Betaine is an important osmoprotectant, synthesized by many plants in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidations of choline, recently a novel biosynthetic pathway of betaine from glycine, catalyzed by two N-methyltransferase enzymes, was found. Here, the potential role of N-methyltransferase genes for betaine synthesis was examined in a fresh water cyanobacterium Synechococcus sp. PCC 7942 and in Arabidopsis plants. Co-expression of N-methyltransferase genes in Synechococcus caused accumulation of significant amount of betaine, and conferred sufficient salt tolerance to a fresh water cyanobacterium. Arabidopsis plants expressing the N-methyltransferase genes also accumulated betaine to a high level in roots, stems, leaves, and flowers, and improved the seed yield under stress conditions. Betaine levels were higher than those produced by choline-oxidizing enzymes. These results demonstrate the usefulness of glycine N-methyltransferase genes for improvement of abiotic stress tolerance in crop plants.

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Ascorbate oxidase is an apoplastic enzyme in plants which catalyzes the oxidation of ascorbate to monodehydroascorbate. Its biological function remains largely unclear. Transgenic tobacco plants expressing AAO gene in sense and antisense orientations and an Arabidopsis mutant were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing AAO gene in sense and antisense orientations were about 16-fold and 0.2-fold of wild-type's ones. Under high salinity condition, the germination yields were highest in antisense plants, then wild-type and sense plants followed. When tobacco plants were subjected to salt stress during the developmental- and reproductive-stage, the sense plants were most severely damaged whereas the antisense plants exhibited the modest inhibition. Number of ovaries and seed weight was high in the antisense plants, but significantly reduced in the sense plants. These results will be presented.

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Abiotic stresses such as high salinity are limiting factors for plant productivity. Upon exposure to those stresses, a polyamine biosynthetic pathway in plants is enhanced, resulting in the change of the polyamine contents. However, it is difficult to establish a direct cause-and-effect chain between polyamine levels and the ability to adapt to the stresses.
This time, we address a role of spermine in the adaptive response to high salt stress in Arabidopsis using a spermine-deficient mutant. Two Arabidopsis gene products, ACL5 and SPMS, have been proven to exhibit spermine synthase activities. The double knock-out mutant, acl5/spms, producing no spermine is viable and sets seeds under non-stressed condition but features hypersensitivity to high salt. This phenotype was specifically suppressed by adding spermine to the growth media. We will refer the analysis of differential gene expression in this mutant plant under high salt stress and the impact of the polyamine spermine.

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We have described the lip19 subfamily of basic region-leucine zipper protein (bZIP)-encoding genes from plants. In Arabidopsis, there are 75 bZIP-encoding genes (AtbZIP1-AtbZIP75) which, based on criteria of associated common domains, have been subdivided into 10 groups designated A to I and S. Here, we characterized the three group S members, AtbZIP2, AtbZIP11, and AtbZIP53, showing high sequence similarity to lip19 subfamily. Those gene products were localized to the nucleus and showed strong DNA binding activity to hexamer and C/G box hybrid sequences. Also, transactivation activity was examined in yeast and plant cells. The expression of the genes upon exposure to various abiotic stresses and to hormone treatments was examined in Arabidopsis. We found that AtbZIP11 and AtbZIP53 were responsive to cytokinin and high salt stress, respectively. To fully understanding the regulatory roles of AtbZIP11 and AtbZIP53 in gene transcription, we have commenced detailed analyses of AtbZIP11- and AtbZIP53-overexpressing plants.

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The plastid genome contains more than one hundred genes and the expression is regulated depending on cell- and plastid-types. Sigma factors determine the specificity of the eubacteria-type plastid RNA polymerase (PEP), but roles of each sigma factor for chloroplast gene expression remain largely unknown. During oilseed development, chloroplasts play a critical role for the fatty-acid biosynthesis. We focused on this chloroplast function in Arabidopsis, and searched for sigma factors (SIG1-6) which involve in this, by using sig mutants. As a result, greening of seed chloroplasts was inhibited in newly identified sig2-2 and sig6-2 mutants, whereas thylakoid development was only inhibited in sig6-2. In situ hybridization analysis showed the expression of SIG2 and SIG6 in developing seed embryo, and measurement of fatty-acids contents showed the reduction of triacylglycerols (TGs) and the alteration of constitution of TGs. These data suggest differential roles of SIG2 and SIG6 for oilseed development.

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Chloroplast genes are transcribed by two types of RNA polymerase, termed NEP and PEP. PEP is eubacteria-type RNA polymerase, which is composed of plastid-encoded core subunits and one of nuclear-encoded sigma factors. In Arabidopsis thaliana, there are six sigma factors, SIG1 to SIG6. It is assumed that each sigma factor is involved in transcription of different plastid genes in response to both developmental and environmental signals. However, the roles of individual sigma factor in chloroplast transcription remain to be determined.
We are studying the function of sigma factors under stress conditions, and have already demonstrated that expression of SIG5 is induced by several environmental stresses. In this work, we found that expression of SIG6 is also, but specifically induced under low-temperature condition. In transposon-tagged sig6-2 mutant, growth inhibition and etiolation of leaves were observed after low-temperature treatment. Results including microarray analysis to identify low temperature-specific SIG6-dependent genes, will be reported.

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Chloroplasts are endosymbiotic resultants of an ancient photosynthetic prokaryote, related to modern cyanobacteria. The modern chloroplasts utilise the inherent prokaryotic and the acquired eukaryotic systems for their development and functions in cells. Chloroplast division, an essential cellular process in plants, also represents a "hybrid" that involves the co-ordinated action of the prokaryotic division proteins and the eukaryotic membrane fission proteins.
Recent studies have revealed that the conserved system is essential for the initiation of chloroplast division and the products of Min that determine the division site in prokaryotic cells also participate in chloroplast division. Here, we investigated the role of AtMinE1, a nucleus-encoded MinE orthologue, in the model plant, Arabidopsis thaliana. We constructed transgenic Arabidopsis plants which overexpress or downexpress AtMinE1. Detailed microscopic characterisations of chloroplasts in the transgenic plants revealed that AtMinE1 is an indispensable plastid division factor and is involved in division site placement of chloroplasts.

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Most of the chloroplast proteins are nuclear-encoded. For the functional analysis of the nuclear-encoded chloroplast proteins in Arabidopsis, we systematically collected these tag-lines. 2,090 chloroplast proteins are identified by typical 4 predictors. Base on database of RIKEN, NASC, Wisconsin, CSHL and SALK, we collected 3,416 tag-lines disrupted by transposon or T-DNA. From this collection, we have not only isolated mutants with visible phenotype (apg mutants), but also collected homo-lines without clear phenotypes when plated on agar medium. We identified 55 mutants showing abnormal seedling by screening of 704 of RIKEN Ds tag-lines. To determine whether these mutations are caused by a Ds insertion, we examined mutant alleles existed in other tag-lines. Among 33 genes, 24 genes existed two or more alleles, and mutant alleles of 17 genes showed similar phenotype. These results suggest that at least 17 genes tagged with Ds and have an important role for chloroplast development.

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External Ca²⁺ promotes stomatal closure by triggering Ca²⁺ oscillation in guard cells. Elevation of cytosolic Ca²⁺ concentration is dependent on Ca²⁺ entry from extracellular space and/or inner Ca²⁺ stores, such as ER and vacuole. We found that chloroplast localized CAS (Calcium sensing receptor) is involved in the external Ca²⁺-induced stomatal closure in Arabidopsis. CAS is a plant-specific putative Ca²⁺ binding protein that has a single transmembrane domain. CAS carries a potential chloroplast transit peptide at N termini. To confirm its subcellular localization, stable transgenic plants that express CAS-GFP fusion proteins were examined with confocal laser microscopy. The CAS-GFP fusion proteins are localized in chloroplasts in all tissues. Furthermore, CAS is identified in several chloroplast proteomics studies. Ca²⁺-induced stomatal closure was abolished in CAS knock out plants, suggesting that chloroplast localized CAS might be involved in the regulation of stomatal movement.

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In chloroplast, many characteristics of the gene expression system resemble those of bacteria because chloroplast has an evolutionally prokaryotic origin. To characterize the molecular mechanism of gene expression in chloroplast, we screened several Arabidopsis mutants for chloroplast RNases homologous to bacterial ones from the SALK institute and identified three putative RNase mutants (RNase E, PNPase or RNase II/R) with defect in chloroplast. RNA gel blot analysis confirmed the disrupted expression of the corresponding genes by the T-DNA insertion in these mutants. We also determined that each phenotype of mutants was single, recessive and linked with antibiotic resistance, indicating that the mutant phenotype was derived from the disruption of corresponding genes. We report here more detailed characterization of a putative chloroplastic RNase E mutant, which showed pale-green leaves, retarded growth and significantly reduced photosynthetic activities. Modification of RNA metabolism in the mutant was suggested by RNA blot analysis of plastid-encoded genes.

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A large gene family encoding proteins with pentatricopeptide repeat (PPR) motif consisting of 35 degenerate amino acids exists in flowering plants but not in algae, fungi, or animals. For instance, the Arabidopsis thaliana genome has 470 PPR protein genes and the rice (Oryza sativa) genome encodes 670 PPR proteins. In contrast, the moss Physcomitrella patens genome is estimated to encode 70 to 100 PPR proteins. The most interesting feature of the plant PPR proteins is their cellular localization; 80% of Arabidopsis PPR proteins are predicted to localize in either the mitochondria or plastids. To investigate the function of plastid PPR proteins, we have generated and characterized the plastid-localized PPR protein gene disruptant of P. patens, and characterized the Arabidopsis T-DNA mutants with insertion in PPR protein genes. PPR513-11 disruptant mosses displayed abnormal phenotypic characters such as the growth of protonema colonies, the number and shape of chloroplasts, and so on.

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A mutation in CRUMPLED LEAF (CRL) of Arabidopsis thaliana causes a severe defect in plastid division, resulting in 1.5 chloroplasts in mesophyll cell in average. Besides the inhibition of plastid division, the mutation causes distortion of cell division planes and abnormality in cell differentiation (Asano et al., Plant J., 38, 448-459, 2004). FtsZ1-1 is one of the other genes that is involved in chloroplast division. Here we show that ftsz1-1 crl double mutants showed severer defetcs in gross morphlogy compare to the each single mutants before and after germination. Embryos of the double mutant contained enlarged shoot apical meristem and increased number of cotyledon primordia. The seedlings of the double mutants showed aberrant gross morphology of plants: small in size, pale, and leaves with narrow and asymmetric leaf lamina. The number of chloroplast in embryonic cells was reduced in the double mutant compared to either crl or ftsZ1-1.

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Using talin actin-binding domain fused to tdTomato, a new RFP variant (Shaner et al. 2004), actin filaments were visualized in both moss and fern cells. We have been studying actin dynamics during photoresponses in GFP-talin-expressing line of Physcomitrella. Blue excitation of GFP, however is problematic because it interferes blue-light responses. Therefore we tried to visualize actin by green-excitable label. The same actin dynamics as in GFP-talin transformant could be observed in tdTomato-talin-expressing line. Further, by transforming tdTomato-talin into the line stably expressing GFP-tubulin, actin filaments and microtubules were observed in the same cell. In fern Adiantum cells, actin filaments had been scarcely observed by expression of GFP-talin. Actin filaments were clearly observed in fern cells by tdTomato-talin expression

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Chloroplasts show intracellular photomovements, regulated by phototropin1 and 2. Here in Arabidopsis, we show evidence that during photomovement, chloroplasts migrate using a unique mechanism involving short actin filaments localized to parts of their surfaces. In the absence of such light signals, chloroplasts are anchored by non-localized surface filaments. Mutants analyses reveal that CHUP1 functions in chloroplast-specific actin organization and that phototropins localize filament assembly to the region facing movement direction on the chloroplast surface.

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Actin microfilaments (MFs) are known to play important roles in plant morphogenesis and cell cycle progression. During the process of cytokinesis, MFs organize a phragmoplast-like structure, however, its role is unclear yet. To examine the function MFs in cytokinesis, we performed time-sequential observations of MFs or MTs and the cell plate dynamics visualized with transgenic cell lines of tobacco BY-GF11 or BY-GT16, respectively, in addition to a staining with a styryl dye, FM4-64. As a result we observed that the MF phragmoplast-like structure appeared later than that of MT and located farther from the cell plate than the MTs. When the MFs were broken by an actin polymerization inhibitor, bistheonellide A, cell plate oscillation was suppressed and it took more time to fuse the cell wall. These results suggested that MFs were involved in the cell plate development.

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The actin microfilaments (MFs) have essential roles in plant cell division and morphogenesis. However, little is known about the dynamics of the MF structure during cell division. To observe MFs in living plant cell, we have already established a transgenic BY-2 cell line expressing GFP-Fimbrin (BY-GF). Using this cell line, we have found that MF structure at cell cortex in mitosis appeared to form two bands, and named the structure MF twin peaks (MFTP).
In this study, we examined the roles of MFTP in cell plate formation. Disruption of MFTP by pulse-treatment of cytochalasin D induced distorted cell plate, while disruption of actin phragmoplast had little effect on cell plate guidance. These results suggested that MFTP play important roles in cell plate guidance.

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In higher plants, large vacuoles occupy a great portion of the cell volume and have a role in its morphogenesis. Tobacco BY-2 miniprotoplasts which were mechanically removed their large vacuoles regenerate these structures in a proper culture medium. Since in vacuolar and subsequent cell morphogenesis, cytoskeletons are suggested to be involved, we simultaneously observed dynamics of actin microfilaments (MFs) and vacuolar structures in tobacco BY-GF11 miniprotoplasts stained with FM4-64 in detail. MFs localized adjacent to the network-like vacuoles. Treatment of an actin polymerization inhibitor disrupted their network-like structures, but little effects on structures of large vacuoles. A microtubule polymerization inhibitor did not affect the network-like and large vacuolar structures, essentially. These results suggest that the MFs are involved in maintenance of the network-like vacuoles.

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In plant cells, cytoplasm is partitioned by a cell plate during cytokinesis. The cell plate expands by centrifugal expansion of the phragmoplast, which transports cell wall materials. Because assembly of microtubules drives phragmoplast expansion, molecular mechanism of the microtubule assembly in the phragmoplast has been analyzed. We have shown that microtubules nucleate on existing microtubules as branches in interphase cortical arrays of microtubules (Murata et al. 2005). In the present study we tested if microtubules nucleate on existing microtubules during phragmoplast development by direct observation of microtubules in living cells. We found that microtubules were extended from periphery of the phragmoplast and then incorporated into the outermost sides of the phragmoplast. These microtubules seemed to be nucleated on phragmoplast microtubules. We propose that the phragmoplast is expanded by addition of newly formed microtubules, formed on existing microtubules as branches, onto the sides of the phragmoplast.

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Microtubule (MT) structure is highly conserved in eukaryotes. Stable heterodimers of α-tubulin and β-tubulin are stacked longitudinally to form a tubulin protofilament and 13 protofilaments are associated laterally to form a hollow MT. Interactions between tubulins are considered to be important for microtubule structure and dynamics.
Previously, we isolated 40 Arabidopsis mutants that show herical growth phenotypes and have missense or deletion mutations in their tubulin gene. These mutations act dominant-negatively, since transgenic plants expressing mutated tubulins showed skewed elongation growth. Quantitative analysis of cortical MT arrangement demonstrated that all right-handed mutants have left-handed MT arrays and left-handed mutants have right-handed MT arrays. Thus, the direction of cell elongation is tightly correlated with cortical MT array organization.
Currently, we are analyzing cortical MT dynamics in these mutants by using GFP-TUB6 and EB1-GFP transgenic lines which label whole MTs and the plus end of elongating MTs, respectively.

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It has been generally accepted that the motility of cellulose synthase on the plasma membrane is regulated by the cortical microtubules. However, the mechanism for the regulation is no known To solve this problem, we tried to synthesize polysaccharides using the plasma membrane vesicles (PMVs) isolated from tobacco BY-2 cells. As reported previously, the PMVs retained the cortical microtubules. In intact PMVs, synthesis of small amount of polysaccharides was detected by calcoflour staining. Synthesis of polysaccharides was highly enhanced when the CHAPS insoluble fraction was used. In electron microscopy, many fibrous structures associated with the cortical microtubules were observed. These results suggested association of glucan synthase with the cortical microtubules.

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Higher plants respond to environmental stresses by a sequence of reactions which include a reduction of growth by affecting cell division. It has been shown that calcium ions play a role as a second messenger in mediating various defence responses under environmental stresses. In this study, the role of calcium ions on cell cycle progression under abiotic stresses has been examined in tobacco BY-2 suspension culture cells. Using synchronized BY-2 cells expressing the endogenous calcium sensor aequorin as experimental system, we could show that oxidative and hypoosmotic stress both induce an increase of intracellular calcium concentration and cause a delay of the cell cycle. Depletion of calcium ions in the culture medium suppressed the effect of the stimuli tested. These results demonstrate that calcium signalling is involved in the regulation of cell cycle progression in response under abiotic stress.

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Ferrous (Fe²⁺) is a plant micro nutrient. On the other hand, excess ferrous is known to be toxic in cells. In this study, we examined a mechanism to keep a moderate ferrous uptake into tobacco BY-2 cells. After transfer of the tobacco BY-2 cells to a medium omitted divalent cations and applied 1.0 mg/L FeSO₄, cell death in about 80 % of the cells were observed in 18 h. The Fe²⁺ influx is known to be mediated by Fe²⁺ transporters, which, its selectivity of Fe²⁺ is rather low and other divalent cations can be transported. Therefore, in addition to 1.0 mg/L FeSO₄, when we applied 1.5 mM MgCl₂ and 3.0 mM CaCl₂ into the medium, DNA degradation and cell death is decreased and the amount of Fe²⁺ uptake was reduced. These results suggested that divalent cations such as Mg²⁺ and Ca²⁺ in the medium antagonized Fe²⁺ influx into cells.

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Lethal effect of Al on cultured tobacco cells was investigated by treating tobacco cells (Nicotiana tabacum L. cv. Samsun) at the logarithmic phase of growth with AlCl₃ in a simple calcium medium containing 3 mM CaCl₂ and 3% sucrose. Propidium iodide and Fluorescein diacetate staining revealed the maintenance of the integrity of the plasma membrane even after 18 h of Al treatment. However, BCECF-AM and Neutral red staining showed the evidence for the loss of function in the vacuole. Pre-treatment with Caspase-1 inhibitor, Ac-YVAD-CHO, significantly reduced the Al-induced vacuole collapse in tobacco cells. Furthermore, a strong negative correlation was found between the ratio of collapsed protoplasts scored after 18 h of Al treatment and growth rate of tobacco cells 6 days after post treatment. These findings suggest that Al can cause vacuole collapse before final death and caspase-1 activity might be involved in Al-induced cell death process in tobacco cells.

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Programmed cell death (PCD) is crucial in development, stress responses and homeostasis in all multicellular organisms. In plants, homologs of most major apoptosis regulators in animals have not been found and molecular mechanisms for regulation and execution of PCD are mostly unknown in plants. Ectopic expression of mammalian inhibitor of apoptosis proteins (IAPs) has recently been shown to affect plant PCD, suggesting possible presence of unknown functional IAP homologs in plants. By applying an improved motif searching method, we recently identified a novel gene family including Arabidopsis thaliana IAP like-protein1 and 2, which contains conserved domains similar to BIR domains of IAP (Higashi et al., 2005). We here report identification of a novel Arabidopsis gene with a mitochondrial localization signal, AtSmac, in which the IAP binding motif of the mammalian apoptosis regulator Smac/DIABLO was conserved. Possible physiological functions of AtSmac will be discussed.

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AtBI-1 (Arabidopsis thaliana Bax Inhibitor-1) is considered to be associated with the resistance against ROS (reactive oxygen species) stresses such as H₂O₂^- or salicylic acid. AtBI-1 is known to localize in ER membranes, and has seven transmembrane domains. The AtBI-1 lacking the C-terminal 14 amino acid residues lost the activity as a cell death suppressor. Several studies have suggested that cellular calcium levels played an important role in determination of the cells' sensitivity to various cell death-inducing stresses. Using yeast split-ubiquitin system, we discovered that AtBI-1 interacted with barley calmodulin (CaM) protein. Furthermore, we demonstrated that the C-terminal region of AtBI-1 could directly interact with calmodulin. Namely, Arabidopsis has 16 CaM genes (AtCaMs) in its genome. The overlay assay showed that at least two CaMs (AtCaM7 and AtCaM6) were able to interact with C-terminal 14 amino acids. The biological meaning of the interaction between CaM and AtBI-1 will be discussed.

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Bax inhibitor-1 (BI-1) is a widely conserved ER membrane protein known as cell-death suppressor. Overexpression of AtBI-1 (Arabidopsis BI-1) suppresses the H₂O₂-, SA- or elicitor-induced cell death in plant cells. To investigate the function of AtBI-1 in detail, isolation of interacting factors of AtBI-1 was performed by screening of Arabidopsis cDNA library. Using yeast split-ubiquitin system, we found AtCb5 (Arabidopsis cytochrome b₅) as a candidate of such factor. Cb5 is known to be a ubiquitous electron transport protein whose function is formation or modification of fatty acid. Furthermore, AtBI-1 was supposed to interact with AtFAH (Arabidopsis fatty acid hydroxylase) via AtCb5. Arabidopsispossesses several homologs of AtCb5, of which such interaction was found in AtCb5s localized in ER and mitochondria. In particular, AtBI-1 interacted with the cytosolic N-terminal region of AtCb5 containing the catalytic heme-binding domain. Possible role of such proteins in lipid metabolic pathway will be presented.

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Plants use sophisticated strategies to balance their responses to oxidative stress. Programmed cell death is one outcome that is regulated by reactive oxygens in various cellular contexts, including the hypersensitive response (HR) associated with successful pathogen recognition. We focus on the Arabidopsis bZIP transcription factor AtbZIP10 that binds G- and C-box consensus sequences. AtbZIP10 shuttles between nucleus and cytoplasm, likely via interaction with the export receptor AtXPO1. Surprisingly, AtbZIP10 can be retained outside nucleus by LSD1, a protein that protects Arabidopsis cells from death in the face of various oxidative stress signals. Our genetic analyses demonstrate that AtbZIP10 is a positive mediator of the uncontrolled cell death observed in lsd1 mutants. Additionally, AtbZIP10 and LSD1 act antagonistically in both pathogen-induced HR and basal defense responses. LSD1 likely functions as a cellular hub, where its interaction with AtbZIP10 and additional, as yet unidentified, proteins contributes significantly to plant oxidative stress responses.

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It has been revealed that cytosolic Hsp90 is essential for steady-state accumulation of R proteins. Reduction of Hsp90 level compromises R gene-dependent defense responses. p50, which is a truncated gene product of TMV, is the Avr component of N resistance gene inducing HR like cell death in NN tobacco. The specific inhibitor geldanamycin (GDA) for Hsp90 inhibited TMV- and p50-mediated cell death but not the cell death by Bax which is a mammalian pro-apoptotic protein inducing HR like cell death in plants. TMV- and p50-mediated cell death was accompanied with the activation of WIPK and SIPK, but Bax-mediated cell death was not. In WIPK and SIPK double silenced plants, p50- but not Bax-mediated cell death was suppressed. These results indicate that p50 induces cell death via activation of WIPK and SIPK but Bax induces cell death in the pathway which is independent on WIPK and SIPK.

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Phytotoxin-induced cell death is pathogen strategy for infection. To clarify the executioner of the toxin-induced cell death, we examined a fumonisin B1 (FB1)-induced cell death of Arabidopsis plants (1). FB1-induced cell death was accompanied with disruption of vacuolar membrane followed by lesion formation. The features of FB1-induced cell death were completely abolished in the Arabidopsis vacuolar processing enzyme (VPE)-null mutant, which lacks all four VPE genes. Recombinant VPE recognized a VPE substrate with K_m = 30.3 μM and a caspase-1 substrate with K_m = 44.2 μM, which is comparable to mammalian caspase-1. We show that VPE is a key molecule in toxin-induced cell death. Our findings suggest that a susceptible response of toxin-induced cell death is caused by the VPE-mediated vacuolar mechanism similar to a resistance response of hypersensitive cell death (2).

(1) Kuroyanagi et al., 280, 32914-32920 (2005) J. Biol. Chem.
(2) Hatsugai et al., 305, 855-858 (2004) Science

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Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been mainly studied by morphological analyses, accordingly, the physiological roles and molecular mechanism of autophagy in plants had not been made clear. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeasts and plants. It has not been proved, however, that Arabidopsis ATG genes are required for plant autophagy. To evaluate this requirement, we established autophagy-monitoring systems in Arabidopsis roots and leaves using concanamycin A, a V-ATPase inhibitor and clearly proved that T-DNA-insertion mutants of the ATGs (atgs) are defective in autophagy. The atg mutants exhibited a reduction in the growth rate of roots under nitrogen-starved conditions and early senescence phenotype even in nutrient-rich conditions. In addition, hypersensitive response cell death was accelerated in the mutants during the plant innate immune response. We will discuss the physiological roles of plant autophgy.

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In this study, we examined biological effects of malondialdehyde (MDA) formed by polyunsaturated fatty acid peroxidation on protein modification and gene expression in heat stressed plants. In vitro analysis showed that Protein modification by MDA was effectively caused by peroxidation of linolenic acid and was promoted over 33℃ in the presence of reactive oxygen. When intact plants were stressed at 40℃ under light illumination, chloroplast proteins was modified by MDA. To assess the effect of MDA formed during heat stress on gene expression, changes in transcript levels of representative protein family known as heat-shock proteins (HSPs) were investigated. Transcript levels of HSPs increased in correlation with transient increase of MDA in heat stressed Arabidopsis. Furthermore, exogenous MDA increased transcript levels of HSPs in Arabidopsis plants grown under normal condition. In conclusion, MDA might have not only proteotoxic effects but regulatory role in HSP genes expression.

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In plants, tocopherols (vitamin E) have long been assumed to protect photosynthetic membranes from oxidative stress by acting as lipid-soluble antioxidants. However, this hypothesis has not been rigorously tested. Here we subjected and analyzed the response of a series of Arabidopsis vitamin E-deficient (vte) mutants to various abiotic stresses. Surprisingly, these mutants were virtually identical to wild-type during high light, salinity and drought stresses. Also, the photosynthetic response of these mutants did not differ significantly from wild-type during high light stress, suggesting tocopherols have a limited photoprotective role in plants. In contrast, these mutants exhibited a dramatic chilling susceptible phenotype; the mutant in comparison to wild-type grew slower, accumulated anthocyanins in mature leaves, and produced less seeds during the treatment. Detail biochemical characterization of the phenotype provided evidence that tocopherols play a critical role in chilling adaptation by affecting carbohydrate metabolism, which is independent of photoprotective function of tocopherols.

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A multidomain cystatin cDNA (TaMDC1) was isolated from cold acclimated winter wheat. TaMDC1 contains a highly conserved N-terminal cystatin domain (DI) and a C-terminal cystatin-like domain (DII). Northern and western blot analyses showed elevated expression of TaMDC1 mRNA and protein during cold acclimation. Recombinant mTaMDC1 exhibited an inhibitory activity against papain. We found that mTaMDC1 inhibits mycelium growth of the snow mold fungus Microdochium nivale. Hyphae growth was totally inhibited in the presence of 50 μg/ml mTaMDC1 and morphological changes such as swelling, fragmentation and sporulation of the fungus were observed. Domain separation experiments demonstrated that both DI and DII display the antifungal activity, while proteinase inhibitory activity resides within DI. This suggested that proteinase inhibitory activity is not associated with antifungal activity of TaMDC1. The mechanisms of the in vitro antifungal effects and the possible involvement of TaMDC1 in cold induced snow mold resistance of winter wheat are discussed

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Fructan, fructose-based polymers, is accumulated in many C3 plants. Fructan is considered to play important roles not only as a reserve carbohydrate but also as a biologically regulatory substance of cytoplasm against osmotic stress caused by freezing and drought in plants. Therefore, an understanding of the metabolism of fructan is important to clarify the mechanisms of plant cold tolerance.
Six genes, prft1-prft6 encoding fructosyltransferases, were isolated from the cDNA library that was derived from crown tissues of cold-acclimated perennial ryegrass plants. Functional characterization of these genes indicated that the prft3 and prft5 encode 6G-FFT (fructan:fructan 6G-fructosyltransferase) and the prft4 encodes 1-SST (sucrose:sucrose 1-fructosyltransferase). Real-time RT-PCR showed that the mRNA levels of these genes increased, in different pattern, in both leaf and crown tissues during cold acclimation. These results indicated that changes in the mRNA level of these genes coincided with those in the fructan contents of perennial ryegrass plants.

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An application of low temperature or ABA induces freezing tolerance in Arabidopsis plants. In this study, we analyzed responses of Arabidopsis cultured cells (T87) to low temperature or ABA to understand molecular mechanisms of freezing tolerance acquisition at cellular level. Increase in freezing tolerance occurred only with cells at late lag phase when exposed to a low temperature for 2 days or ABA for 1 day. Osmotic concentration, sugar content and cold-inducible gene expression (COR15a and RD29A) had no apparent relationship with changes in freezing tolerance. Microarray analysis to determine the freezing-tolerance-associated genes of cultured cells revealed that there were 448 up- or 438 down-regulated genes after cold treatment. Among these genes, 17 and 14% were up- and down-regulated, respectively, only with cultured cells. These results suggest that perception and signal transduction of low temperature or ABA varies between at cellular and whole plant level.

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Upon cold acclimation, freezing tolerance of Arabidopsis thaliana increases significantly. Cold acclimation results in changes in protein composition and decreases in sphingolipids (i.e., cerebrosides) in the plasma membrane. In the plasma membrane of animal cells, proteins associated with sphingolipid- and cholesterol-rich detergent-resistant membrane (DRM) microdomain, are known to play important roles in signal transduction and pathogenesis. To investigate whether DRM-associated proteins involve in freezing tolerance in plants, we here determined expression patterns of DRM-associated proteins during cold acclimation. A DRM fraction was obtained by sucrose density gradient centrifugation of the plasma membrane fraction prepared from Arabidopsis seedlings after treatment with 1% (w/v) Triton X-100. When separated with SDS-PAGE and 2D SDS-PAGE for differential protein expression analysis, DRM-associated proteins showed quantitative changes during cold acclimation, suggesting the involvement of DRM in cold acclimation in plants. We are now identifying cold-responsive DRM-associated proteins by MALDI-TOF-MS.

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Environmental stresses such as low temperature have significant impact on various metabolic activities in chloroplasts. Cold stress also leads to the injury of membranes and the inactivation of proteins. Under low temperature stress, plants accumulate COR (cold-regulated) proteins targeted to chloroplasts, which may protect chloroplasts from dysfunction. However, biochemical functions of these proteins remain to be determined. Here, we report biochemical analysis of Arabidopsis thaliana Cor15a protein. As a first step of our analysis, we produced the Cor15a-His recombinant protein in E. coli and generated antibody against Cor15a. Using this antibody, we examined the expression, localization and immunoaffinity purification of Cor15a. To purify the Cor15a complex, we also generated Arabidopsis overexpressing COR15a-PROTEIN A fusion construct by Agrobacterium-mediated transformation. We show the analysis of these plants and discuss biochemical and physiological roles of Cor15a under cold stress.

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Plastids are surrounded by envelope membranes that allow compartmentalization of plastids from the cytosol. The envelope structure of plastids is often damaged by abiotic stresses, such as low temperature. Despite the fact that plants somehow acquire the mechanism avoiding the injury of plastid envelope during a process known as cold acclimation, envelope proteins that may protect plastids from freezing injury remain to be characterized. We examined the function of a cold inducible gene encoding a putative plastid protein of unknown function by in vitro and in vivo approaches. Biochemical characterization revealed that this protein is an integral membrane protein that localizes to the inner envelope of plastids. We will also show the contribution of this protein to the freezing tolerance of A.thaliana.

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Isoprene is a volatile hemiterpene emitted from leaves of many plants. Despite its massive emission, the physiological function of isoprene in plants is not well understood. In this study, we have cloned isoprene synthase (PaIspS) cDNA from Populus alba to analyze the environmental response of gene expression, and PaIspS-expressing Arabidopsis was characterized to clarify the physiological roles of isoprene emission from plants.
PaIspS cDNA was isolated by RT-PCR, and its enzymatic function was characterized in Escherichia coli. Then, we analyzed the response of its gene expression to environmental stresses. PaIspS was strongly induced by heat treatment and light irradiation, and was decreased in the dark, suggesting that isoprene emission was regulated at the transcriptional level.
We have prepared transgenic Arabidopsis overexpressing PaIspS gene, which is thought to be a non-isoprene emitter. The isoprene production by these transgenic plants and its influence on the Arabidopsis phenotype will be also reported.

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Tobacco EIN3 like (TEIL) was isolated as the factor binding the promoter sequence of acidic tobacco PR1 gene, sharing 60% identical amino acids with EIN3 from Arabidopsis. TEIL exhibited transcriptional activation binding the specific sequence tebs (TEIL binding sites) (Kosugi and Ohashi, 2000). TEIL was expressed in all tobacco tissues examined. The transcript was found at higher levels in lower leaves than upper leaves, and accumulated after wounding preceding basic PR gene expression. In TEIL-overexpressed transgenic plants, expression of basic PR genes was constitutive, whereas wound-induced expression of basic PR was reduced in TEIL-suppressed transgenic tobacco. Further we found that lesion size originated by TMV-infection was decreased in TEIL-suppressed tobacco indicating enhanced resistance. Senescence of detached leaf was delayed in TEIL-suppressed lines than wild-type tobacco. Triple response in the presence of ACC in TEIL over-expressed or suppressed line was more sensitive or insensitive than wild-type, respectively.

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AP2/EREBP gene family consists one of the largest groups of transcriptional factors in plants. Previously we showed that AtEBP, one of AP2/EREBP proteins, functioned as a transcriptional factor and suggested that AtEBP controlled plant defense genes transcriptionally.
In this study, to evaluate whether or not AtEBP plays a key role on the resistance to pathogens, we treated Arabidopsis plants with Pseudomonas syringae pv. tomato DC3000 with an avirulent gene, AvrRpm1. The results showed that the level of cell death during hypersensitive response (HR) increased in an AtEBP knockout line and decreased in AtEBP over-expressing lines. These results indicate that AtEBP plays a cell death suppressor on plant during HR. We have been analyzing these plants in terms of the general resistance to pathogens. On the basis of these results, we will discuss the role of AtEBP on plant cell death.

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Trichothecenes are a family of closely related phytotoxins produced by several phytopathogenic fungi, and acts on eukaryotic ribosome as inhibitors of protein synthesis. We revealed that T-2 toxin, one of trichothecenes induce defense response in Arabidopsis plants. We also found that the AtNF-X1 gene, which is an Arabidopsis homolog of the human transcriptional repressor NF-X1, was up-regulated in T-2 toxin-treated plants. The AtNF-X1 protein contains a RING finger domain and a NF-X1-type Zn finger domains. An atnf-x1 mutant was highly sensitive to T-2 toxin compared with the wild type. Western blot analysis revealed that AtNF-X1 protein levels increased by T-2 toxin treatment. Expected molecular weight of AtNF-X1 protein is about 130 kD , but protein bands of 80 kD and 62 kD were detected by αAtNF-X1-N (60-142 a.a.) and αAtNF-X1-C (766-1188) antibody, respectively. These results suggest that the AtNF-X1 protein was cleaved by a protease.

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Plants contain a large number of ATP-binding cassette (ABC) transporters belonging to different subclasses. AtPDR8 is the only member of the pleiotropic drug resistance ABC transporter (PDR) subclass in Arabidopsis that is constitutively highly expressed. Subcellular fractionation and immunochemical analysis showed that AtPDR8 was localized in the plasma membrane. When a knockout mutant of AtPDR8 (atpdr8) was infected with bacterial and oomycete pathogens, the plants exhibited chlorotic lesions and hypersensitive response (HR)-like cell death. The growth of virulent Pseudomonas syringae in the leaves of the atpdr8 mutant was reduced to 1% of that in the wild type. The defense response genes, PR-1, PR-2, PR-5, VPEg, AtrbohD, and AtrbohF were highly expressed in atpdr8 mutants. Our results indicate that AtPDR8 is a key factor controlling the extent of cell death in HR and suggest that AtPDR8 transports some substance(s), which is closely related to the response of plants to pathogens.

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To date, many genes which are transcriptionally activated upon wound- and defense-responses have been identified, but their biological functions are not necessarily clear yet. In the present study, we focused on a wound- and pathogen-responsive gene encoding a novel bHLH protein, WINB (Wound-Induced bHLH protein), which localizes to plastids in tobacco plants.
In leaves, expression of WINB was induced by wounding and TMV infection. WINB expression was also clearly activated by H₂O₂. Plastid localization signal of WINB spans about 70 amino acids at the N-terminus, and contains DNA binding domain of a bHLH motif. Using the yeast two hybrid assay, WINB was found not to form a homo-dimer. These results suggest that WINB is up-regulated by ROS, and is localized to plastids by signal sequence containing DNA binding domain. Moreover, WINB might bind to DNA by forming a hetero-dimer with other protein(s), thereby regulating plastid function(s).

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The molecular basis has been proposed as a gene-for-gene system, in which plants recognize the pathogen molecule by specified protein(s), this being called the R factor. However, mechanisms of interaction between proteins from the host and the pathogen are not completely understood. Here, we analyzed the mode of interaction between the N factor, a tobacco R factor, and the helicase domain (p50) of tobacco mosaic virus (TMV). To this end, domain dissected proteins were prepared and subjected to Agroinfiltration into intact leaves, followed by yeast two hybrid and pull-down assays. The results pointed to three novel features. First, the N factor was found to directly bind to the p50 of TMV, second, ATP was prerequisite for this interaction, with formation of an ATP/N factor complex, and third, the N factor was shown to possess ATPase activity, which is enhanced by the p50.

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Piriformospora indica is an endophytic fungus of the heterobasidiomycetous Sebacinales colonizing roots of different plant species. We investigated the interaction of P. indica with Barley (Hordeum vulgare) roots with respect to growth and pathogen resistance of the plants. P. indica infestation of the roots increases fresh weight of barley plants up to 60% and enhances resistance of the leaves against the biotrophic barley powdery mildew fungus Blumeria graminis f.sp. hordei. As a first step towards elucidating the mechanism of resistance induction, we checked mRNA levels of pathogenesis-related and hormone-induced genes in leaves of P. indica infested barley plants. Interestingly, mRNA of both jasmonic acid and salicylic acid induced genes, JIP-23 and PR-5, were not elevated in leaves of P. indica infested plants without challenge, indicating a possible involvement of new resistance signaling mechanisms.

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Plants are exposed to many types of abiotic or biotic stresses. Many researchers have analyzed the mechanism of these stress responses using Arabidopsis plants. At the present day, creation of plants having tolerance for several abiotic or biotic stresses has reported. On the other hand, insect damage is very serious problem that decrease the crop yields. However, the mechanism of plant response to feeding damage has not been well understood. Thrips are cell content feeding insect that penetrate single cells with stylet to suck out the contents. In addition, thrips transmit the virus from plant to plant. We used western flower thrips (Frankliniella occidentalis) in our analyses. In this time, we performed the GeneChip analyses (Affymetrix: ATH1 chip) of Arabidopsis plants attacked by thrips. We report here the function of Jasmonic acid (JA), Ethylene (ET) and Salicylic acid (SA) in thrips feedind.

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Plant latex is widely found in plant species (ca. 12,000-35,000 species). We here show two novel examples from our studies where latex ingredients, proteins and chemicals, play crucial roles in plant defense against insect herbivory.
Leaves of the Papaya tree (Carica papaya) showed strong toxicities against lepidopteran larvae such as Samia ricini, Mamestra brassicae, and Spodoptera litura, though no apparent toxic factors from these species had been reported. Our results suggested that cysteine proteases in latex, papain, is responsible for the defense activity.
Mulberry leaves (Morus spp.) showed strong toxicities to lepidopteran larvae other than the silkworm, Bombyx mori, (such as S. ricini and M. brassicae). The high concentrations (1.5-2.5% to wet latex, 8-18% to dried latex) of anti-diabetic sugar-mimic alkaloids that we found in mulberry latex, such as 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1), 1-deoxy nojirimycin (DNJ), and 1,4-dideoxy -1,4-imino-D-ribitol, accounted for the defense activities of mulberry leaves against insect herbivory.

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We reported formerly that cyclamen plant grown under excess nitrogen tends to induce Fusarium wilt disease. The present investigation was undertaken to examine the nitrogen compounds, which promote the growth of infected Fusarium and accelerate wilt symptom of cyclamen shoot. High levels of asparagine, glutamine and arginine were detected in cyclamen tubers, which were stained with Fusarium. All of these amino acids showed in vitro stimulation of Fusarium growth. Treatment with high ammonium sulfate resulted in the accumulation of glutamine in underground part of cyclamen plant. Furthermore, ¹⁵N-labelled glutamic acid, when absorbed from cyclamen root, was immediately transformed to glutamine in the root and transported to the tuber possibly through xylem vessel. These results strongly suggest that glutamine, asparagine and arginine, which are synthesized with the assimilation process of excess nitrogen, promote the growth of Fusarium infected within xylem tissue and finally induce wilt symptom of cyclamen shoot.

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Microarray analyses have provided opportunity to investigate quantitative information coded in the genome by measuring transcriptome, which records the decoded information from the genome. State of a cell and differences between other states can be studied through genome information, by comparing a set of transcriptome data to others. Clearly, those data should be shared and compared with researchers, and the knowledge should be integrated. However, there have been difficulties in data comparisons between experiments and laboratories. The barrier must be the lack of an universal framework that provide a set of common standard for data analysis.

A new framework that use thermodynamic model is introduced, by explaining the bases of the framework and the process of scientific testing. Additionally, differences of obtained results by the frameworks are presented by showing stability of log-ratio measurements and reproducibility of analyses. Artificial decoding of genome information is also mentioned.

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