The fruit vacuole is a large organelle accumulating sugar or organic acid in high concentration. To clarify the role of vacuolar H⁺-pump in fruit growth and solute accumulation, we generated antisense-transgenic tomatoes of the V-ATPase A subunit, which is under the control of a 2A11 fruit-specific promoter. Miniature-dwarf tomato (Micro-Tom) was used to transformation mediated by A. tumefaciens.
A subunit mRNA level in fruit decreased in antisense-transgenic lines, while that in leaves showed no difference compared with the GUS control line and non-transformant. This suggests that suppression is limited to fruit. The antisense-transgenic plants had smaller fruits and had few seed, although the plant growth was normal. These results considered to correlates with A subunit mRNA level. Sucrose concentration in fruits increased, although glucose and fructose concentration did not change. These results show the V-ATPase expression affect not only in fruit growth but also in seed formation and sugar composition.

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It is well known that vacuole has various functions for inorganic metabolisms in plant cell. Although vacuolar membrane proteins must be playing important roles for this, molecular works of those proteins are quite limited.
In the previous study we have succeeded in isolating highly purified tonoplast from Arabidopsis cultured cells and conducted the proteome analysis of membrane-bound proteins by LC-MS/MS. We identified certain unannotated transmembrane proteins besides the major tonoplast proteins. In order to find phosphate metabolism-related proteins, we selected possible candidates having phosphate responsible specific sequences in promoter regions and analyzed them in detail. At first to confirm the subcellular distribution of these proteins, we transformed GFP fusion proteins transiently to Arabidopsis cultured cells. To analyze the function of these proteins, we constructed transgenic BY-2 cells which overexpressed these proteins or GFP fusion proteins. We are measuring physiological responses of these cells to several phosphate conditions.

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In plants, mechano-sensitive channels are supposed to be a sensor for mechano-stimulation and gravi-stimulation. Using the transgenic intact plants and cultured cell line expressing aequorin, a Ca²⁺-reporting protein, the mechano- and gravi-induced increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c), implying that the possible involvement of mechano-sensitive Ca²⁺-permeable channel (MSCC), were observed. Nevertheless, the molecular mechanism for its activation and the direct physiological roles are still unknown. In this study, Electrophysiological analysis on MSCC in plasma membrane of guard cells of Vicia faba was performed. In the attached patch configuration, suction into the pipette reversibly induced channel opening. In whole cell patch configuration, corresponding kinetics of channel activity was recorded when cells were exposed to TNP, which specifically activate MSCC by generating the distortion of plasma membrane. Similar channel activity and a transient, TNP-induced increase in [Ca²⁺]_c were also observed in mesophyll cells of A. thaliana expressing aequorin.

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Heat stress in plant results in repression of most mRNA translation and the preferential translation of heat shock protein (hsp) mRNAs. 5' untranslated regions (5'-UTR) of hsp mRNAs are known to confer the preferential translation during heat stress. In this study, we showed that the m⁷G-capped reporter mRNA with 5'-UTR of Arabidopsis hsp90 gene was translated efficiently during heat stress by using Arabidopsis T87 protoplast transient expression assay. To investigate the function of the hsp90 5'-UTR in more detail, we performed the same transient expression assay using uncapped monocistronic reporter mRNA or capped bicistronic mRNA. As a result, hsp90 5'-UTR conferred temperature dependent increase in the expression from uncapped monocistronic mRNA and from second cistron of bicistronic mRNA. These data suggest that cap-independent translation via internal ribosome entry site (IRES) in hsp90 5'-UTR contributes the efficient translation of hsp90 during heat stress, which inhibits cap-dependent translation.

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The genes related to response of various stresses seem to be particularly prone to alternative splicing in higher plants. Ser/Arg-rich (SR) proteins are important splicing regulators at post-transcriptional level. Arabidopsis contains 20 types of SR protein homologue. We previously showed that the SR41a was rapidly induced by the high-light intensity. Here we aimed to clarify the alternative splicing mechanism regulated by the SR41a.
The RT-PCR analysis showed that 5 types of splicing variant are produced from the SR41a gene by the alternative splicing. By the transient assay using tobacco BY-2 protoplasts, the SR41a-GFP fusion protein was detected only in nucleus, suggesting its function as a splicing factor. To identify genes regulated by the SR41a, the analysis of expression levels studied using the transgenic plant over-expressing SR41a- or SR41a-KO mutant. Interestingly, transcript levels of SR34b and SCL33 were decreased in SR41a- KO mutant compared with those of wild-type plants.

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Cystathionine γ-synthase (CGS) of Arabidopsis catalyzes the key step of methionine biosynthesis. Expression of the CGS gene is negatively feedback-regulated at the step of mRNA stability in response to S-adenosyl-L-methyonine (SAM). The exon 1-coding region of CGS is necessary and sufficient for this regulation. The regulation was reproduced in the in vitro translation system of wheat germ extract. Studies using the wheat germ extract revealed that, prior to the CGS mRNA degradation, translational pausing was induced in response to SAM application. In this study, we developed an in vitro translation system from Arabidopsis callus cultures. A reporter assay analysis using the Arabidopsis cell extract system showed CGS exon 1-mediated downregulation of reporter activity in response to SAM. Also SAM-dependent translational pausing and mRNA degradation were reproduced in this system. Furthermore we optimized conditions to detect the response to SAM.

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Cystathionine γ-synthase (CGS) catalyzes the key step of methionine biosynthesis. Expression of AtCGS1 is down-regulated at the step of mRNA stability in response to S-adenosylmethionine (SAM). A short amino acid sequence within the first exon coding region of AtCGS1 is important. Translation arrest is observed prior to mRNA degradation in the cell-free translation system of wheat germ extract, and Trp-93 located right before the arrest site is important. Although Trp-93 is conserved among angiosperms, the two CGS sequences of Physcomitrella patens have Ala. In vitro translation of PpCGS sequences corresponding to the AtCGS1 exon 1 was performed. The PpCGS sequences responded to SAM, although the response was weaker than in AtCGS1. When the Ala codon was substituted with Trp, accumulation of mRNA degradation intermediate and translation arrest product was promoted. These results suggest that Trp-93 promotes the translation arrest, and that additional elements are required for the efficient arrest.

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Expression of the Arabidopsis CGS1 gene that codes for cystathionine γ-synthase (CGS) is feedback-regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (SAM). This regulation occurs during translation and a short stretch of amino acid sequence (MTO1 region) encoded by the first exon of CGS1 itself is involved in this regulation. Recently, we found that SAM induces translational pausing prior to the mRNA degradation.
CGS1 regulation is reproduced in a cell-free system, and intermediates of mRNA degradation accumulate that are truncated at their 5' region. Primer extension experiments revealed several different 5' ends of the intermediate that are separated by approximately 30 nucleotides from each other. In addition, multiple translation products that likely corresponded to jammed ribosomes were also detected. These results demonstrate that SAM induces ribosome stacking on the CGS1 mRNA, which likely determines the mRNA degradation points.

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Expression of Arabidopsis cystathionine γ-synthase (CGS), the key step in methionine biosynthesis, is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (SAM). The first exon coding region of CGS is necessary and sufficient. A short amino acid sequence (MTO1 region) is particularly important for this regulation. However, Cys-80 and Ser-81 within the MTO1 region were able to change to alanine without affecting the response to SAM. SAM has sulfer-linked adenosyl and methyl group. S-adenosyl-L-ethionine (SAE), which has ethyl group in place of methyl, also works as an effector. We analyzed substitutions of amino acids of MTO1 region, and the role of Cys-80 and Ser-81 will be discussed.

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Chloroplast gene expression in higher plants is thought to be regulated mainly at the post-transcriptional level. To study mRNA translation in the chloroplast, we have improved the in vitro translation system derived from tobacco chloroplasts. The new system had about 100-fold higher translation activity when compared to the previous system. Using GFP reporter gene, we established non-radioactive detection system to measure mRNA translation activity. We are currently analyzing cis-elements and trans-acting factors that are required for efficient translation initiation, and the results will be presented.

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In general, it has been thought that the codon usage is correlated with the translational efficiency, and that adjusting the codon usage is important in the high level expression in the other organisms. Recently, we have several reports to express useful proteins in the chloroplast. Since the chloroplast genome lacks tRNA genes corresponding to the most frequent alanine and proline codons, and the expression of chloroplast genes is generally regulated at the post-transcriptional level, the translational efficiency of synonymous codons in the chloroplast might be different from their usage in the genome. Here, we analyzed the translational efficiency of several synonymous codons using the tobacco chloroplast in vitro translation system.

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We have identified a rice mutant, fish bone (fib), which exhibits pleiotropic abnormalities in most of above- and under-ground organs. fib plants show abnormal phyllotaxy, disrupted vascular systems, aberrant floral organs, reduced numbers of crown roots and lateral roots, agravitropism and so on. The wild type plants treated with polar auxin transport (PAT) inhibitor mimicked most of the fib phenotypes, suggesting that fib has defects in PAT. Then, we have measured PAT activities in root and inflorescence stem. The results indicate that PAT activity is impaired in fib plants. In addition, fib plants also show reduced auxin contents and increased auxin sensitivity, but detailed analyses suggest that these are secondary effects caused by PAT defects. Thus, many abnormalities in fib are caused by defects in PAT. The FIB gene encodes novel factor, and it would play important and novel roles in controlling PAT.

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The Arabidopsis loss-of-function mutant of MSG2/IAA19 is defective in tropistic responses of hypocotyl. We examined staining pattern of MSG2 promoter::GUS in 4-day-old etiolated seedlings in response to unilateral irradiation with 0.06 umol/m²/s blue light. Without the photostimulus hypocotyls of 83% seedlings were uniformly stained. However, number of seedlings with differential staining increased in proportion to phototropic curvature. After 24-hr stimulation, 58% of seedlings showed differential staining, which appeared to develop by disappearance of the staining mainly in epidermis and cortex of the irradiated side of hypocotyls. Similar results were obtained in gravitropism. Because it was difficult to detect change of staining with the 90° reorientation, seedlings were reoriented 135° to induce stronger gravitropism. After 24-hr stimulation, 49% of seedlings exhibited differential staining. We also carried out the same experiments with the DR5::GUS seedlings. GUS staining was too faint to detect asymmetric staining in this case.

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massugu2 (msg2/iaa19) mutant grows similarly to wild type with an exception of slightly reduced fecundity. But, hand self-polination recovers infertility of msg2 flowers. In this study we observed development of flower organs to find physiological basis of reduced fecundity in msg2. In wild type flowers, anthers deposited pollen grains on stigma papilla when the length of stamens exceeded the length of a pistil. On the other hand, most stamens did not exceed pistils and failed in pollinating in msg2 flowers. Stamens elongated because of elongation of stamen filaments. Number of epidermal cells in a cell file of stamen filaments remained constant irrespective of length of filaments. It was also not changed by msg2 mutation. Analysis with MSG2promorter-GUS showed that MSG2 was expressed in stamen filaments after they started to elongate faster than ever. These results suggest that MSG2 plays an important role in cell elongation of stamen filaments.

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To investigate the molecular mechanism of gravitropism in higher plants, we have isolated Arabidopsis mutants with altered shoot gravitropism. When the wild-type inflorescence stems were placed horizontally, they curved upward about 90^o within 90 min. By contrast, a newly isolated sgr9 mutant exihibited only about 50^o curvature even after 8 hours gravi-stimulation, and the curvature never reached 90^o. No visible morphological phenotype was oveserved in sgr9, except for lateral shoots growing horizontally. The endodermal cells containing sedimentable amyloplasts are the gravity-perceptive cells in Arabidopsis shoot. In Wild-type plants, each endodermal cell has ten to twenty amyloplasts and most of them sedimented to direction of gravity. Besides endodermal cells containing sedimented amyloplasts, there are considerable number of endodermal cells containing a few floating amyloplasts in sgr9 mutant. Interestingly, these phenotypes are distinct from those of other gravitropic mutants including sgr1-8. Molecular cloning of SGR9 is currently underway.

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The formation and degradation of IAA-amino acid conjugates (IAA-aa) play an important role in IAA homeostasis. Hence the inhibition of IAA-aa synthesis is a key to understanding the mechanism of IAA homeostasis. Recently, GH3s, early auxin-inducible genes were reported to have IAA-aa synthetic activities. However, due to the presence of multiple pathways of IAA-aa synthesis catalyzed by several GH3s, a chemical approach using specific inhibitors of GH3s is expected to be more promising than a genetic one such as gene knockout. We therefore synthesized specific inhibitors of GH3 on the basis of its proposed catalytic mechanism. This chemical approach is expected to inhibit all the GH3s involved in IAA-aa synthesis, and the inhibitors are expected to serve as a highly effective tool to study IAA homeostasis. Here, we report the characterization of GH3-5 and GH3-6, and the inhibitory activities of synthesized inhibitors.

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Synthetic peptides corresponding to the C-terminus of auxin-binding protein 1 (ABP1) have been shown to function as auxin agonists. The identification of a C-terminal receptor would be key importance in analyzing the auxin-signaling pathways. To define the receptor, photoaffinity crosslinking studies were performed and isolated two types of maize proteins as candidates for the receptor. One was a GPI-anchored plasma membrane protein (termed C-terminal peptide-binding protein 1, CBP1). CBP1 was found to be a copper-binding protein, and is highly homologous to the proteins functioning on cell elongation processes relating to plant cell polarity such as Arabidopsis SKU5, SKS6 and tobacco NTP303. Furthermore, a null mutation of the ABP1 gene has been shown to disturb the directional cell elongation at the early embryogenesis. The present results indicate that an ABP1-CBP1 pathway may contribute to directional cell growth processes relating to cell polarity among the auxin-mediated cell expansion processes.

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Yeast VAM3 is a Q_a-SNARE that is involved in vacuolar transport of proteins and vacuolar assembly. Previously, we showed that atvam3 mutants accumulate large amounts of thioglucoside glucohydrolase (TGG), which hydrolyze glucosinolates to produce toxic compounds for repelling pests. Myrosin cells in Capparales plants are idioblasts that accumulate TGG. An immunogold revealed TGGs were specifically localized in the vacuole of myrosin cells in atvam3 mutants. This result indicates that TGGs are normally transported to vacuoles in these mutants and that AtVAM3 is not essential for vacuolar transport of TGG. Myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in atvam3 leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observed independent of leaf veins. Our results suggest that AtVAM3 has a plant-specific function in development of myrosin cells.

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In higher plants, leaves initiate in constant spatial and temporal patterns. Although the pattern of leaf initiation is a key element of plant shoot architecture, little is known about how the time interval between initiation events, termed plastochron, is regulated. plastochron2 (pla2) exhibits shortened plastochron and precocious maturation of leaves during the vegetative phase and ectopic shoot formation during the reproductive phase. PLA2 gene encodes a MEI2-like RNA binding protein, and is an orthologue of maize TERMINAL EAR1 (TE1). We show that PLA2 normally acts to retard the rate of leaf maturation, but does so independently of PLASTOCHRON1, which encodes a member of P450 family. Based on these analyses, we propose a model in which plastochron is determined by signals from immature leaves that act non-cell-autonomously in the shoot apical meristem to inhibit the initiation of new leaves.

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KNOX genes play an important role in formation and maintenance of the shoot apical meristem (SAM), and their ectopic expression in leaf results in the abnormal morphology of the leaf. Thus, the SAM-specific expression of the KNOX genes is essential for normal development of plants. OSH1 (a rice KNOX gene) promoter has activity in leaf, and introduction of OSH1 cDNA induced ectopic expression of endogenous OSH1 in leaf. These results indicate that the regulatory sequence of OSH1 is not limited in the promoter, and the extra copy of OSH1 perturbs its regulation. To understand a suppression mechanism of OSH1 in leaf, onion mutant was identified which resembled a severe phenotype of an OSH1-overexpressor. The onion mutant showed the OSH1 expression in leaf. ONION is a novel negative regulator of the KNOX expression, since no homologue of known KNOX negtaive regulators was mapped in the onion locus.

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Higher plants make a shoot apical meristem, a root apical meristem, epidermis and vascular system during embryogenesis. Thus, embryogenesis is a process of making basic body plan, and biologically interesting processes such as generation of positional information, axis establishment and differentiation of organs and tissues occur successively during this period. In order to understand molecular mechanisms governing organ differentiation during embryogenesis, we screened organogenesis defective mutants from embryogenesis lethal mutant stocks of rice. Because these mutants are derived from Tos17 mutagenized line, we conducted linkage analysis using Tos17 probe. In one line with globular embryo phenotype, complete linkage between the genotype of the mutation and the presence of newly transposed Tos17 was observed and recovering flanking region revealed the causal gene of this mutation. In this presentation, we report the putative function of the gene product.

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Asymmetric cell division generates two different daughter cells. Although unequal distribution of mRNA or proteins has been known to play a pivotal role to specialize each daughter cell, such molecules in plants remain largely unknown. The moss, Physcomitrella patens assures a good system for the study of molecular mechanisms for asymmetric cell division. The protoplast divides asymmetrically to generate apical stem cell and differentiated protonemal, non-stem cell, thereafter the apical stem cell continues to divide asymmetrically to generate a row of differentiated protonemal cells. We have devised a systematic overexpression screening and identified 58 cDNAs as candidates for genes that are involved in asymmetric cell division. For those candidates, we made cDNA-YFP knock-in transgenic plants by using gene targeting technique to investigate protein localization during asymmetric cell division under a control of native promoters. We found several fusion proteins preferentially accumulated in apical stem cells.

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We isolated RNA from pea seeds (Pisum sativum L. var. Alaska) at various stages of germination of pea seeds, synthesized cDNA, and used RT-PCR and western blots to study expressions of major cytoskeleton-associated proteins, 49 kDa apyrase (APY1). Neither APY1 transcript nor protein were detected during imbibition, but appeared after 16 hr germination and then increased sharply with germination time. This result was confirmed using immunohistochemistry, which showed no cells stained with antibody to 49 kDa apyrase earlier than 10 hours, a few cells were stained at 10 hours, while the entire tissue became heavily stained after 16 hours germination. APY2, a paralog of APY1, was also induced after 16 hours germination and increased similarly to APY1, but at much lower levels than APY1. We will discuss this in relation to expression of the major structural cytoskeleton proteins, actin and tubulin, during imbibition and early stages of germination of peas.

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Aldrovanda vesiculosa is an aquatic carnivorous plant, close to extinction world-wide. Its carnivorous leaves bear numerous ‘digestive’ glands, which are believed to secrete digestive enzymes. Development and secretion of protease, one of the main digestive enzymes was studied on in vitro propagated plants. Gelatin film was used to detect protease activity. No protease activity was found in leaf tissue or in liquid secreted before prey was actually trapped. However, 2 h after prey was caught strong activity was detected both in carnivorous leaves and the secreted liquid. Protease activity was also detected in several bands after Native PAGE in tissue extract and the secreted liquid 2 h after trapping and in the secreted liquid 6 h after trapping. The accompanying dynamic ultrastructural changes observed by TEM in digestive gland cells will be discussed in relation to the synthesis and secretion of the enzymes.

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Control of leaf size is central for its appropriate function. To understand mechanisms controlling leaf size, we focused on 'compensation' in which a mutation such as angustifolia3 (an3) causes a decrease in cell number in leaf primordia, which in turn induces an increase in the final size of leaf cells, thereby compensating the reduction of leaf size. In this study, we generated a series of mutants in which leaf cell number is reduced to various levels using the an3 mutant and two mutants, grandifolia1-D (gra1-D) and jaw-D, that increase the number of leaf cells. Characterization of these plants showed that compensation is induced only when the number of leaf cells is substantially reduced, suggesting a threshold level of leaf cells for the induction of compensation. This threshold is found to be associated with a specific pathway of cell proliferation, since jaw-D but not gra1-D was able to suppress compensation.

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Compensation is a phenomenon in which cell expansion is enhanced in association with decrease in cell proliferation in leaf primordia. This phenomenon suggests that intrinsic mechanisms might exist by which expansion of differentiating cell is regulated by proliferating cells in a leaf primordium. To reveal the mechanisms of the compensation, we subdivide compensation into two processes, namely, "induction (stage)" and "response (stage)". For the induction, we reported previously that induction of compensation needs reduction in cell number below a threshold level. However, details of response stage are still unclear. To address this issue, we isolated several mutants having reduced cell size but normal cell number in leaves. Then, we introduced theses mutations into angstifollia3, a typical compensation-exhibiting mutant. Interestingly, some mutations strongly suppressed the enhanced cell expansion induced by compensation. This observation indicates that the responsible genes of these mutants are involved in the response processes of the compensation

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Leaf shape is though to be determined by polar cell expansion and polar cell proliferation along the leaf axes. The genes controlling the polar cell expansion and the proliferation during the leaf morphogenesis are largely unknown.
We identified a dominant mutant of Arabidopsis thaliana, mco1-D ,which possess expanded rosette leaves( screened from T-DNA transformed activation tag lines made by W. Scheible). Another phenotypes of this mutants are short hypocotyls and increased lateral root formation.The mutant which exhibited the above phenotypes was heterozygote of the T-DNA insertion (confirmed by PCR). Then we detected the T-DNA insertion site using the plasmid rescue method. Expression levels of the four genes neighboring the T-DNA insertion site of mco1 have been detected using Real Time RT-PCR.One gene was enhanced in mco1-D. This gene encodes glucose-methanol-choline (GMC) oxidoreductase family protein and contains the FAD binding domain (from TAIR http://www.arabidopsis.org).
Now we analyse the function of this gene.

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In Arabidopsis thaliana, leaves have adaxial side (upper side) and abaxial side (lower side), which differ in shapes of cells and number of stomatas or trichomes. Recently, some genes are shown to play important roles in the establishment of the adaxial/abaxial identity. The expression regions of these genes are restricted to the either side. It has been suggested that the adaxial- or abaxial-specific expression patterns require the pre-exising polarity in the leaf primordia. What mechanisms determine the polarity is not well known. To understand the mechanisms, we try to find new genes involved in the adaxial/abaxial specific expressions. To obtain mutants in which the expression pattern alter, we screened several mutants from mutagenized FILp::GFP transformants, which express GFP on abaxial side. #1-63 mutant express GFP on adaxial side also. We will report analyses of phenotypes and expression patterns of adaxial makers in #1-63. Cloning of the gene is in progress.

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A heterophyllous plant of Ludwigia arcuata, displays developmental plasticity; ethylene-treated young leaves matured into spoon-shaped leaves, while untreated leaves matured into round-shaped aerial leaves. Comparison of developmental process between such spoon-shaped and control leaves revealed that the alternation of distribution, orientation, and frequency of cell divisions was induced. Here we intend to clarify the relationship between cell division pattern and leaf shape formation and try to establish a dynamic model of leaf development. This model follows subsequent three conditions: preservation of cell-cell relationships (1), recovery of cell size to original ones after division (2) and parameters such as distribution, orientation, and frequency of cell division being considered (3). Computer simulation on these was reflected in the growth for the leaf transverse directions. However, this consideration is not sufficient to the longitudinal direction, implying growth for longitudinal direction needs more considerations. Details on modeling will be explained by the next speaker.

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To clarify how cell division patterns affect ultimate leaf shape, we intended to establish a dynamic model of leaf development. To do this, input data are measured values with L. arcuata, while outputs are their final shape. This was conducted under following assumptions: 1) cell sizes in meristematic regions being identical, 2) sizes of daughter cells tending to recover to original, 3) numbers of cells not decreasing and 4) intercellular relationship being preserved. To prove how our model would reflect actual data, after data of varying situations of L. arcuata are incorporated, deviation from calculated values is compared with actual ones. Currently, the growth of leaf lamina for the leaf transverse direction was observed, however, that for the leaf longitudinal direction needs some more considerations. We are looking for factors that regulate the longitudinal growth, with speculating that a possible candidate for such factor is the cell shape.

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ASYMMETRIC LEAVES2 gene encodes a plant specific protein with cystein repeats, conserved-glycine and leucine-zipper-like sequence in the N-terminal half. AS2 is involved in the development of a flat and symmetric lamina. In the seedlings of ProAS2:GUS transgenic plants, GUS expression was observed not only in the shoot apex and the developing leaves but also in the roots. Although GUS expression was observed in the root, the root of as2 mutant resembled that of wild-type. To understand the function of AS2 in the root, we investigated the root tips of AS2-GR transgenic plants which induced nuclear translocation by DEX treatment. AS2-GR transgenic plants grown on the plate containing100 nM DEX exhibited severe phenotypes and died on several days. We investigate the root of AS2-GR transgenic plants grown on the plate containing low concentrations of DEX.

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We isolated a recessive mutant defective in root hair elongation from RIKEN Arabidopsis Ds transposon insertion lines. In the mutant, 15-1096-1, the tip growths of almost all root hairs are stopped and their tips are ruptured. The transposon is inserted into a gene encoding a glycosylphosphatidylinositol-anchored protein. Genetic analysis and DNA sequencing revealed that 15-1096-1 is allelic to shaven3 whose responsible gene has not been identified yet (Parker et al., Plant Cell, 2000, 12, 1961-). Histochemical staining of transgenic plants harboring a SHV3 promoter::GUS gene showed that SHV3 is predominantly expressed in the root hair cells. In addition, SHV3-GFP fusion protein was localized to plasma membrane. The burst of root hair tip suggests that SHV3 is essential for modulation of cell wall strength during the tip growth. The progress of our study will be presented and the possible function of SHV3 will be discussed.

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O. corniculata exhibits a wide range of variations.Some have decumbent branches, and another have ascendant ones.Also some petioles are oblique and the others arrange horizontally.
When excised leaves with oblique petioles placed horizontally in the dark, the petioles bend upward.Excised petioles set on a 1D clinostat with rotating lamp, also,bend toward to the light source.But, petioles treated with NPA, did not respond to the gravitropic stimuli or photo stimuli.These observations indicated that the gravitropic and phototropic response of the O. corniculata petioles were associated with polar auxin transport in them.
The elongation zone of the petioles shifts from the basal ends to the top ends, during the elongation period.Amyloplasts in the starch sheath disappeared as the elongation zone move toward the top ends of the petioles.The amyloplasts finally disappeared from the bending region to ca. 5mm above the abscission layers.

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It is believed that there are not enough number of mutants available for understanding molecular mechanism of gravity response in root. That is because root gravitropic mutants currently available do not include ones defective in the signalling pathway. Such mutants of the signalling pathways may be less defective in gravitropism. We therefore need to develop a screening system to isolate such mutants. It has been described that gravitropism in Arabidopsis roots interacts with phototropism. In this study, for isolating novel Arabidopsis mutants of root gravitropism by screening of the M₂ seedlings, we developed a screening system to detect less defective phenotype in root gravitropism by using the interaction between gravitropism and phototropism. Then, we screened approximately 100,000 ethylmethanesulfonate (EMS)-mutagenized M₂ seedlings of Arabidopsis and obtained 44 lines that are defective in gravitropism. In addition, we will present genetic analysis of our isolated root gravitropic mutants.

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The mechanism of the gravitropic bending was studied in azuki bean epicotyl. It is well known that orientation of cortical microtubule (cMT) changes during the bending. However, we reported that the change of cMT orientation was not caused by gravity situation, but by bending. In the present study, we analyzed the change of extensibility of epicotyl upon gravi-stimulation. We measured cell wall extensibility in the bending part for gravitropism. The extensibility of the lower side was higher than that of the upper side in epicotyl bending upward. After an epicotyl was fixed to a hard metal rod to prevent bending, gravi-stimulation was applied. It was found that the extensibility of lower part was increased, although bending did not occur. Thus, it is concluded that increase in extensibility of lower part is induced via a process other than cortical microtubules at least in the early step of gravi-response.

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As plant roots explore, they sense the physical hardness of the soil. Little is known about the genes involved in touch sensing, or the subsequent signal transduction pathways and responses in plants. We therefore isolated Arabidopsis weak touch response mutants 1 and 3 (wtr1 and 3), whose primary roots were defective in the bending reaction to physical hardness of the growth medium. The proposed roles of ethylene in thigmomorphogenesis led us to test the responses of wtr1 and 3 to exogenously applied ACC. However, the response of their roots was similar to that of wild-type. Both wtr1 and 3 roots showed normal gravitropic response. On the other hand, the root growth pattern of wtr3 showed an increased tendency to produce coils on inclined agar medium, compared to the wild type. Characterization of the mutants is now in progress to elucidate the molecular mechanism by which roots sense soil hardness.

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Rice is the most Al resistant species among small-grain cereal crops, but the mechanism underlying its high Al resistance is still poorly understood. In the present study, a gene which is associated with Al tolerance in rice was cloned and characterized using an Al-sensitive rice mutant (als1). Rough mapping shows that Als1 was localized on chromosome 6. Fine mapping resulted in identification of Als1. By comparing the sequence of Als1 between the wild type and the mutant, a 15 bp deletion was found in the mutant. Als1 consists of four exons and three introns, encoding a 291 amino acid protein that is predicted to be a putative ABC transporter. The expression of this gene is relatively higher in the roots than in the shoots in the absence of Al and the expression was up-regulated by Al in the root tips but not basal roots.

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Barley is one of the most aluminum sensitive species in the small cereal grains. However, a wide genetic variation in Al tolerance has been observed. It was reported that Al tolerance was tightly linked to the Al-induced citrate secretion from the roots. To identify the mechanisms leading to Al-induced citrate secretion, gene expression patterns of Al-tolerant cultivar (Murasakimochi) and -sensitive cultivar (Morex) were investigated using a barley genechip containing 22840 probes. Total RNA was extracted from root tips (1cm) exposed to 0 or 5 μM Al for 6 h. Results show that 149 and 211 genes were up-regulated more than 2-fold by Al, respectively, in Murasakimochi and Morex. In Murasakimochi, genes related to transporter were not induced by Al, but some transporter-related genes show high and constitutive expression pattern compared to Morex. Time and tissue-dependent expression of these genes are now under investigation.

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In an effort to understand the molecular mechanisms of an Al-induced malate efflux from Brassica napus, we isolated two Al-induced cDNAs named BnALMT1 and BnALMT2. Analysis of expression pattern showed that the transcript of the genes is accumulated in response to Al treatment. This was observed after 2 h Al treatment. However, exposure to Al for longer than 4 h did not further increase gene expression. Transcript accumulation in P-sufficient plants increased with increasing Al concentration up to 100 μM whereas in P-deficient plants expression did not increase above 50 μM Al. Furthermore, these genes were expressed exclusively in the roots. Over-expression of BnALMT1 and BnALMT2 in cultured tobacco cells (Nicotiana tabacum L. nonchlorophyllic) significantly induced malate efflux in the presence of 100 μM Al. Other lanthanides (La, Er and Yb) could only slightly induce malate efflux, indicating that the transporter is primarily activated by Al.

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Plants resistant to Al stress were isolated from Arabidopsis enhancer-tagging lines. RT-PCR experiments indicated that one of the resistant candidates (#355-2) showed a higher expression of two genes on chromosome 1, F9E10.5 and F9E10.6, compared with Col-7 control line. Transgenic plants over-expressing the F9E10.5 gene showed a higher resistance than a control line.
The #355-2 line always showed a lower Al content in whole roots, and the root hair length of this line was approximately 30% of that of Col-7. Both Al influx and associated oxidative stress occurred in root hairs, as well as in root tips of Col-7, however staining was seen only in root-tips of #355-2. Moreover, transgenic plants over-expressing the F9E10.5 gene showed shorter root-hairs than control.
Resistance of #355-2 line may be due to a lower Al uptake from the short root hair derived from an over-expression of F9E10.5 gene caused by activation tag.

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AtGST11 is one of GST gene family that was induced by various stresses. Precise response mechanism of AtGST11 gene at transcriptional level during Al stress is now investigated in this study. A cDNA library was constructed from Arabidopsis treated with 0.1 mM Al and cloned in T7 phage dispay library. Phage lysate was hybridized with biotynelated AtGST11 promoter. This DNA/protein complex was captured using streptavidin paramagnetic particles. Fifty plaques were selected to determine the insert by PCR amplification followed by DNA sequencing. Ten plaques were determined to contain the desired inserts. Homology search against several databases revealed that one of these (#43) is a transcription factor HD-Zip encoded by ATHB6 gene which has known to be up-regulated to various abiotic stresses. This putative transcription factor is being confirmed by gel-shift assay. The existence of other proteins may as well suggest the transcription of AtGST11 involving complex factors acting as regulators.

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Phytochrom B (phyB) is a dimeric chromoprotein that detects the quantity, quality, and duration of red or far-red light throughout the entire life cycle of plants. Upon absorption of red light, phyB translocates from the cytoplasm to nucleus, and regulates gene expression through interaction with transcription factors such as basic-helix-loop-helix proteins. The PAS domain within the phyB C-terminal domain contains determinants necessary for nuclear translocation and signal transduction. Here we showed the solution structure of PAS1 domain, one of two PAS domains, determined by multidimensional NMR spectroscopy. Both the core region of a loss of function missense mutations (referred as the "Quail-box") and dimerization site were mapped on the β-sheet side, which was constituted of conserved residues among all phyAs and phyBs. Stable dimer formation of two PAS1 domains on the β-sheet side will be linked to phytochrome function.

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We have isolated all phytochrome mutants (phyA, phyB and phyC) from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyBphyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant de-etiolation responses. The responses to Rc were completely canceled in phyAphyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control de-etiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired de-etiolation and phyAphyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyBphyC double mutant displayed clear R/FR reversibility in the pulse-irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression.

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Based on its domain organization, fern phytochrome 3 looks like a chimeric protein composed of both red- and blue-light photoreceptors. N-terminal half of PHY3 contains a chromophore-binding domain of phytochrome and its C-terminal half shows a remarkable similarity to phototropin, containing two LOV domains and a Ser/Thr kinase domain. This unconventional domain organization implies that this single chromoprotein has both phytochrome and phototropin properties. We have demonstrated that red-light induced chloroplast accumulation response of fern was mediated by this photoreceptor, however, it is still unclear whether this photoreceptor can work as a blue-light photoreceptor or not. To address this question, we introduced CaMV 35S promoter driven PHY3 gene into Arabidopsis phot1-5/phot2-1 double mutant line. Here we report that PHY3 plays a role for both red- and blue-light mediated phototropic response in Arabidopsis. This result shows that fern PHY3 acts as not only red-light photoreceptor but also blue-light photoreceptor.

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Phytochrome photoreceptors regulate various physiological light responses in plants. In Arabidopsis, phytochrome interacting factors (PIFs) directly bind to both phytochromes and specific DNA elements in vitro, and regulate phytochrome-dependent gene expression to control seed germination, chlorophyl biosynthesis, and possibly, flowering time. Arabidopsis PIFs contain a phytochrome-interacting domain termed APB at N-terminus and a bHLH domain at C-terminus. Here, we identified 10 PIF-like genes based on this structure in rice genome. Interestingly, some rice PIF-like genes have only the APB, but no bHLH domain. Detailed motif analysis further revealed that several novel protein motifs were conserved in PIF-like proteins between rice and Arabidopsis, suggesting their evolutional relationship. Quantitative RT-PCR analysis indicated that some PIF like genes including the solo-APB proteins, showed distinct diurnal expression patterns between wild-type and se5, a phytochorme- deficient mutant of rice. This indicated that some PIF-like genes are regulated by phytochrome signaling in rice.

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Regulatory mechanisms of stress responses in rice largely remain unclear, compared to those in Arabidopsis. Using rice microarray, we identified many abiotic stress-responsive genes. Among them, a gene for a bHLH transcription factor down-regulated by drought stress has been studied. The bHLH protein showed a high sequence homology with Arabidopsis PIF1, driving us to name the transcription factor OsPIF1. Transgenic Arabidopsis plants overexpressing OsPIF1 and dominant loss-of-function Arabidopsis mutants with the chimera repressor changed growth rates and drought stress tolerances, suggesting that OsPIF1 functions as an important growth regulatory factor in response to drought stress in rice. Here, we generated transgenic rice plants overexpressing OsPIF1 and dominant loss-of-function rice mutants with the chimera repressor. The former displayed long length grains and giant culms with elongated internodes. The latter exhibited conversely short length grains and dwarf culms with short internodes. Currently, we are analyzing target genes for OsPIF1.

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In the root of Arabidopsis thaliana, the transcript of a gene encoding putative pathogenesis-related protein 1 (PR1) increases under FR light illumination to the whole plant, while an aquaporin gene, TIP2;2, decreases. We analyzed a gene expression manner of the two FR-light-responsive genes in a Ler plant and various mutants regarding phytochromes when only shoots or roots were illuminated by FR light. The induction of PR1 and the repression of TIP2;2 in roots were caused by FR light illumination to only shoots of Ler plants. When only roots were illuminated by weak FR light, however, both PR1 and TIP2;2 mRNAs increased. The two mRNAs also increased under FR light illumination to only hypocotyls of Ler plants from which leaves and buds were removed. The results suggest that one signal transported from the shoot and another generated in a root cell exist in light-regulated gene expression in roots.

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Light is perceived by three types of photoreceptor, red/far-red light receptor phytochromes (phy), UV-A/blue-light sensors phototropins (phot) and cryptochromes (cry). In red-light, hypocotyls of A. thaliana exhibit a random growth pattern due to the inhibition of gravitropism and random hypocotyl-bending. To understand the mechanism which induces the random hypocotyl-bending, we isolated an A. thaliana mutant, flabby (fby), which shows enhanced random hypocotyl-bending in red-light. FBY encodes the AtPGP19 protein which belongs to the multi-drug resistance subfamily of ABC transporters involving the auxin distribution. phyA phyB fby triple-mutant did not exhibit the red light-induced random hypocotyl-bending. The activity of polar auxin transport was inhibited by red-light or the defect of fby. Furthermore, phot1 phot2 fby triple-mutant showed the random hypocotyl-bending under blue-light. Western analysis of PGP19 revealed that red-light-activated phytochromes and blue-light-activated cryptochromes reduce PGP19 accumulation in hypocotyl. These results suggest phytochromes and probably cryptochromes negatively regulate PGP19, resulting in hypocotyl-bending.

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RSG is a bZIP transcriptional activator that regulates endogenous amounts of gibberellins. The endogenous level of GA decreased in the transgenic tobacco expressed of dominant negative form of RSG (dnRSG). The transgenic tobacco exhibited dwarf phenotypes.We investigated the expression levels of six GA biosynthetic genes. RSG regulates GA biosynthesis through the transcriptional control of ent -kaurene oxidase gene.
dnRSG inhibits the feedback regulation of the GA20-oxidase (Ntc12) gene. GAs regulates intracellular localization of RSG. To search where is necessary to feedback regulation in Ntc12 promoter, we produced transgenic tobacco that GUS gene fused to Ntc12 promoter. We identified a cis element that is important of GA feedback regulation. RSG binds this element directly. The point mutation of this element in Ntc12 promoter is disrupting the feedback regulation. These results indicate that RSG regulates feedback regulation of GA in Ntc12 promoter.

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Gibberellins (GAs) are essential regulators of many aspects of plant development. GA 3-oxidase catalyzes the final step of the biosynthetic pathway to produce the active GA. The expression of AtGA3ox1 (GA4) encoding GA 3-oxidase is regulated by GA levels in a feedback manner. To understand the molecular mechanism of feedback regulation, we have identified a cis-acting sequence responsible for GA-negative feedback of AtGA3ox1. Furthermore, we isolated an AT-hook protein AGF1 as a DNA-binding protein for the cis-acting sequence. The mutation in the cis-acting sequence abolished both GA-negative feedback and AGF1 binding. The expression of AGF1 is not regulated by GA levels. This suggested an involvement of post-transcriptional and/or post-translational modification of the transcription factor. We have identified Zn finger protein ZAF1 as an AGF binding protein by a yeast two-hybrid screen. In vitro pull-down assay shows that ZAF1 specifically binds to AGF1 and its binding is direct.

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