumulation of photosynthates in leaves. Maximum activity of RuBPcase did not differ from that of control plants, indicating that the girdling treatment resulted in the sink-limited state. Leaf extracts prepared from the plants girdled were mixed with activated RuBPcase to bind inhibitor to this enzyme. Then, the RuBPcase was collected by PEG treatment, and the inhibitor bound to RuBPcase was dissociated with heat treatment. The highly purified inhibitor could be obtained through these treatments.

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Biomass production is determined by respiration as well as photosynthesis. In addition, respiration is also strongly affected by temperature. We examined the effects of night temperature on biomass production and respiration in rice during different stages. During the vegetative stages, high night temperature led to increases in biomass production and respiration. Photosynthesis was more stimulated and this was mainly caused by an enlargement of leaf area. During the ripening stages, final biomass production was not affected by night temperature. Although respiration was increased by high night temperature, a delay of leaf senescence and a retardative decrease in green leaf area were found for the high night temperature treatment.

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We identified Arabidopsis mutant SS93 having short root. SS93 shows disorganization of cell layers not only in RAM but also in SAM, abnormal phyllotaxy and leaf morphology, and fasciated stem. We mapped SS93 mutation on chromosome4 by using genome information and found a deletion of 2.7kb in a gene which may encode a protein with the polymerase domains of prokaryotic DNA polymerases and conserved motifs characteristic of helicases. Proteins having these two structures were previously identified in various eukaryotes. Mutation in the Drosophila homolog, mus308, gene confer specific hypersensitivity to DNA-crosslinking agents as a consequence of defects in DNA repair. SS93 phenocopies fasciata (fas) mutant which shows abnormal meristem structures. FASCIATA gene encodes a subunit of the Arabidopsis counterpart of chromatin assembly factor-1 (CAF-1), and previous studies in animals show that CAF-1 is involved in DNA repair and replication. It seems that SS93 and fas have common defect in replication.

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To elucidate the molecular mechanisms of auxin-regulated lateral root formation, we are studying solitary-root (slr) mutants, which have gain-of-function mutations in IAA14, a member of Aux/IAA family in Arabidopsis (Fukaki et al., Plant J. 2002). The slr mutants have no lateral roots, few root hairs, and reduced gravitropism in roots and hypocotyls. To identify genes that genetically interact with SLR/IAA14 in lateral root formation, we isolated the extragenic suppressor mutants of slr-1 from EMS-mutagenized slr-1 seedlings. The ssl2 (suppressor of slr 2) is a single recessive mutation, and we have four ssl2 alleles (ssl2-1~ssl2-4). The ssl2 slr-1 double mutants produce lateral roots but still have few root hairs and reduced gravitropism, indicating that the ssl2 mutations specifically suppress the slr-1 defect in lateral root formation. These results indicate that the slr phenotype in lateral root formation is partially dependent on SSL2. Positional cloning of SSL2 gene will be presented.

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In general, it is thought that organogenesis is composed of three sequential phases; acquisition of developmental competence for regeneration, commitment to differentiate shoot or root, and formation of determined organs. Arabidopsis ESR1, a member of AP2/ERF transcriptional factor, is thought to regulate induction of shoot regeneration after the acquisition of competence for organogenesis since expression of ESR1 is transiently induced during the shoot regeneration process and ESR1 overexpression greatly increases the efficiency of shoot regeneration. To dissect the subsequent chain of events that culminates in shoot regeneration, we are screening the genes under ESR1 regulation. For the purpose, a cDNA library was constructed by subtraction between cDNAs from root cultures in which overexpression of ESR1 was induced and those in which the overexpression was not induced. At present we are examining expression of cDNAs in the library during shoot regeneration.

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In flower buds, three floral organs, petals, stamens, and carpels are enclosed by sepals. During the process of floral organogesis, four sepal primordia are generated first at the outermost whorl of a floral meristem, and then the other floral organ primordia appear. This reflects that sepals have evolved to protect the other floral organs. For this purpose, sepals are required to have enough width to cover the young bud. In the fl51 mutants, four sepals can not cover completely, because the sepals are narrower and longer than those of the wild type, though sepal identity is normal. It suggests that the FL51 gene is involved in the sepal development in both lateral and proximal-distal directions.We mapped the FL51 gene on the upper part of chromosome 1. Mapping data and possible functions of the FL51 gene will be presented.

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In monocots, the mechanism of floral organ specification is less understood. In rice, mutations in the dl locus show homeotic conversion of carpels into stamens. We have reported previously that DL encodes a YABBY family protein and is expressed specifically in carpel primordium.
In this study, we dissected the genetic relationships of DL with the ABC MADS-box genes by molecular genetic approaches using floral homeotic mutants and transgenic plants. Our results indicate that DL is essential for carpel specification and interacts antagonistically with the B-function genes.
Approaches to isolate knock-out lines of C-function genes such as AG homologs is in progress to reveal the roles of C function genes in rice carpel specification and the relationships with DL.

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Garden asparagus has homochlamydeous flower like tulip and lily. To explain the floral morphology of Liliaceae, van Tunen et al. (1993) proposed a modified ABC model, exemplified by tulip. In this model, class B genes are expressed in whorl 1 as well as whorls 2 and 3, thus the organs of whorl 1 and 2 have the same petaloid structure. To clarify the perianth identity in asparagus, we isolated one DEF-like gene (AODEF) and two GLO-like genes (AOGLOA, AOGLOB) from asparagus. Northern blot analysis showed that these genes are specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that these genes are expressed in whorl 2 and 3 (inner tepal and stamen), but not in whorl 1 (outer tepal). These results suggest that modified ABC model would not be applicable in asparagus and class B genes would not be involved in perianth identity in asparagus.

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Temperature is one of the critical environmental cues for seed germination. Germination of Arabidopsis seeds is inhibited by high temperature and induced by low temperature. We showed alleviation of thermoinhibition by abscisic acid (ABA) biosynthesis inhibitor and enhancement of ABA sensitivity at high temperature, and suggested that regulations of both ABA biosynthesis and signaling are involved in thermoinhibition. To see the effect of temperature on expression of ABA-related genes, we adopted quantitative PCR method. Expression of ABA biosynthesis genes, ZEP, NCED, ABA2 and AAO3 was induced remarkably after 12 h at 34^oC, but decreased to low levels at 22^oC. Expression of ABA signaling genes, ABI3 and ABI5, was induced at 34^oC but diminished at 22^oC. These results suggest that the induction of ABA biosynthesis and signaling genes by high temperature is responsible for the enhancement of ABA content and the sensitivity in Arabidopsis seeds.

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Seed germination is dependent on dormancy and environmental conditions. Temperature plays a decisive role on germination. Germination of Arabidopsis seeds is inhibited by high temperature (thermoinhibition) and promoted by low temperature treatment. We analyzed the genes expressed in Arabidopsis seeds by microarray, and found that expression of gibberellin (GA)-related genes was affected by temperature. Quantitative PCR analysis showed that expression of AtGA3ox2 gene, which converts inactive precursors to active GAs, was evidently lower at 34^oC than at 22^oC. Expression of RGL2 gene, a member of the GRAS gene family, peaked at 12h after imbibition and decreased to a lower level after 36h at 22^oC, whereas it remained at high levels even after 48h at 34^oC. These results suggest that the inhibition of active GA synthesis and the accumulation of a negative regulator of GA signaling play a role in inhibiting germination at high temperature.

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