Ferritin is a class of iron storage proteins. In the present study, two full-length cDNA clone, NtFer1 and NtFer2, encoding ferritin from cDNA library of 3-old tobacco seeding, constructed in the lambda TriplEx2 vector have been isolated and characterized. NtFer1 and NtFer2, were 1214 and 1125 nucleotides long encoding 251 and 259 amino acid residues, respectively. The mature subunit encoded by NtFer1 shares 78.3% and 70.6% homology with two soybean ferritin subunits, while NtFer2 shares 71.7% and 68.4%. Southern blot indicated that NtFer1 was encoded by a two-copy gene and NtFer2 was encoded by a single-copy gene in the tobacco genome. Northern blot analysis showed that NtFer1 and NtFer2 were expressed in leaves as well as in roots, and NtFer1 was well responsive to excess iron then NtFer2. The abundance of NtFer1 mRNA was increased in leaves by exogenous ferritin gene everexpressing, while the abundance of NtFer2 mRNA was stably.

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Ferritin is a multimeric iron storage protein. We presented that ferritin in the soybean seed contained two types of subunits, H-1 and H-2. The C-terminal domain of H-1 was cleaved and degraded completely, whereas the H-2, which we newly identified, was not subject to such cleavage in vitro. To examine the stability and capacity in iron storage of them, we use two types of transgenic tobacco, which express the H-1 and H-2 ferritin, respectively.
The iron content of the H-1 transgenic tobacco is higher than that of the H-1 when they grow in iron rich conditions. The stability assay is performed by the western blot using the antibody which recognize both H-1 and H-2 subunit. As the result, H-2 subunit was stable than the case of H-1 subunit. These results suggest that the H-2 subunit can be retained more stably and store iron efficienctly in the transgenic tobacco.

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Nicotianamine (NA) is a biosynthetic precursor to mugineic acid family phytosiderophores (MAs) and thus nicotianamine synthase (NAS) is a critical component in the iron acquisition strategy of graminaceous plants. NA, which is structurally similar to MAs and is also a strong chelator of various transition metals, is ubiquitously present in higher plants. Therefore, it is thought that NA is a key component in transition metal homeostasis in non-graminaceous plants. We isolated three NAS genes (AtNAS1, 2 and 3) from Arabidopsis. To elucidate the physiological functions of the three AtNAS genes in Arabidopsis, we examined the expression of AtNAS1, AtNAS2 and AtNAS3 by promoter:β-glucuronidase analysis and quantitative RT-PCR. Expression of AtNAS1 and AtNAS2 were induced by iron deficiency. In contrast, expression of AtNAS3 was suppressed by iron deficiency. Therefore, three NAS genes were differentially regulated by iron in Arabidopsis.

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Serine acetyltransferase (SATase) catalyzes the formation of O-acetylserine (OAS) from L-serine and acetyl-CoA, connecting serine metabolism to cysteine biosynthesis. SATase and OAS are proposed to be regulatory factors in the biosynthesis of cysteine in plants. In the genome of Arabidopsis thaliana, there are five predicted SATase-like genes. To elucidate the temporal and spatial expression patterns of the different putative SATase isoforms (SAT-p, -c, -m, -#5, and -106) during the development of wild type A. thaliana, real-time quantitative PCR was performed. The results show that SATases mRNAs have distinct expression levels between aerial and root regions during plant development.
A. thaliana plants carrying fusions of SATase promoter region and green fluorescent protein (GFP) were produced to verify SAT-m, -c, and -p tissue-specific expressions. The result showed similar tissue-specific expressions of SAT-p and SAT-c, differing with SAT-m that is expressed ubiquitously.

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Serine acetyltransferase (SATase) catalyzes the formation of O-acetyl-L-serine (OAS) which is the essential precursor of cysteine biosynthesis. There are five SATase isozyme genes on Arabidopsis genome. The activity of cytoplasmic SATase (SAT-c) is highly sensitive to the feedback inhibition by cysteine, therefore, SAT-c seems to play an important role in sulfur assimilation in Arabidopsis. To clarify the role of SAT-c in sulfur assimilation pathway, we first identified T-DNA insertion knock-out mutant in SAT-c gene (KOC). The content of cysteine in KOC was reduced by approximately 50% as compared to that of wild-type Arabidopsis, although no significant changes in OAS and glutathione contents were observed in KOC plant. We are currently dealing with the DNA array analysis and comprehensive non-targeted metabolome analysis by FT-MS to investigate the impact of SAT-c mutation.

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Acylamino acid-releasing enzyme (AARE) catalyzes the N-terminal hydrolysis ofN-acylpeptides to release N-acylated amino acid. In the genome sequence from Arabidopsis thaliana,a gene that was homologous to the mammalian gene for AARE was found by BLAST search. The gene was amplified by RT-PCR and subcloned into an expression vector, pQE32. Cloned cDNA coded 694 amino acids, and this amino acid sequence showed 28% identity with those of AAREs from mammalians. Especially, the proposed active residues (Ser, Asp, and His) of AARE, called "catalytic triad residues" were completely conserved. Overexpressed AARE protein in E.coli protein showed AARE activity, and it was similar to those of native AARE prepared from cucumber and Arabidopsis leaves. Furthermore, native AARE degraded glycated riblose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) but not native Rubisco. These results indicate that plants have AARE, and plant AARE might have roles in elimination of glycated proteins as well as mammalian AAREs.

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During the purification of Clp protease from chloroplast stroma of spinach, we found other enzyme that degrades Suc-Leu-Tyr-MCA, the substrate of Clp protease. This enzyme was purified by five steps of chromatography and molecular weight was estimated as 35,000. The N-terminal sequence of this protein was determined to be AVSLSPP and this sequence completely agreed with that of O-Acetylserine (thiol) lyase (OAS lyase) derived from the spinach chloroplast. OAS lyase is the last enzyme in the cysteine biosynthetic pathway. This enzyme distributes in cytosol, mitochondria, and chloroplast. The enzyme had an absorption peak at 407 nm due to the presence of pyridoxal phosphate, which is a cofactor of OAS lyase. Our purified enzyme also showed the absorption peak at 407 nm. From these results, it is likely to conclude that the purified enzyme that degrades Suc-Leu-Tyr-MCA is OAS lyase. Kinetical analysis of the enzyme is now in progress.

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Chlamydomonas reinhardtii has two matrix metalloproteases (MMP), Mmp1 and Mmp2. The Mmp1 is named gametolysin, which is responsible for lysis of gametic cell walls during mating, and the Mmp2 appears to function at the zygotic stage. In this study, we identified the third MMP, Mmp3 from the Chlamydomonas EST library and analyzed the structure and function at the nucleotide and protein levels.
The ORF of Mmp3 cDNA codes for 646 aa, shows 70 and 59% identity with Mmp1 and Mmp2, and contains the hallmark motif characteristic of metzinsin-type MMP. Northern blot analysis suggests that Mmp3 is expressed in vegetative cells, especially during G₁ phase. Two antibodies raised against N-terminal and C-terminal regions of the mature Mmp3 recognized a common 65-kDa protein, which was detected significantly at the G₁ phase of cell cycle. It is therefore possible that Mmp3 is related to lysis and/or loosening of cell wall for proliferation.

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UDP-D-galacturonate, the precursor for the galacturonic acid residues in pectic polysaccharides, is synthesized through the activity of UDP-D-glucuronate 4-epimerase. The enzyme from pea or radish has been reported to be associated with membranes. A BLAST search with the protein sequence of Streptococcus pneumoniae UDP-D-glucuronate 4-epimerase identified six putative isoenzymes in Arabidopsis. The predicted cytoplasmic domain of one isoform (At4g30440) was expressed in Escherichia coli. The recombinant protein catalyzed the conversion of UDP-D-glucuronate into UDP-D-galacturonate, confirming that the gene encodes UDP-D-galacturonate in Arabidopsis. The subcellular localization of the enzyme is now under investigation using the antibody raised against the recombinant protein.

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Synechocystis sp. PCC6803 extracellularly produced a hemolysin homolog (gene product of sll1951) by culturing in BG-11 medium supplemented with 3 μM CuSO₄. No hemolytic activity was found in the culture medium. The protein was purified by DEAE-cellulose and Superose 6 chromatography. On the latter, the protein was eluted at the position of 560 kDa. When this value is compared with the molecular weight of the monomer, deduced from the DNA sequence, 178 kDa, the protein is likely to form a trimer. The mobility of the protein upon SDS-PAGE largely and reversibly changed by heat: 84-98 kDa when not heated and 224 kDa when heated. This mobility change suggests that the hemolysin homolog underwent a reversible structural change by heat, and is likely to be due to the Ca-binding β-roll motifs in the protein molecule. The Ca content in the protein is now being measured.

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In Arabidopsis, phosphatidylcholine (PC) is thought to be synthesized via the so-called nucleotide pathway, in which two isogenes for CTP:phosphorylcholine cytidylyltransferase, AtCCT1 (At2g32260) and AtCCT2 (At4g15130), play a regulatory role. We recently reported that PC accumulation in the rosettes of Arabidopsis at low temperature could be supported by selective enhancement of the expression of AtCCT2 (Inatsugi et al. (2002) Plant Cell Physiol. 43:1342-1350). To dissect further the differential roles of AtCCT isogenes, we isolated T-DNA-tagged mutants of AtCCT1 and AtCCT2 and designated cct1 and cct2, respectively. Construction of a cct1 cct2 double mutant that exhibited < 2% of the wild-type CCT activity in the rosettes revealed that such a low level of CCT activity is sufficient for normal growth and normal lipid composition. Alternatively, it is suggested that Arabidopsis contains uncharacterized pathways for PC biosynthesis that could compensate for the defect in the nucleotide pathway.

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Cold acclimation induces lipid changes in plant cells. Especially, the increase in the content of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) has attracted our major interest in understanding the lipid function at low temperature. We recently reported that selective enhancement of the expression of AtCCT2 (At4g15130), one of the two isogenes for Arabidopsis CTP:phosphorylcholine cytidylyltransferase, could account for the biosynthesis of PC in the rosettes of Arabidopsis at low temperature (Inatsugi et al. (2002) Plant Cell Physiol. 43:1342-1350). We herein found that accumulation of PE in Arabidopsis at low temperature started a few days later than that of PC and that the delayed PE accumulation could be accounted for by delayed enhancement of the transcript levels of AtECT1 (At2g38670) encoding Arabidopsis CTP:phosphorylethanolamine cytidylyltransferase. The result clearly demonstrates that Arabidopsis has evolved different modes of gene expression in the biosyntheses of PE and PC (Supported in part by a grant form PROBRAIN).

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Many temperate plants, such as temperate grasses, synthesize fructan that is considered to play an important role in the response to environmental stresses as an osmoregulation factor. Perennial ryegrass is one of the most important temperate grasses. Perennial ryegrass, however, is sensitive to environmental stresses in comparison with other temperate grasses. To improve cold tolerance of perennial ryegrass, we have made transgenic plants carrying wft1 and wft2¹⁾, which encode wheat sucrose-fructan 6-fructosyltransferase and sucrose-sucrose 1-fructosyltransferase, respectively.
Of 16 transgenic plants analyzed, two plants containing wft1 and three plants containing wft2 showed significant increase in fructan accumulation compared with non-transgenic plants. Freezing tolerance of transgenic plants were tested by electrical conductivity method. Some transgenic plants were more tolerant to cold stress than non-transgenic plants, suggesting that an increase in fructan accumulation might be effective in enhancing cold tolerance in perennial ryegrass.
¹⁾Kawakami and Yoshida, Biosci, Biotechnol. Biochem. 66, 2297-2305 (2002)

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Increase in freezing tolerance of temperate plants during cold acclimation is associated with accumulation of compatible solutes. To understand the cryoprotective mechanisms of compatible solutes in cold acclimation process, we initiated to determine the subcellular localization of compatible solutes in wheat and its changes during cold acclimation after leaves were fractionated using a nonaqueous fractionation technique. The results revealed that (1) sugars are the most accumulated compatible solutes in all subcellular compartments both before and after cold acclimation, (2) the total amount of compatible solutes is greatest in cytosol after cold acclimation, and (3) all of mono- and disaccharides, proline and glycinebetaine are accumulated most in cytosol after cold acclimation. With the volume of each organelle determined using electron microscopy, these results will be discussed in terms of relationship between changes in freezing tolerance and subcellular compatible solute concentrations in each organelle. (This study is in part supported by BRAIN.)

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A cDNA microarray was utilized to screen for clones that are induced by chilling stress in rice roots. Two cDNAs clones induced at 12oC and involved in trehalose biosynthesis, TPS and TPP, were identified. Full-length cDNA clones were isolated through library screening. Southern analysis indicates, OsTPP1 is a single copy gene while OsTPS1 may have similar genes. Northern blotting showed that both clones were rapidly and transiently induced within 1-4h and the signals disappeared after 6-10h of chilling stress. Fructose and glucose levels in cold-treated rice roots consistently increased after a lag of 5h. Sucrose levels consistently increased during the 24h of chilling treatment. Concurrent increase of trehalose levels was apparently faster and earlier than the other sugars, and followed the patterns of OsTPP and OsTPS mRNAs accumulation. It appears that trehalose biosynthesis is transiently induced and maybe involve in the regulation of sugar metabolism in chilling-stressed roots in rice.

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Cold-stress treated and normally grown rice seedlings were compared by differential screening method. One of the differential clones was characterized as a partial S-adenosylmethionine decarboxylase (SAMDC) cDNA by sequencing analysis and homology search. Two full-length cDNA clones, OsSAMDC1 and OsSAMDC2, were isolated from rice cDNA library with the differential clone as probe. Deduced amino acid sequences of OsSAMDC1 and OsSAMDC2 are identical with those of rice YO7766- and AJ251899-SAMDC released in GenBank databases, respectively. Southern blot analysis with 3'-UTR gene-specific probes indicated that a single-copy of each OsSAMDC gene is present in Yukihikari rice genome. Northern blot analysis with 3'-UTR probes showed that OsSAMDC1 mRNA level is increased in response to cold stress, while OsSAMDC2 mRNA level is increased in response to cold, salt as well as drought stresses. Results on the analysis of polyamines in transgenic rice introduced with OsSAMDC1 was also discussed.

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Transgenic Arabidopsis overexpressing spermidine synthase (SPDS) genes under the control of 35S CaMV promoter exhibits increased tolerance to environmental stress over the wild-type plants. Since stress injury of plants often correlates with excess generation of reactive oxygen species, the antioxidant enzyme activities in chilled leaves were compared between the transgenic and wild-type plants. Chilling the plants at 5C in a moderate light caused a reduction in Fv/Fm and an increase in H₂O₂ in leaves, both of which were less pronounced in the transgenic plants. During chilling, SOD and APX activities increased over time particularly in the transgenic plants. An SPDS inhibitor applied to the transgenic plants suppressed the chill-induced increase of SOD activity while spermidine application to the wild-type plants enhanced it. The results suggest that high cellular levels of spermidine may promote the induction of antioxidant enzyme activities in chill-stressed leaves.

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In northern part of Japan, rice crop production is occasionally damaged severely by low temperature in summer due to the disturbance in pollen formation. Using cDNA microarray system, we analyzed rice anthers cooled at the young microspore stage, which is the most cool temperature-sensitive stage, in order to identify and characterize genes responsive to cool temperature and are involved in cool temperature damage process in rice pollen formation. We found that many genes are up- or down-regulated by cool temperature in rice anther, and among these, a gene for jasmonate biosynthesis were significantly up-regulated and a gene for polyamine biosynthesis were significantly down-regulated, respectively. We will report the gene organization and expression pattern under cool temperature stress in pollen development stage.

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Freezing resistance of birch (Betula platyphylla L.) xylem tissue is induced during seasonal cold acclimation with the development of supercooling ability. Both intracellular and extracellular components may contribute to the supercooling ability of winter xylem tissue. In this study, identification of winter-induced extracellular proteins (WCWPs) was examined to study the physiological roles of WCWPs in the freezing resistance mechanism of birch xylem tissue.
Extracellular proteins were eluted with acidic solution from xylem tissues of birch twigs harvested in the field. SDS-PAGE analysis showed that the extracellular protein composition changed drastically after seasonal cold acclimation. Levels of proteins with molecular masses of about 30 kDa, namely WCWPs, increased markedly in winter. The N-terminal amino acid sequencing revealed that some of WCWPs are similar to each other and are homologous to a pathogenesis-related protein. Immunoblot analysis also showed the similarity between the WCWPs.

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During seasonal cold acclimation, 18 kD proteins, designated WAP18, were accumulated in the soluble protein fractions in mulberry (Morus bombycis Koidz.) tree. The purified WAP18 showed cryoprotective activity against freeze-labile lactate-dehydrogenase, LDH, suggesting that WAP18 has roles to contribute freezing tolerance in cortical parenchyma cells of mulberry tree in winter. The cDNA cloning revealed that WAP18 has high sequence similarity to pathogenesis-related (PR)-10/Bet v 1 protein family. Northern blot analysis showed that transcript levels of WAP18 increased not only by cold treatment at 4^oC but also by wounding, ethephon and salicylic acid.
It has been predicted that PR-10/Bet v 1 proteins localize in the cytosol based on the absence of apparent signal sequence. Immuno-electronmicroscpe analysis showed that gold particles against anti-WAP18 antibodies localized not only in cytosol but also in nucleus in mulberry tree. This result indicates that PR-10/Betv1 proteins localize in cytosol and nucleus.

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Rice plant is quite sensitive to low temperature at its seedling stage. Bleaching (discoloration) of the leaves is one of the typical symptom of chilling stress. This is mainly caused by the generation of active oxygen species (AOS) in chloroplasts. Thus, reinforcement of the scavenging system of AOS by introducing genes coding for stable scavenging enzymes would be a good remedy for the improvement of the chilling-stress tolerance of rice plants. From this point of view, we introduced the plastidal ascorbate peroxidase gene (apx) from Chlamydomonas sp. W80 into rice plants. Transgenic plants had 2-5 fold higher APX activity. These transgenic lines were more tolerant to 10 microM of methylviologen. Improvement of the tolerance to low temperature-stress of these transgenic plants was also confirmed.

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Cold deacclimation (DA) is a phenomenon that is genetically controlled to lose the degree of freezing resistance acquired during cold acclimation (CA) and is triggered by spring warmth. DA is also accompanied by a restart of plant growth arrested during CA. We hence set out to establish an experimental system of Arabidopsis for studying DA at molecular level, which may enhance our understanding of the regulation of freezing resistance and growth. We demonstrated that an application of the protein synthesis inhibitor cycloheximide at the onset of DA treatment prevented significantly the subsequent loss of freezing resistance, suggesting that novel gene expression is required for the development of DA. We therefore conducted macroarray and differential display analyses to list up genes that showed altered transcript levels during DA. These results provide us with a future guideline for studying DA at molecular level. (Supported in part by a grant from PROBRAIN.)

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Rice is a typical chilling sensitive plant. Cell suspension cultures of rice also suffer injuries at chilling temperatures. However, we found out that exogenous application of ABA induces freezing tolerance in rice cell cultures in a similar manner as in other cell cultures of Gramineae plants that have genetically cold hardiness. We analyzed physiological factors involved and cytosolic proteins expressed during ABA induced freezing tolerance by 2D electrophoresis.
At the optimum conditions, the rice cells tolerated -12C and some cells even -20C after 7 days of incubation in the presence of 75 μM ABA at 25C. More than 80 CBB-stainable protein spots were either increased or induced by ABA treatment. Some of the major ABA responsive spots were detected as early as 6h of incubation with ABA containing medium while after one day incubation most ABA responsive spots were detected when the rice cells tolerated moderate freezing.

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Freezing tolerance can be induced in bromegrass (Bromus inermis Leyss) suspension cultures by exposure to low temperature or by exogenous application of abscisic acid (ABA). To understand the mechanisms of freezing tolerance in bromegrass, we analyzed the genes responsive to low temperature and ABA using rice cDNA microarray.
Ten genes induced by cold and ABA were cloned and sequenced. One of them has homology to a chitinase reported as an antifreeze protein in cold-hardened winter rye plants.
At present, we are constructing transgenic bromegrass in an attempt to identify contribution of the chitinase to antifreeze activity detected in the culture medium of bromegrass exposed to low temperature.

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Plastids perform a number of essential biochemical functions. Since most of the proteins are encoded in nucleus, they are synthesized as precursor proteins and are imported into plastids by preprotein translocases. The translocon at the outer membrane of chloroplasts (Toc complex) and the translocon inner membrane of chloroplasts (Tic complex) act co-operatively during the import process. Although putative components of the import apparatus have been identified biochemically using isolated pea (Pisum sativum) chloroplasts, their role in import remains to be proven in vivo. With the completion of the Arabidopsis genome sequencing project, it is now possible to identify putative homologs of the import components in this species. Arabidopsis mutants lacking one of the Tic component has been isolated. The content of chloroplast pigments were reduced proportionally. Additionally, morphological and biochemical data demonstrate that the effects on leaf development by the translocon mutation.

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The chloroplast characteristic of plants has photosynthetic activity and develops in restricted organs such as leaves and stems. To reveal the mechanisms of building up chloroplasts in an organ-specific manner, activation tagging was applied to a parental line transformed with hygromycin B hopsphotransferase gene under the control of RBCS-3B promoter, resulting in selection of ces (callus expression of RBCS) mutants. The calli of ces101 were green, where transcripts for RBCS were approximately 700 times higher and those for SIG1 encoding σ_factor responsible for transcription of chloroplast photosynthesis genes were also higher in comparison with those in the parental line. A receptor-like kinase gene closed to the integrated T-DNA was driven by the enhancer sequences in transformants, which exhibited the same phenotype as ces101 line, leading the conclusion that the CES101 is the receptor-like kinase. We would also report results of the analysis of ces102 line.

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The heat shock protein 70 (Hsp70) molecular chaperone is known to be involved in protein folding and protein translocation across membranes in an ATP-dependent fashion. GrpE, an essential co-chaperone which stimulates ADP/ATP exchange on Hsp70, has been conserved in various bacteria and in mitochondria of eukaryotes. Although higher plant chloroplasts also have been known to possess Hsp70 molecular chaperone system, detailed characterization of co-chaperone proteins acting with chloroplast Hsp70 has not yet been achieved. By analyzing genomic and EST databases of Arabidopsis thaliana, we found two candidate genes potentially encoding chloroplast homologs of GrpE which contained an amino-terminal transit peptide-like domain for chloroplast localization and a mature domain which closely resembled to the cyanobacterial GrpE. Both GrpE homologs are actually localized in the chloroplast stroma evenly and form a protein complex with chloroplast Hsp70. Analysis of knockout mutant and functional cooperation of two chloroplast GrpE are under investigation.

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RubisCO of higher plants is a hexadecamer of eight large subunits (LSU) and eight small subunits (SSU). RubisCO is quickly and abundantly synthesized in the leaves during greening. Moreover, it is disintegrated in senescence and stress. The details of the mechanism of its biosynthesis and degradation are still unclear. In this study, we analyzed the composition of RubisCO subunits of the extracts of greening radish cotyledons. 2D-PAGE revealed that the insoluble fraction obtained by centrifugation of the leaf extracts contained SSU without LSU. The amount of the insoluble SSU decreased during the progression of greening. We reason that there may be an imbalance in the rates of synthesis of LSU and SSU during greening. This study was partly supported by the Research Association for Biotechnology subsidized by the Ministry of Economy, Trade and Industry of Japan.

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We transposed Dissociation elements from 3 start loci on chromosome 5 and 4 loci on chromosome 1 in Arabidopsis thaliana and made 9425 Ds-tagged lines of F3 plants carrying independent transposition events. To study function of nuclear genes involved in chloroplast development and photosynthesis, we have screened 9425 lines to isolate 87 mutants with albino or pale green (apg) phenotypes. Forty-two albino phenotypes out of 87 mutants were tagged with Ds insertions. We estimated that 50 % mutants from Ds transposon-mutagenized population are tagged with Ds. Our Ds tagging population is more efficient for gene isolation than T-DNA tagging populations. Twenty-one of the apg mutants were simple albino, 15 were shown pale green, 4 were white cotyledon and 3 were variegated. In 40 apg mutants, Ds element was inserted into protein-coding genes. Twenty-two genes in the 40 mutants have sequence homology with reported proteins whereas 18 are unknown genes.

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Regulation of cytoplasmic male sterility (CMS) by fertility restorer (Rf) genes is one of the suitable systems for molecular-genetic analysis of the interactions between mitochondria and nuclear genome. In Kosena CMS, presence of a CMS-associated gene, orf125, in the cytoplasm and the accumulation of ORF125 protein are strongly associated with the CMS phenotype. The fertility restoration was accompanied by a decrease in the amount of the mitochondial protein. To understand the molecular mechanisms by which the Rf gene acts on the expression of orf125, the gene was cloned by a positional cloning strategy. The cloned gene, orf687, encodes a protein that comprises sixteen repeats of pentatricopeptide repeat motif and a putative mitochondrial target sequence. Analysis of the temporal and developmental expression of orf687 and its homologues are in progress. Based upon these results, we will discuss possible mechanism of orf687 gene on the expression of the CMS gene.

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It is widely believed that chloroplast was generated by endosymbiosis of ancient photosynthetic bacterium into ancient eukaryotic cell. arc3 (Accumulation and Replication of Chloroplast) mutant of Arabidopsis thaliana has a few abnormally large chloroplasts in cell. Fine mapping analyses showed ARC3 gene is a fusion gene of prokaryotic, FtsZ, and eukaryotic, phosphatidylinositol-4-phosphate 5-kinase (PIP5K), genes. The fusion protein of the full-length ARC3 and GFP (Green fluorescent protein) was located exclusively in chloroplast. The PIP5K activity of ARC3 protein was not detected. The ARC3 protein, however is phosphorylated by A. thaliana protein in vitro. These results show that ARC3 is a functional prokaryotic and eukaryotic fusion protein, and is a linkage point in the signal transduction by which eukaryotic nucleus regulates prokaryotic chloroplast division.

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Two types of ftsZ genes (MpftsZ1 and MpftsZ2) were isolated from the liverwort Marchantia polymorpha using degenerate RT-PCR. Northern analysis confirmed that both genes are active. Transformation of a sense MpftsZ2 overexpression construct into M. polymorpha produced a large chloroplast phenotype in one transgenic plant. The epidermal cells of wild-type plants contained a mean ± SD of 38.2 ± 21.4 chloroplasts, whereas the chloroplast number was 7.4 ± 4.4 in the transgenic plant. Southern analysis showed that the CaMV 35S promoter-MpftsZ2 construct was inserted in at least three positions. Northern analysis suggested that the high accumulation of MpftsZ2 mRNA blocked plastid division. Determination of the chlorophyll content and chlorophyll fluorescence parameters suggested that the macrochloroplasts function like chloroplasts in wild-type plants under normal light conditions. However, the transgenic plant grew more slowly than did wild-type plants.

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Previously, we characterized an Arabidopsis dynamin-like protein, ADL2b, that functions in the division of plant mitochondria. The Arabidopsis genome has another highly homologous gene, ADL2a, which has been reported to localize in chloroplasts. To confirm the localizations of ADL2a and ADL2b, we constructed GFP fusion proteins with the N-terminal portions of these proteins. However, in our experiment, no signals were detected in chloroplasts. When the full length of ADL2a was attached to GFP, a strong GFP signal was localized at the tips and at the constriction sites of mitochondria. This localization pattern was probably the same as that of ADL2b. We confirmed that ADL2a and ADL2b had the same localization by using GFP and DsRed (RFP). Furthermore, overexpression of ADL2a that had a point mutation in its GTPase domain caused mitochondrial elongation. Consequently, we suggest that not only ADL2b but also ADL2a is involved in the division of plant mitochondria.

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Peroxisomes are single membrane organelles that have a variety of functions in eukaryotes. In higher plants, peroxisomes have vital roles in fatty acid degradation and photorespiration. To better understand peroxisome biogenesis, we attempted to generate mutants having peroxisomes with different sizes or shapes, as these mutants would be expected to be defective in peroxisomal division. We used seeds of a transgenic Arabidopsis termed GFP-PTS1 in which peroxisomes can be visualized with GFP. GFP-PTS1 seeds were mutagenized with EMS and allowed to germinate. Of about 37,000 M2 plants under the fluorescence microscope, we identified eighty-two candidates as having abnormal peroxisome morphology. apm mutants exhibited the tissue- and/or organ-specific patterns of GFP as follows, (1) long peroxisomes, (2) giant peroxisomes, (3) GFP fluorescence in the cytosol as same as in peroxisomes, and (4) different distribution. In this presentation, we would like to discuss the phenotypes of apm mutants.

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Plant peroxisomes are known to differentiate into at least three classes, namely glyoxysomes, leaf peroxisomes and unspecialized peroxisomes. These different peroxisomes possess a different set of enzymes that is related to special function in various developmental stages of higher plants. In the early germination, glyoxysomes are developed in storage tissues of oilseeds, which contain enzymes for fatty acids β-oxidation and the glyoxylate cycle that implicate to lipid metabolism. After greening, leaf peroxisomes are function in the photosynthetic organs, which contain glycorate pathway in photorespiration. Interestingly, these peroxisomes are known to be transformed into one another under plant growth. In recent study from other organism, peroxisomal biogenesis factors are identified by mutant analysis and designated peroxins. We found 18 Arabidopsis peroxins in the genome using homology search and analyzed in detail by employing various strategy. Based on these results, we discuss functional roles of these factors in the plant peroxisomal biogenesis.

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In higher plants, glyoxysomes and leaf peroxisomes are present in etiolated and green cotyledons, respectively. Peroxisomes are directly transformed from glyoxysomes to leaf peroxisomes in greening cotyledons of fatty seedlings.
It is deduced from the genome search that 281 genes for proteins with peroxisomal targeting signals (PTS1 and PTS2) or proteins related to the functions of peroxisomes are present in the Arabidopsis genome. Since only a few genes were identified functionally among them, there seem to be many functionally unknown peroxisomal proteins in cotyledons of Arabidopsis. In addition to the proteomic analysis of leaf peroxisomal proteins, we report here a method of isolating glyoxysomes from cotyledons of Arabidopsis in high purity and the proteomic analysis of glyoxysomal proteins. We identified 19 proteins including 13 novel glyoxysomal proteins by MALDI-TOF MS. We will discuss on the peroxisomal transformation of glyoxysomes to leaf peroxisomes by the proteomic analysis.

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All organisms synthesize heat-shock proteins (HSPs) in response to supraoptimal temperatures and other types of stress. Most of them act as molecular chaperones aiding other proteins to maintain or regain their native conformation by stabilizing partially unfolded states. The small HSPs (sHSPs) that range in size from approximately 15-30kDa have a conserved C-terminal region called alpha-crystallin domain. Small HSPs found in a distinct cellular compartment including the cytosol, chloroplast, ER and mitochondrion. However, the peroxisomal proteins that are homologous to sHSPs were not identified.
We found a protein with the molecular mass of 15.7 kDa in Arabidopsis, which has the putative PTS (peroxisome targeting signal) in its C-terminal end. The 15.7-kDa protein has alpha-crystallin domain and its mRNA is accumulated in heat-shock treated leaves. Now, we are analyzing about its localization in cell and its functions.

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Peroxisomes are known to possess several metabolic pathways, such as fatty acid β-oxidation, glyoxylate cycle and glycolate metabolism, and play important roles for lipid degradation and photorespiration. We have isolated Arabidopsis mutants that had defect in fatty acid β-oxidation. Of those, we succeeded in identifying three alleles, ped1, ped2 and ped3, that have defects in 3-ketoacyl CoA thiolase, AtPex14p and peroxisomal membrane ABC transporter, respectively. Lipid degradation of all these mutants is reduced because of the termination of fatty acid β-oxidation at a different step in each mutant.
Since degradation of seed reserved lipid in peroxisomes is an important process, defects in fatty acid β-oxidation may affect gene expression ocurring during postgerminative growth. To investigate regulation of the gene expression, we compare gene expression of the mutants. We discuss function of genes that alter their expression in the mutants.

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To dissect vascular pattern formation genetically, we have isolated and analyzed van mutants of arabidopsis that are characterized by fragmented minor veins. Here we report putative auxin distribution in relation to vein development of the van3 mutant and genetic interaction between VAN3 and auxin-related genes.
Putative auxin distribution was analyzed using a fusion gene of auxin-inducible promoter and reporter, DR5::GUS. In the formation of tertiary veins of wild type plants and the van3 mutant, GUS staining was observed as a dotted pattern in the provascular cell and staining cells were interspersed between secondary veins but the number of staining cells of the van3 mutant was reduced substantially. Next, we generated double mutants of van3 with pin1, gn and mp. The van3pin1 double mutant displayed the sum of two mutant phenotypes, suggesting that VAN3 and polar auxin transport system do not act in the same pathway toward vascular pattern formation.

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To understand molecular mechanisms underlying temporal and spatial regulation of vascular pattern formation, which has provided a model for studying pattern formation and tissue differentiation in plants, we have been screening genes involved in vascular formation using Arabidopsis gene trap lines established in Kazusa DNA institute. We screened 93 lines having GUS staining along with vascular network in leaves from 47800 gene trap lines. We are now trying to determine GUS insertion locus using methods of TAIL-PCR and/or 5RACE. In the 30 lines, which have GUS staining pattern specific to vascular systems, KG09040 showed fragmentation of lateral vein, which is a typical phenotype of van mutants, indicating that the KG09040 gene may be responsible for provascular tissue patterning. Here we report histochemical and molecular genetic analyses of KG09040 and some other gene trap lines.

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Tracheary element (TE) differentiation involves the morphological events such as secondary wall formation and programmed cell death which are spatiotemporally regulated. To elucidate the regulation mechanism of TE morphogenesis, we paid attention to the generation of reactive oxygen species (ROS) which are well-known signal molecules in the process of hypersensitive response. We found, by nitroblue tetrazolium (NBT) staining, that superoxide was generated on the cell surface at an apex of immature TEs and TE precursor cells. The area of superoxide generation was consistent with the initiation area of secondary wall formation recognized by FITC-WGA. When DPI, a suicide inhibitor of NADPH oxidase, was added prior to the secondary wall formation, superoxide generation at the apex and subsequent TE differentiation were suppressed, suggesting that local superoxide generated by NADPH oxidase plays a role in the initiation of secondary wall.

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We have proposed that the two-dimensional growth of leaf blade is controlled, at least in part, by polar cell expansion. On the other hand, contribution of cell division orientation in the determination of leaf shape has been unclear. To further understand the molecular basis of leaf morphogenesis, we characterized an Arabidopsis leaf-shape mutant, angustifolia3 (an3). Wild-type and an3 leaves were similar in length, while the width of an3 leaves was narrower than that of wild-type leaves. Leaf cell shape (ratio of cell length versus cell width) in an3 is not significantly different from that of wild-type plants. In contrast, the number of leaf cells was reduced preferentially in the leaf-with direction when compared to that of wild-type plant. These observations suggest that the cell division orientation in the leaf-with direction and the leaf-length directions are controlled by different mechanisms. Progress of positional cloning of AN3 gene will also be presented.

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ANgene of Arabidopsis encodes the homolog of the human CtBP which regulates polar elongation of the leaf cells. AN homologs from Marchantia polymorpha(MAN) and Ipomoea nil(MAN) rescued the an-1 mutation in terms of leaf morphology. MAN does not conserve the LXCXE domain which were contained in the AN and IAN. Moreover, although LXCXE domain is known to have ability to interact with Rb, AN does not interact with Rb in Yeast two-hybrid system. Taken together, we concluded the LXCXE domain of AN is not necessary to regulate the leaf width. Another yeast two-hybrid analysis indicated that AN can not interact with Ela, unlike mammalian CtBP. RT-PCR suggested that the transcriptional expression level of AN is important to regulate the leaf width. We analyzed relationship between translational level of AN and the an phenotype. Based on these results, possible role of AN will be discussed.

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Plant leaves have polarities such as axes along the leaf length and width directions, symmetry and dorsoventrality. However, the molecular mechanisms by which flat structure of leaves is developed are largely unknown. To understand such mechanisms, we isolated and characterized an Arabidopsis activation T-DNA tag line, K346, associated with unusual leaf morphogenesis. K346 showed the dwarfism, and its leaves were depressed and round-shaped. K346 showed semi-dominance, suggesting that the over-expression of a gene caused the phenotypes. The T-DNA insertion site in K346 was found between At2g36980 and At2g36990. But the expression levels of these genes were similar to those of wild type. By a fine analysis of the genomic sequence around the T-DNA insertion site, we discovered an ORF encoding 53 amino acid residues and named it At2g3698x. At2g3698x in K346 expressed at a higher level than in wild type, suggesting that over-expression of At2g3698x is responsible to K346 phenotype.

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The Arabidopsis SPIRAL1 (SPR1) gene encodes a novel plant-specific protein. spr1 mutants show right-handed twisting in the epidermis of roots and etiolated hypocotyls presumably due to impaired organization of cortical microtubules (cMTs). Both SPR1::GFP and SPR1::His fusion proteins were able to compliment spr1 mutants, and the GFP fluorescence co-localized with cMTs. Bacterially expressed SPR1::His, however, failed to bind taxol-stabilized tubulin polymers, suggesting indirect interaction between SPR1 and cMTs. The Arabidopsis genome contains five SPIRAL1-LIKE (SP1L) genes. While double mutants of spr1 and some sp1ls showed novel twisting phenotype in the inflorescence, CaMV35S::SP1L fusion genes were able to rescue the spr1 defects. SPR1 and SP1Ls share high identity in both N- and C-terminal regions. GFP fusion experiments indicated that these regions alone are sufficient for targeting SPR1 to cMTs. These results suggest that the conserved N- and C-regions of SPR1 and SP1Ls indirectly act on cMTs through as yet unidentified molecule(s).

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Recessive mutations in Arabidopsis thaliana SPIRAL1 locus, SPR1, reduce anisotropic growth of endodermal and cortical cells in roots and etiolated hypocotyls, and induce a right handed helical growth in epidermal cells of these organs. It was proposed that a microtubule dependent process and SPR1 act antagonistically to control directional cell elongation. SPR1 encodes a novel protein of low molecular weight. We identified two genes, SPI1 and SPI2, encoding proteins that interact specifically with SPR1 in the yeast two-hybrid system assay. SPI1 and SPI2 define a new family of putative type Ib membrane proteins. We have isolated plants harboring T-DNA insertions in SPI1 and SPI2 genes. Characterization of spr1/spi mutants, together with the SPR1 and Myc-SPI tagged proteins association to the Arabidopsis microsomal protein fraction, strongly suggest that the putative SPR1/SPI membrane bound protein complex is involved in a cell expansion process that might be related with cortical microtubule arrays.

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Newly divided cells in plants undergo significant elongation before their differentiation. Because plant cells are enclosed in rigid cell walls, cellulose microfibrils, which are deposited in cell walls in a transverse direction to the axis of elongating cells, are critical for the directionality of cell elongation. Because of their coaliment with microfibrils, transversely oriented cortical microtubles lying underneath plasma membrane have be proposed to regulate the microfibril orientation.
Arabidopsis spiral2 (spr2) mutant is defective in directional cell elongation and shows right-handed helical growth in axial organs. We reveled that the SPR2 gene encodes a novel protein with HEAT repeats. The fusion protein of SPR2 and GFP localized to the cortical microtubles in various tissues. Recombinant SPR2 proteins were sedimented in vitro with taxol-stabilized microtubles. Our results suggest that SPR2 is a novel microtuble-associated protein and might be required for proper organization of cortical microtubles.

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ZIM is a putative transcription factor in Arabidopsis with zinc-finger and CCT motifs. ZIM mRNA was detected from the vegetative phase and was more abundant in apices than leaves. To elucidate ZIM function, ZIM-overexpressing transgenic plants (ZIM-ox) were generated. In ZIM-ox, hypocotyls and petioles were elongated due to increased cell elongation. Competence in cell elongation was not affected, since ZIM-ox hypocotyls were as long as wild-type in the dark. The hypocotyl elongation phenotype was also observed in red, far-red, and blue light. This indicated that ZIM functioned as a negative regulator in the signal transduction pathway after convergence of photoreceptor-mediated light signals. The hypocotyl and petiole elongation in ZIM-ox was inhibited by brassinazole, a brassinosteroid biosynthesis inhibitor, which indicated that the elongation required brassinosteroids. These results suggest that ZIM is a regulator of photomorphogenesis via brassinosteroids. We are analyzing microarray data from ZIM-ox to identify genes responsible for elongation.

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The ACAULIS5 (ACL5) gene, whose inactivation causes a severe defect in the elongation of stem internodes, encodes a polyamine biosynthetic enzyme, spermine synthase. In order to define further the function of polyamines in plant development, we have isolated five suppressor of acl5 (sac) mutants. Although they exhibited various degrees of the recovery in the growth of stem internodes, stem sections and expression analyses of several genes revealed that the recovery of the plant height was resulted from that of the cell length in all five sac mutants. In addition, expression levels of ACL5, which is upregulated in acl5-1 mutants, were restored even before bolting in these sac mutants. Fine mapping located the Sac51 mutation in the 5' leader sequence of the MSJ1.18 gene, which encodes a putative bHLH transcription factor. The possible regulatory mechanism leading to the dominant Sac51 phenotype will be discussed.

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The plant cell wall is constructed outside the plasma membrane by many kinds of proteins, most of which are encoded by multi-gene families. Although many cell-wall-related gene families have been identified, little is known about their expression profiles. By means of the microarray technology and the quantitative real-time RT-PCR procedures, we have analyzed expression patterns of individual members of the cell-wall-related gene families in Arabidopsis. The results reveal that each member of a gene family exhibits its own expression profile in terms of tissue specificity and responses to hormonal signals, with some members exhibiting similar expression patterns. Phylogenetic studies suggested that most of the gene-pairs, which had been generated by gene duplication, show similar expression patterns and share common sequences in their promoter regions. By focussing on root-specific members of xyloglucan endotransglucosylase / hydrolase (XTH) gene family, we discuss evolutionary implication of diversification and conservation of the expression patterns.

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