Plant gene expression involves plant-specific classes of transcription factors, which are intimately associated with a variety of plant-specific phenomena. The Dof family is a typical example of such transcription factors. We discovered the first Dof transcription factor (maize Dof1) and identified a novel DNA-binding domain within Dof1. Because this domain appeared to form a single zinc finger, I named it the Dof (DNA-binding with one finger) domain. We also showed that Dof1 is a transcriptional activator involved in leaf-specific and light-responsive expression of the C4 photosynthetic phosphoenolpyruvate carboxylase gene. Other Dof transcription factors also appear to play vital roles in diverse plant-specific biological processes. I talk the overview of the Dof family, highlighting the function of maize Dof1. I also show our recent successful application of maize Dof1 to biotechnology, that is, enhancement of photosynthetic nitrogen assimilation in transgenic plants expressing Dof1.

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A novel compartment with a characteristic structure of ~0.5 m diameter x ~10 m long that accumulated stress-inducible proteinases was found in Arabidopsis thaliana. They are surrounded with ribosomes and are directly derived from the endoplasmic reticulum. Therefore, we propose to call them the ER bodies. Fluorescent ER bodies were observed in transgenic plants of Arabidopsis thaliana expressing GFP fused with an ER retention signal. ER bodies were widely distributed in the epidermal cells of whole seedlings. In contrast, rosette leaves had no ER bodies. We found that wound stress induced the formation of many ER bodies in rosette leaves. ER bodies were also induced by treatment with methyl jasmonate, a plant hormone involved in the defense against wounding and chewing by insects. The induction of ER bodies is a novel endomembrane system in the response of plant cells to environmental stresses.

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(1) The maximum rate of photosynthesis on leaf area (Pmax) increases with leaf development and peaks at around the full leaf area expansion (FLE) in annual herbs. But, in evergreen trees, Pmax at FLE is very low and continues to increase for another two to three weeks. Cell division and chloroplast division continued to the later stages in leaf development and thereby chloroplast maturation occurs well after FLE. Competition for resources and energy among these developmental processes would explain the delayed maturation in evergreen tree leaves.
(2) Sun leaves have thicker palisade tissue. In flush-type deciduous trees, number of cell layers in the palisade tissue is determined by the light environment when leaves develop in the winter bud. Similarly, in annual herbs, the layer number in developing leaves is determined by light environment of the mature leaves. But, sun/shade chloroplast development depends on current, local light environment.

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To analyze the molecular mechanisms for salt tolerance in a mangrove plant, Bruguiera sexangula, and a member of the family Chenopodiaceae, Suaeda japonica, functional screening of cDNAs encoding proteins which may play an important role for salt tolerance in these halophytes was done using Escherichia coli as a host organism. In this screening, cDNAs encoding allene oxide cyclase homolog (mangrin) (Yamada et al. 2002 Plant Cell Physiol, 43 903-910), alfa subunit of chapenonin containing TCP-1 (CCT alfa) (Yamada et al. 2002 Plant Cell Physiol, 43 1043-1048), and RelA/SpoT homolog (Yamada et al. Plant Cell Physiol, in press) were successfully isolated. The role of these proteins in the mechanisms for salt tolerance will be discussed.

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The initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles are catalyzed by the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by combining CO₂ and O₂, respectively, to ribulose-1,5-bisphosphate. Since Rubisco limits photosynthesis and thus plant growth, an understanding of the structure/function relationships of this enzyme is extremely important to design Rubisco superior in catalysis and to improve crop productivity. Rubisco from an acido-thermophilic unicellular red alga, Galdieria partita (optimal growth conditions are pH 2 and 40 °C) has the highest specificity for carboxylation reaction among the Rubiscos reported so far. In order to clarify the structural basis of the excellent property of the Galdieria enzyme, X-ray crystallography of this enzyme has been conducted. In this symposium, we show crystal structures of Galdieria Rubisco complexed with some different ligands, and propose the mechanism in which the enzyme acquired the high functionality.

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Drought is one of the major abiotic stresses affecting plant productivity. Under water deficit conditions, generation and proliferation of reactive oxygen species increases and results in oxidative damage to plant cells. In contrast to this idea established with domesticated or mild environment-adapted plants, wild watermelon, a C3-plant from the Kalahari desert, Botswana, had very unique mechanisms for surviving drought. Under stress conditions, the plants accumulated large amount of a novel compatible solute, citrulline, in the leaves. Citrulline is one of the most efficient hydroxyl radical-scavengers among natural compounds examined so far. Moreover, the plants induced unique set of proteins such as DRIP-1, metallothionein and cytochrome b-561 in response to drought. Some of these factors are thought to be involved in antioxidative defense system of the plants. Molecular analysis of drought-tolerant wild watermelon provides new hints and tools to improve the stress tolerance of crop plants.

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The spadix of skunk cabbage, Symplocarpus foetidus, maintains an internal temperature of around 20^oC even when the ambient air temperature drops to freezing. To understand the mechanism and the way of thermoregulation, detailed analyses of the temperatures of spadix and ambient air were made under field and artificial conditions. Based on a series of temperature measurements, temperature-triggered thermogenic oscillation in the spadix was discovered. The identified thermogenic oscillator had an accurate periodical cycle (ca. 60 min per cycle) that apparently responds to changes in the spadix temperature with a threshold of less than 0.5^oC. These results suggest that periodical thermogenic oscillations may have a critical role in thermoregulation of the spadix. Based on these results, we propose a time-dependent thermogenic oscillatory model that acts as a precise thermal regulator under dynamic environmental temperature changes. This work was supported by a grant from the PROBRAIN.

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The domestication of wheat and barley was the most important step in the emergence of farming communities that later led to the ancient civilisation in Mesopotamia. Several lines of evidence indicated that emmer wheat (Triticum turgidum ssp. dicoccum, genome: AABB, 2n = 28) was the earliest domesticated wheat derived from the wild emmer (ssp. dicoccoides) and that it was domesticated within the Fertile Crescent of southwest Asia. Emmer was the main crop during the spread of Neolithic agriculture from the Fertile Crescent to Europe, Eurasia, and Africa. However the precise location and history of the emmer domestication remain unknown. Here we show that a major chloroplast haplotype of domesticated emmer was found in only 4% of wild emmer accessions, all collected from the northern Fertile Crescent. The result shows that the domestication of emmer wheat belonging to this major lineage occurred in this region.

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It is well known that the molecular biological approaches using Arabidopsis gave very big impacts on plant sciences. This is also the case of inorganic metabolisms in plant cells. Most membrane transport proteins were found from Arabidopsis and other few model plants. However, we have still possibilities to find new physiological phenomena from many wild plants, because such wild plants are surviving in the ultimate environments.
Recently, we are investigating salt tolerance of cells of mangrove plants, which are surviving in high salt condition, and phosphate metabolisms of algal cells, which are in the very low level of inorganic phosphate. From these materials, we found new physiological mechanisms for inorganic ion metabolisms. In the present paper, I would like to discuss how we approach survival strategies for the environmental adaptation of plants, using classical physiology of wild plants and modern genome sciences.

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We screened rice mutants impaired in coleoptile phototropism from gamma-ray-mutagenized M₂ caryopses of cv. Nihonmasari. Until now, three genetically separable mutants, designated as cpt1, cpt2, and cpt3 (cpt: coleoptile phototropism), have been isolated. We characterized cpt1 and cpt2 in detail. The cpt1 mutant shows no coleoptile phototropism over a wide range of blue-light fluence rates. This mutant is also impaired in root phototropism. The cpt2 mutant shows a reduced phototropic response of coleoptiles; root phototropism is normal. Tip-shading experiments revealed that, in this mutant, the tip-specific light perception is selectively impaired. In the two mutants, gravitropism and growth of the two organs are normal. Therefore, the products of CPT1 and CPT2 are probably involved in the signal transduction pathway specific to phototropism. Map-based cloning of these genes is in progress. The most recently isolated cpt3 mutant is phenotypically similar to the cpt2 mutant, but additionally shows a leaf curling phenotype.

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Chloroplast positioning is dependent upon environmental light conditions. In Arabidopsis, chloroplast relocation movement is controlled by the phototropin gene family. While chloroplast accumulation movement is controlled by phot1 and phot2, chloroplast avoidance movement is mediated only by phot2, Experiments using a phot1 mutant show that this phototropin family member does not contribute to chloroplast movement.
In the fern Adiantum capillus-veneris, phototropin family genes has also been found. Mutants deficient in chloroplast avoidance movement have been isolated, and the defects traced to mutations in phot2 genes. When phot2 cDNA were introduced into the mutant cells by particle bombardment, the avoidance movement was recovered, indicating that phot2 is the photoreceptor of the response in Adiantum, as it is in Arabidopsis.
In this symposium, we will talk about the roles of phototropin family genes in chloroplast relocation movement.

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We have investigated galacturonosyl- [1] and methyltransferase [2] participating in the synthesis and modification of pectic polymers of soybean (Glycine max Merr.). We report the properties of a galactosyltransferase (GalT) involved in the synthesis of β-1,4-galactan side chains of pectins. A membrane preparation from etiolated soybean hypocotyls transferred [¹⁴C]Gal from UDP-[¹⁴C]Gal into β-1,4-galactans as exogenous acceptor, while activity to endogenous acceptor was negligible. The reaction occurred maximally at pH 6.5 and 25ºC in the presence of 25 mM Mn²⁺ and 0.75% Triton X-100. Digestion of the radiolabeled product with endo-β-1,4-galactanase released mainly radioactive β-1,4-galactobiose and Gal, indicating that the transfer of [¹⁴C]Gal occurred through β-1,4-linkages. On HPLC analysis, the enzyme also catalyzed incorporation of Gal into pyridylaminated (PA) β-1,4-galactooligomers (DP=5-7). The chain elongation of Gal₇-PA occurred and led to the formation of Gal_8-10-PA.
[1] Biosci. Biotechnol. Biochem., 65 (2001) 1519-1527
[2] Planta, 210 (2000) 782-791

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Rhamnogalacturonan II (RG-II) is a pectic polysaccharide present in the primary cell walls of all seed plants examined to date. RG-II exists in the primary wall predominantly as a dimer (dRG-II-B) that is cross linked by a 1:2 borate-diol ester. Cross-linking of pectin by this ester regulates wall porosity and thickness and is required for normal plant growth. Little is known about the occurrence of dRG-II-B in the walls of Pteridophytes and Brophytes. We have used LC-ICP-MS to determine the amounts of B and dRG-II-B in the walls of Pteridophytes (lycopods, ferns, whisk ferns, and horsetails) and Bryophytes (mosses, liverworts and hornworts). Pteridophytes and seed plants contain comparable amounts of dRG-II-B. The structures of their RG-IIs are similar but not identical. In contrast, Bryophyte walls contain about 60% less B and <1% of the amount of dRG-II-B present in the walls of seed plants.

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Intercellular attachment is an essential process in the morphogenesis of multicellular organisms. A novel mutant, nolac-H18 (non-organogenic callus with loosely attached cells), generated by T-DNA transformation using leaf-disk cultures of haploid Nicotiana plumbaginifolia, lost the ability to form tight intercellular attachments and adventitious shoots. The mutation caused defects in the glucuronic acid of rhamnogalacturonan II (RG-II) of pectin, which drastically reduced the formation of borate cross-linking of RG-II. The gene tagged with T-DNA, named NpGUT1 (glucuronyltransferase 1), which encodes a novel glucuronyltransferase, is the first glycosyltransferase gene identified in pectin biosynthesis and is essential for intercellular attachment in plant meristems and tissues (PNAS, in press). The GUS activity in transgenic tobacco plants with pNpGUT1::GUS was detected in immature embryo, cotyledon, phloem, vascular cambium, tapetum, pollen and pollen tube. These results indicate that these meristematic tissues and pollen are required for a high level of NpGUT1 expression.

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Xylem sap contains macromolecules such as proteins and polysaccharides. We have analyzed the root specific gene expression of a lectin-like xylem sap protein (XSP30) in cucumber. The expression of XSP30 in roots oscillates in a diurnal pattern. The amplitude of the diurnal expression is regulated by leaf gibberellin. In this report, we histologically investigated gene expression and lectin activity of XSP30. The XSP30 promoter directed specific expression of a glucuronidase reporter gene in xylem parenchyma and pericycle cells in the central cylinder of mature transgenic hairy roots. The lectin activity of XSP30 was analyzed by lectin blot coupled with immunological detection of XSP30. The recognition site of XSP30 was N-acetylglucosamine of the glycoproteins. We also found that the glycoproteins having the recognition site of XSP30 was abundant in leaf parenchyma cells. From these result, we will discuss about the possible functions of XSP30.

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When sporamin is expressesd in tobacco BY-2 cells, it is glycosylated at the 36th Pro with an arabinogalactan-type glycan. Taking advantage that this site is the only proline hydroxylation and glycosylation site for arabinogalactan-type glycan in sporamin, we are analyzing sequence requirement for this type glycosylation using sporamin. We have reported previously that five amino acids each before and after the glycosylation site, respectively, is indispensable for this modification. We analyzed the nature of each amiono acids in this region that are required for the glycosylation. Secretory sporamin with different amino acid substitutions were expressed in BY-2 cells and the extent of glycosylation was estimated. Mutant sporamins that were less glycosylated than wild-type were purified and analyzed by Edman degradation. Based on results of this analysis, we will discuss the sequence motifs for the Pro hydroxylation and for the glycosylation of Hyp.

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Sporamin is a vacuolar storage protein of sweet potato. This protein, when expressed in tobacco BY-2 cells, is hydroxylated at Pro³⁶ residue and further modified by O-glycosylation. In the present study, we analyzed the primary structure of the glycosyl residue motif using MALDI-TOF MS.
Sporamin was first denatured in SDS solution, and subsequently digested by 0.5 pmol of Trypsin or Endoproteinase Asp-N, respectively. The digested sporamin was directly spotted on sample plate. Both secretory mutant sporamin and vacuolar wild-type sporamin were hydroxylated and glycosylated in contrary to recombinant sporamin prepared from E. coli. Both glycosylated sporamins contained degree of polymerization up to 9 hexoses with one to three pentoses, as judged from MALDI-TOF MS results.We are currently analyzing the glycosylation pattern of several mutant sporamins that have replaced single amino acid residues around Pro³⁶ with alternative amino acids.

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Boron deficiency causes various symptoms such as growth suppression and unusual development. Recently, we reported the adapted mechanism for boron deficiency by using poplar cells (1/20-B and 0-B) grown in 5 and 0 μM boron concentration medium. In 1/20-B cell, we proposed that pectin methylesterase (PME) activity increased to cleave methyl esters of GalA residues and that newly formed free GalA residues were cross-linked by Calcium bridges. These calcium bridges would compensate for the insufficiency of borate bridging between pectic polysaccharide. In this study, we report the PME activity in normal cell (1/1-B), 1/20-B, 0-B, and 1/20-B cell transferred into MS medium containing 100 μM boron (1/20-BR). The PME activity of 1/20-B and 0-B was 1.6- and 1.5-fold higher than 1/1-B, respectively. The activity of 1/20-BR gradually decreased every subcultures to 1.2-fold of 1/1-B in 5th subculture. These results indicated that the PME activity is affected indirectly by boron supply.

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To understand how boron (B) deficiency damages plant cells, responses to B-deprivation were studied in tobacco BY-2 cells.
The genes of which expression are up-regulated in low B-tolerant cells (acclimated to a medium containing 0.05 mg B liter^-1) were screened with cDNA differential subtraction method. The low B-specific genes include putative antioxidant enzymes such as catalase, glutathione S-transferase, and a glucosyltransferase of phenylpropanoid biosynthesis pathway. This suggests that the acclimated cells have enhanced activity to tolerate oxidative conditions, and that B deficiency may cause oxidative damages to the cells. The generation of reactive oxygen species under B deficiency is now under examination.
Some of the low B-specific genes were induced within 30 minutes after the removal of B from the medium. The result indicates that the absence of B from the surrounding medium immediately affects the cellular functions.

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Monodehydroascorbate reductase (MDAR) isoforms exist in mitochondria, chloroplasts, cytosol and microbodies. Two MDAR amino acid sequences with an extended N-terminal region are found in Arabidopsis. They differ in the length of the extension by 7 amino acid residues. We have shown that these two isoforms arise from a single gene by the use of multiple transcription starts. Green fluorescent protein was fused to each extension, revealing that the longer and shorter isoforms were imported into mitochondria and chloroplasts, respectively. These results demonstrate that MDAR is a dual-targeting protein transported into mitochondria and chloroplasts. The 7 amino acid residues extend in the longer one was predicted to form a potent positively charged amphiphilic α-helix often seen in mitochondrial presequences. Although there have been several reports of dual targeting of proteins to mitochondria and chloroplasts, this is the first example in which it is achieved by the use of multiple transcription starts.

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Core promoter plays a crucial role in determining the transcription initiation site. Recent studies revealed that the diversity of its architecture is sometimes involved in the gene-specific transcriptional regulation. Generally, core promoters are composed of modules, such as TATA box and initiator element, the latter of which covers the transcription initiation site (+1), and has a weak consensus motif in mammalian genes. However, we still have limited knowledge about the transcription initiation signals operative in the plant genome. In this study, we carried out a systematic survey of the plant transcription initiation signals in in vitro transcription systems, with special reference to the tri-nucleotides covering the transcription initiation site (-1 to +2). Possible 64 combinations of the tri-nucleotides were subjected to this analysis in the nuclear extracts from both tobacco and Hela cells. The obtained results revealed the unexpected feature of the transcription initiation signals in the plant genome.

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Cystathionine γ-synthase (CGS) is the key enzyme for methionine biosynthesis. CGS mRNA stability is down regulated in response to S-adenosylmethionine (SAM). 11-13 amino acid region (MTO1 region) in the first exon 1 of the CGS gene is important for this regulation. As the exon 1 polypeptide acts in cis, it is likely that the regulation occurs during translation. This regulation has been successfully reconstituted in the in vitro translation system of wheat germ extract. In this study, GST tag-CGS exon 1 mRNA was translated in vitro and translation products were analyzed by Western blot using GST antibody. In the presence of SAM, accumulation of partial translation products was observed, which seemed to be translated up to around the MTO1 region. This result raises the possibility that translation is arrested around MTO1 region in the presence of SAM.

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Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis. We have shown that expression of the CGS gene is feedback-regulated at the level of mRNA stability in response to Met or its metabolite in Arabidopsis, and that the amino acid sequence encoded by the first exon of the CGS gene itself is responsible for the regulation. Since the exon 1 polypeptide acts in cis, we have proposed that the regulation occurs during translation when the nascent polypeptide and its mRNA are in close proximity. The in vitro translation system of wheat germ was used to analyze decay intermediates of CGS mRNA. Furthermore, from the CGS exon 1 deletion analysis of reporter activity using in vitro translation system, it has been shown that the conserved region is important for the regulation. To gain additional insight of decay intermediates of CGS mRNA, Northern blot analysis of deletion constructs is underway.

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Cystathionine γ-synthase (CGS) catalyzes a key step of methionine (Met) biosynthesis. It has been revealed that the stability of CGS mRNA stability is autoregulated by S-adenosylmethionine, a direct metabolite of Met, and that the CGS exon 1 region is necessary and sufficient for the regulation.
To identify the factors that are involved in the regulation, transgenic plants carrying the CGS exon 1 and GFP gene under the control of CaMV 35S promoter were mutagenized. 9 mutant candidates were isolated that showed high GFP fluorescence even under Met treatment. Endogenous CGS gene expression was also increased in 2 of the candidates. The fact that the expression of both the transgene and the endogenous gene are increased suggests that the candidates are involved in the CGS regulation. To exclude the possibility that the candidates are involved in Met biosynthesis, free amino acid concentrations of the candidate are now under being measured.

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Two cDNA libraries were constructed from peach fruit: young / suitable for harvest. A cDNA microarray was made using about 1,500 clones randomly picked up. Target mRNAs were purified from fruits of 10 d, 1, 2.5 and 4 months after blooming as well as leaves just after flowering time. Using these system, about one tenth of cDNA clones varying expression levels were studied. i) Down regulating: polyphenol oxidase, proteinase inhibitor etc., ii) up regulating: antimicrobial peptide homologue, invertase inhibitor etc., iii) weak in mid stage: a transcription factor. A cDNA clone belongs to group i), PF10056 encodes a protein having four times of 28 amino acids repeat motif. Although its function is not clear, expression level was very high until 2.5 months.

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A cell capturing system has been developed to isolate particular cells in the limited area of corresponding organs, and is, therefore, a strong tool for analysis on the profiles of gene expression in individual cells. In order to establish the procedure for efficient isolation of rice genes which are expressed in phloem tissues and to analyze the profiles of gene expression of phloem tissues, this system was applied for isolation of these tissues. RNA was extracted from these cells, and used for construction of a specific cDNA library. Sequence analysis of 413 randomly chosen clones from the library revealed that the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of the groups had novel components while approximately 63% were homologues to the known genes. The results of gene expression analysis of the cDNA clones are discussed.

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The activation state of transcription is thought to be related to a loosening of the nucleosome structure. These changes of nucleosome structure occur through histone modifications such as acetylation, methylation and phosphorylation. It is thought that acetylation/deacetylation of histones leads to activation/repression of transcription. Histone deacetylase (HDAC) catalyzes the deacetylation of histones. In animals and yeast, HDACs function as regulators of gene transcription and gene silencing and they also regulate life span. In this study, we identified several HDAC genes in rice and examined the organ specificity and stress responses of their expressions by RT-PCR. In rice, at least 7 HDAC genes are in class 1, 5 genes are in class 2, 2 genes are in class 3 and 2 genes are in plant-specific HD2-type. The RT-PCR analysis revealed that different HDACs are expressed in different organs and that different HDACs are expressed in response to stress.

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Germin and germin like-proteins (GLPs) genes have been isolated from many plant species, so it has been proposed that GLPs are present in all plants. GLP gene are encoded by multigene family, but their physiological functions have not been cleared. Our laboratory isolated for the first time a GLP with Mn-SOD activity outside the cell in a moss, Barbula unguiculata.
To examine molecular evolution of plant GLPs, we used a moss, Physcomitrella patens which is an excellent model for gene analysis. We identified at least 7 GLP genes from Physcomitrella EST Gen Bank. All of 7 gene products contained N-terminal signal sequences, suggesting that these proteins were targeted to the outside of the cell. We sequenced 2 GLP genes from the genome library. Molecular phylogenetic analysis based on GLP genes revealed that Physcomitrella GLP oroteins showed a divergence from those described in higher plant.

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The SCF ubiquitin ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes including flower formation, hormone and light signal transduction, and senescence in plant. The SCF ubiquitin ligase is composed of SKP1, cullin, Rbx1 and F-box protein. The genome of Arabidopsis has been shown to contain at least 19 SKP1 homologue (ASK; Arabidopsis SKP1 homologue) genes. The biochemical properties, expression, and function of the ASK genes are examined. By yeast two-hybrid analysis, ASK proteins exhibited diverse binding specificities for F-box proteins in Arabidopsis. The tissue specificity of expression of ASK genes was also varied by promoter-GUS transgenic plants. Over expression or antisense expression of ASK genes resulted in abnormal morphology in flower, leaves and stem. Our results suggest that through combinatorial arrangements between ASK proteins and F-box proteins, specific types of SCF complexes can be formed.

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F-box proteins regulate diverse cellular processes including cell cycle transition, transcriptional regulation and signal transduction by playing roles in SCF complexes or non-SCF complexes. Our database search revealed that at least 568 F-box protein genes are present in Arabidopsis genome. We found F-box proteins with LRRs, WD40 repeats, Kelch repeats, several unique functional domains and new putative domains by domain search analysis using SMART and Pfam databases. The domain analysis revealed that the Arabidopsis F-box proteins are classified into at least 19 subgroups based on their domain structures. To carry out functional analysis of F-box protein genes, 82 cDNAs of F-box protein genes were collected by RT-PCR and by searching a RAFL collection, and cloned into Entry vector of Gateway system. Using the cDNAs, tissue specific expression was analyzed by macro array analysis, and we are currently analyzing antisense transgenic plants of F-box protein genes.

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The effects of elevated UVB on the relationship between accumulation of anthocyanins and UV-absorbing compounds and levels of cyclobutane pyrimidine dimer (CPD) with leaf age in near-isogenic line (NIL) for purple leaf gene, pl of rice with a genetic background of Taichung 65 (T-65) rice, were examined. The following things were clear. First, the growth and Rubisco content in the leaf of the NIL were significantly retarded by supplementary UVB, despite it contained higher amounts of these pigments. Second, these pigments functioned effectively for reducing susceptibility to CPD induction by a challenge UVB exposure, but not by chronic UVB exposure. Third, anthocyanins functioned effectively lowering of CPD photorepair by challenge UVB exposure to blue/UV-A radiation. In conclusion, accumulation of anthocyanins and UV-absorbing compounds did not effectively function as screening against damages caused by elevated UVB in NIL.

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qUVR-10 shows the largest allelic difference among six QTLs controlling UVB-resistance, which have been detected by QTL analysis using backcross inbred lines derived from a cross between, Nipponbare (resistant) and Kasalath (sensitive). Plants with Kasalath allele at qUVR-10 were more sensitive to UVB compared with those with Nipponbare allele. Fine-scale mapping localized qUVR-10 to a 27-kb locus containing cyclobutane pyrimidine dimer (CPD) photolyase. Analysis of CPDs photorepair in the plants in which recombination was occurred in flanking region of qUVR-10 indicated that the genomic region responsible for ability of CPDs photorepair was same as that of qUVR-10. Sequence comparison between Nipponbare and Kasalath CPD photolyase gene revealed one base substitution in the conserved region of photolyase, resulting an amino acid substitution. These results strongly suggest that qUVR-10 encodes CPD photolyase and the amino acid substitution in photolyase causes reduction of photorepair capacity in the plants with Kasalath allele at qUVR-10.

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UV radiation induces two types of pyrimidine dimers between adjacent pyrimidines on the same strand: the cyclobutane pyrimidine dimer (CPD) and the pyrimidine-(6-4)-pyrimidone photodimer [(6-4)pp]. Such damage can be lethal or mutagenic to organisms. Organisms have developed mechanisms for repairing these photodamages. Photoreactivation, which is mediated by photolyase, is the main pathway in rice. We report here the photorepair of (6-4)pp in cvs UV-resistant Sasanishiki and UV-sensitive Norin 1. The fully expanded third leaves which had been irradiated with UVB were exposed to blue light, and the change in (6-4)pp was measured by ELISA. Higher photorepair of (6-4)pp was found in Sasanishiki than in Norin 1, suggesting a possibility that (6-4)pp photolyase besides CPD photolyase is involved in the different sensitivity to UVB in rice.

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The cyclobutane prymidine dimer (CPD) is one of the major UV-induced photodamage and can be reversed to two monomer pryimidines by photorepair or excision repair. Repair, which is mediated by photolyase, is the major pathway in plants. No photorepair system has been reported to operate in chloroplasts in Arabidopsis and spinach. However, it has still been open in rice. We report here the intercellular localization of CPD photolyase in rice (cv. Sasanishiki) leaves.
Each nucleus- and chloroplast-rich fraction was obtained from leaves by sucrose density gradient centrifugation. Chlorophyll content and NADP-G3P-dehydrogenase activity were used as a marker for chloroplasts. Amounts of rbcS and rbcL measured by QPCR were also applied as a marker for nuclei and chloroplasts, respectively. Higher CPD photorepair activity was observed in nucleus-rich fraction, but not chloroplast-rich fraction. It was evident that little or no CPD photolyase was involved in chloroplasts in rice.

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We have investigated the effects of elevated UVB (1 W/m²), which was provided by UVB-emitting fluorescent lamps, with cellulose diacetate film, and CO₂ atmosphere (700 ppm) on the growth and yield of rice (cv. Sasanishiki) in paddy field or in open-top-chambers in the field at Kashimadai in Miyagi Prefecture since 1993. We report the results obtained from experiments, which had been conducted since 1998. Dry mass and grain yield reduced under elevated UVB although the degree of reduction varied depending on the year. Irrespective of change in weather in each year, grain size became smaller and grain protein content, especially glutelin, significantly increased both in higher CO₂ atmosphere and in ambient CO₂ atmosphere. The ripening stage of seed was most sensitive to elevated UVB to cause grains smaller and to cause grain protein content higher throughout the life cycle of rice.

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In the present experiment, we analyzed the effect of near UV on the growth of Chlorella ellipsoidea(IAM-27). As a resullt, near UV inhibited the growth and physiological activities.
Ethylene, a kind of plant hormons, is known to regulate the growth of plants. So far, ethylene formation by Chlorella has not been reported. Herein, we detected the activities of ACC synthetase/ACC oxidase and the levels of ACC/ethylene. This evidence shows that Chlorella has the system of ethylene formation. Furthermore, these enzymes and substances were regulated to be inhibited by near UV.

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An electron spin resonance (ESR) measurement technique using a spin probe has been developed for the observation of redox states within a plant under stress conditions. Spin probes are reduced in a leaf, and reoxidized through the oxidative transition of the leaf redox states. In this study, we investigated the stress response reactions of a spin probe on purpose to estimate the plant stress-response ability.
A carbamoyl-PROXYL used generally was introduced into a radish seedling and the ESR signal changes induced by light irradiation were monitored. The signal increments in an individual leaf depended on both the light intensities and the introduced carbamoyl-PROXYL amounts. A leaf of the previously photodamaged seedling exhibited significantly large increases of the ESR signals, which corresponded the loss of chlorophyll fluorescence quantum yield. These results indicate that the oxidative-stress adaptation of an individual leaf can be determined with the ESR technique.

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High light stress suppresses expression of photosysnthesis-related genes including CAB, and activates anti-stress factors like scavengers for reactive oxygen species. We are taking a genetic stragegy in order to understand regulatory mechanisms for transcriptional responses to high light. With the aid of a in vivo monitoring system of high light response, we isolated ca. 30 mutants that show altered response to high light. The mutants were classified into two groups: ones with reduced response and the others with consitutive response.

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We are studying the function of a tobacco peroxidase tpoxN1 whose expression is induced by wounding and TMV-dependent hypersensitive reaction. We have reported that the transcript accumulates within 0.5~1h after wounding and the level was maintained for one week (Sasaki et al. 2002). The tpoxN1 gene responded to wounding most sensitively in vascular systems especially in stems, but weakly in leaf blades without veins. Here we generated transgenic tobacco plants overexpressing the tpoxN1. Transgenic plants seemed to exhibit dwarf phenotype. After cutting of stem, the stem sections were incubated at room temperature, and then the weight was measured at various time periods. Decrease in the weight of stems from transgenic plants by drying was significantly smaller than that from wild-type tobacco plants, indicating the possibility that overexpression of tpoxN1 enhance wound-repairing to recover negative xylem pressure for plant survival.

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WIPK (wound-induced protein kinase) and WIZZ (wound-induced leucine zipper zinc finger) encode a MAP kinase and a WRKY transcription factor, respectively. Their transcripts are induced within several minutes after wounding, and encoded proteins are thought to modulate gene expression involved in wound-healing and/or defense. To determine the regulation mechanism of WIPK and WIZZ expression, we isolated corresponding promoter region of each gene from tobacco, and fused to reporter genes. Histochemical analysis of resulting transgenic lines showed that WIPK and WIZZ were localized near the sites of wounding, and along the vascular systems, respectively. Deletion analysis showed that a 430bp of WIPK, and a 290bp of WIZZ promoter regions were sufficient for wound response. In addition to wounding, WIPK transcripts were induced during hypersensitive response (HR) after TMV infection. Promoter analysis indicated that a 430bp promoter region was sufficient for HR, and that WIPK protein was predominantly accumulated around HR lesions.

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Multiprotein bridging factor 1 (MBF1) had been identified as a transcriptional coactivator that bridged between the TATA-box binding protein (TBP) and the DNA binding regulatory factor BmFTZ-F1 in silkworm. MBF1 homologs are conserved from yeast to human. However, it is largely unknown about the function of plant MBF1 homologs.
By differential display approach, we isolated independent 38 cDNA clones that were specifically expressed in calli derived from roots of Arabidopsis thaliana. One of them was a MBF1 homolog. We therefore designated it AtMBF1a. Furthermore, additional two MBF1 homologs, AtMBF1b and AtMBF1c, were found in the A. thaliana genome.
In this report, we analyzed the expression patterns of these three genes in Arabidopsis. Northern blot analysis revealed that these were expressed in various organs, especially in flowers. Furthermore, their promoter-GUS transgenic plants were constructed and these histochemical analysis suggested that AtMBF1c was involved in response to wounding.

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The rice virescent mutants (v₁, v₃) are temperature-conditional and develop chloroplast-deficient leaves under the restrictive temperature. We previously speculated that the virescent genes function at an early stage of leaf development (P4), and are involved in the regulation of chloroplast transcriptional activity. Western blot analysis indicated that in wild-type leaves proteins for plastid transcriptional machinery, including NEP (RpoTp), PEP (RpoA) and the sigma factor (SigA), specifically accumulated at the P4 stages. However, accumulations of these proteins were severely suppressed in the leaves of v₁ and v₃ mutants. In such mutant leaves, chloroplast proteins encoded in the nucleus (LHCP, RbcS), as well as those encoded in the plastid, barely accumulated at P5 stages. These results suggest that the virescent genes function in the expressions of NEP and PEP, and also suggest that the NEP activation at P4 stage is important for the expression nuclear-encoded genes for chloroplast proteins.

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The rice virescent-2 mutant (v₂) is temperature-conditional and develops chlorotic leaves under the restrictive temperature. Previous studies speculated that the V₂ gene functions at an early stage of leaf development (P4), and is involved in the regulation of expression of the plastid-encoded genes. We isolated the V₂ gene using a map-based cloning approach. V₂ gene exhibited an extensive sequence similarity to the genes encoding guanylate kinase (GUK), an essential enzyme in the biosynthesis of nucleotides. We confirmed that the V₂ gene (designated ogkA) complements the yeast guk1 mutation. RT-PCR analysis showed that the ogkA transcripts highly accumulated in the immature leaves at stages from P1 to P4. These results suggest that the functional activation of OgkA, a part of nucleic acid metabolism, is a key process in the early chloroplast biogenesis, and this process may be involved in the construction of chloroplast genetic system.

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We isolated two chloroplast morphological mutants from a mutant library generated by EMS mutagenesis. One line showed variegated leaf phenotype. Electron microscopic observation indicated that the chloroplasts in the green sector have similar structure to that of wild type, but the plastids in the white sector don't have well developed thylakoid membranes. The abnormalities may be due to a lack of some component that is important in the development or maintenance system of the chloroplast. Another line showed that size and shape of chloroplasts were irregular. This mutant contained expanded chloroplasts up to twice in diameter compared with those of wild type, and tiny chloroplasts in the same cells. The machinery controlling correct division of chloroplasts might be missing. Electron microscopic observation revealed further interesting features. Arrangement and direction of thylakoid membranes were disordered and the stromal area was abnormally expanded. The regulatory mechanism for membrane arrangement might be mutated.

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Gene expression of chloroplast is dependent on a large numbers of nuclear-encoded factors. To determine such nuclear-encoded factors for chloroplast gene expression, we isolated high chlorophyll fluorescence mutants of Arabidopsis. Among 59 mutants, a 2627-2 had a T-DNA insertion in a gene encoding an RNase R family member with a putative targeting signal to chloroplasts. Our RNA blot analysis showed that mutant 2627-2 had an impaired processing of ribosomal, and the significant reduction in ribosomal RNA in chloroplasts, suggesting the RNase functions in the maturation of chloroplast ribosomal RNA. The levels of some chloroplasts RNA were higher in 2627-2 than in the wild type, which is possibly due to the transcription rate regulated by nuclear-encoded RNA polymerase as secondary effects of the reduced rRNA levels.

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The chloroplast nucleoid is a complex of chloroplast DNA (cpDNA) and various proteins. Sulfite reductase (SiR) is one of the major proteins of chloroplast nucleoid, which regulates the transcriptional activity of chloroplast nucleoid through changes in compaction of cpDNA. In addition, SiR is known to be a ferredoxin-dependent enzyme catalyzing the reduction of sulfite to sulfide. We analyzed quantitative and qualitative differences in SiR of plastid nucleoid during the greening of pea.
Nucleoids were isolated from pea etioplasts, and SiR was detected by immuno blot analysis. The content of SiR in etioplast nucleoids differed from that in chloroplast nucleoid. Two-dimensional electrophoresis and immuno blot analysis showed that isoelectric point of SiR was different in chloroplast subfractions. Currently, we are investigating the modification of SiR protein.

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Plastid nucleoid is a complex of platid DNA and various proteins, which changes morphologically and molecularly during the development of pea chloroplast. We reported that the 70-kDa protein, which is a major constituent of chloroplast nucleoid, was identified as sulfite reductase (SiR), and this protein has an ability to compact DNA. Tightening of DNA compaction by addition of recombinant SiR and its relaxation owing to release of SiR by heparin were confirmed. In vitro transcription assay using isolated nucleoids showed that the DNA compaction by SiR repressed, while DNA relaxation activated transcriptional activity. We will also present data on the DNA binding of SiR. A SiR-GFP fusion protein was found to localize in both stroma and nucleoids. We are attempting to identify DNA-binding domain of SiR.

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PEND protein is a DNA-binding protein in the plastid envelope membrane of pea. A putative protein (ATF4F15.280) in the Arabidopsis thaliana chromosome 3 and the GSBF1 protein of Brassica napus were found to be PEND homologues. We isolated additional PEND homologues from Brassica napus, Prunus yedoensis and Cucumis sativus. In addition, tomato and wheat cDNAs encoding PEND homologues were found. PEND protein is therefore present widely in angiosperms. Full length PEND-GFP fusion protein was localized to the chloroplasts. The constructs without the N-terminus 15 amino acids was targeted to the nucleus. This was confirmed by immunoblot analysis. We previously reported that the cbZIP-GFP fusion protein was localized in the chloroplasts and also targeted to the nucleus in 50% of transformed cells in a transient expression system. Essentially similar results were obtained for A. thaliana and B. napus homologues. We will also report on the results with stable transformed plants.

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The unicellular red alga Cyanidioschyzon merolae contains a single cell nucleus, a plastid and a mitochondrion, and is thought to be useful in investigation of evolution of plastid in the lineage other than green plants. We isolated plastid nucleoids from C. merolae, and analyzed the basic properties of in vitro transcription. Rifampicin that inhibit transcription of bacterial RNA polymerase have different effects on the nucleoids of C. merolae, green plants and cyanobacteria.
A homologue of bacterial histone-like protein HU is present in the plastid nucleoids of C. merolae, and this protein has ability to compact DNA, while sulfite reductase compacts DNA in the nucleoids of pea plastids. The in vitro transcription activity of plastid nucleoids of C. merolae was activated by the addition of recombinant HU, but the transcription in pea nucleoids was decreased. Nucleoid proteins have different effects in different lineages.

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PpRPOT1 and PpRPOT2 in Physcomitrella patens have two Met residues in N-terminus. When a construct in which GFP is fused to the 5'-UTR and N-terminal sequence containing two Met (M1 and M48) of PpRPOT1 was introduced into the moss protoplasts, the fluorescence of GFP was localized to mitochondria. GFP was localized to plastid when the translation was forced to start at M1 using a translation leader sequence of rbcS 3A in pea, whereas it was localized to mitochondria when the translation was forced to start at M48. We examined the efficiency of translation from the 5'-upstream sequence of M1 and M48 using GUS fusions. The results indicated that the translation starts exclusively at M48. Therefore, in natural context of PpRPOT1 gene, translation starts at M48 and the product is targeted to mitochondria. This is consistent with the tagetitoxin sensitivity of transcription in isolated plastids and mitochondria.

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