Gibberellin (GA), which is synthesized after germination, is involved in expression of a gene for a proteinase, EP-C1 in germinated seeds of common bean. It is known that DELLA proteins are involved in repression of GA-response genes. The genes are activated by degradation of the proteins induced by GA treatment. We isolated two types of cDNA clones for DELLA proteins designated PvGAI1 and PvGAI2, respectively. Because expression level of PvGAI2 was higher than that of PvGAI1 in germinated cotyledons, RNA blot analysis was carried out using cDNA for PvGAI2 as a probe. PvGAI2 mRNA was not detected in cotyledons of dry seeds, and appeared at 1 day after imbibition started (DAI), its level increasing until 3 DAI. In contrast, EP-C1 mRNA appeared at 3 DAI. These results suggest that GA synthesized after germination induces degradation of PvGAI2, which is required for activation of the EP-C1 gene.

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srd2 is a temperature-sensitive mutant of Arabidopsis thaliana that were characterized by defects in various tissue culture responses including hypocotyl dedifferentiation and shoot redifferentiation. The SRD2 gene encodes a nuclear protein similar in sequence with human SNAP50, functioning in activation of snRNA transcription.
Analysis with seedlings indicated that SRD2 is strongly expressed in apical meristems, leaf primordia, and central cylinders of roots, which were largely overlapped with expression of U2.3 snRNA promoter-driven reporter gene. The srd2 mutation caused morphological lesions in these region at the restrictive temperature. In the course of lateral root development, snRNAs, which were abundant in young primordia. disappeared at the end of primordium formation, and then increased again. The srd2 mutation disturbed this increase, which was associated with ill establishment of apical meristems. These findings suggested that snRNA transcription activation by SRD2 is required not only for tissue culture responses but also morphogenesis during normal development.

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AS1 and AS2 regulate development of leaf and floral organs and repress the expression of class 1 knox genes in their organs. AS1 and AS2 encode a myb-like protein and a protein with the AS2/LOB domain, respectively. To understand the molecular mechanisms in which AS1 and AS2 are involved, we examined subcelluler localization of GFP-AS1 and AS2-YFP in Arabidopsis cells. The fluorescence of AS2-YFP was detected as a few dots or bars at the periphery of the nucleolus. The fluorescence of GFP-AS1 was observed as dots in the nucleus. In addition, when AS2-YFP and GFP-AS1 were expressed in the same cells, GFP-AS1 was colocalized with AS2-YFP at the periphery of the nucleolus. It might be possible that AS2 functions with AS1 at the periphery of the nucleolus.

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Leaves are indeterminate organs and possess genes involved in establishing leaf dorsoventrality. These polarities are established relatively early during leaf development and defined relative to the SAM. However, no direct factor has been found in the genetic interaction between meristem and differentiation of leaf organ. Our previous study revealed that. Deformed Root and Leaf1 (DRL1) is involved in the regulation of meristem activity and leaf defferentiation. The DRL1 was found to encode a novel protein showing homology to Elongator-associate protein (EAP) of yeast KTI12. The expression of DRL1 of Arabidopsis can complement KTI12, suggesting that DRL1 may act as an EAP in higher organism. To characterize the function of EAP, we identified the genes encoding RNAPII Elongator subunits (AtELPs) of Arabidopsis. The molecular architecture of Arabidopsis holo-Elongator has investigated by the use of yeast two-hybrid system. Based on these results, we will discuss the putative function of DRL1 and AtELPs.

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Cryptomeria japonica is one of major conifer species. We consider genetic modification (GM) also can be a powerful tool for breeding. But the potential of GM plants to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. For the purpose of creating male sterile GM Cryptomeria japonica, we are trying to identify genes related to male flower formation.
Plants contain a variety of MADS-box genes that play important roles in both the formation of flower meristem and the determination of floral organ identity. We have isolated and characterized 6 MADS-box genes from Cryptomeria japonica using PCR amplification and RACE method. RT-PCR analysis revealed that one of these genes, M8, was expressed stronger in male strobilus and its transcriptional activity was increased according as the development of male strobilus. Now we are trying to determine its localization by in situ hybridization.

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Size of leaves is determined by cell number and cell size. These two parameters are dually affected in several mutants that exhibit "compensation" which consists of increase in cell size in response to reduction of cell number. In this study, to understand leaf size control, we analyzed several compensation-exhibiting mutants, namely fugu1 through fugu5, angustifolia3 and erecta mutants, and KIP-RELATED PROTEIN2 overexpressor. Comparative kinematic analyses revealed that compensation is induced after the exit from mitotic cycle. Ploidy-level analyses in the above-mentioned mutants revealed that endoreduplication is not essential but might partially contribute to increase cell size. Importantly, enhanced cell expansion in compensation-exhibiting mutants is mediated by either increase in expansion duration or expansion rate depending on mutation. These results suggest that compensation is not mediated by a single pathway, but at least two different pathways are involved in it. Positional cloning of fugu2 and fugu5 genes is now in progress.

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BOP1 gene is thought to regulate meristematic activities in leaf primordium of Arabidopsis thaliana, since bop1-1 mutant exhibits indeterminate growth of leaf blade. To understand regulation mechanism of determinate leaf growth by the BOP1, bop1-1 mutation was introduced into yab3/fil mutant, which has defect in establishment of dorsoventrality in leaves. The triple mutant did not exhibit indeterminate growth of leaf, suggesting that the indeterminate growth of leaf in bop1-1 depends on establishment of dorsoventrality. KNAT1, a class I KNOX, is also considered to be involved in determinate nature of leaf growth. When 35S::KNAT1 was introduced to bop1-1 mutant, leaflet-like structures were repeatedly produced on a leaf. This result suggests that BOP1 regulates determinate growth of leaf blade, while KNAT1 permits repeated morphogenesis.
Some plants exhibit indeterminate leaf morphogenesis like the bop1-1 mutant. We will also show results of our analyses on homologs of BOP1 isolated from such plant species.

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Plants form sun or shade leaves depending on light environment. Sun leaf shows thicker lamina and higher photosynthetic rate than shade leaf. It was reported that light environment of mature leaves determines whether novel leaf will develop to sun or shade leaf, suggesting existence of long-distance signaling. We hypothesized that this signal may be sugars as products of photosynthesis. To test the hypothesis, we analyzed 3rd-leaves of Arabidopsis plants cultivated on MS-medium with various sucrose concentrations for 14d. Number of cell layers increased with the increase of sucrose content, indicating that sugar affects on leaf development. Next, plants were transplanted to new medium with various sucrose concentrations at 10d after sowing. Number of cell layers was affected only when the plants transplanted to medium with higher sucrose concentration. Thus, leaves have plasticity at this stage. We will also discuss on results of transplantation at 8d and 12d.

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The loss-of-function mutations in the ASYMMETRIC LEAVES1 (AS1) or in the ASYMMETRIC LEAVES2 (AS2) genes of Arabidopsis thaliana cause pleiotropic phenotypes such as downward curling of leaves and cotyledons, reduced complexity of leaf venation pattern, generation of lobes, and slightly shorter petiole. The AS1 encodes a myb-like transcription factor. The AS2 gene encodes a protein with a novel domain (AS2/LOB domain) that contains cysteine repeats (C-motif), conserved glycine, and a leucine-zipper-like sequence. The genes might function in maintaining leaf cells in a developmentally determinate state, probably by repressing expression of class I KNOX homeobox genes. To clarify the function of AS2, we performed a genetic screen for the enhancer of as2. We will report several candidate mutants and its phenotypes.

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In model eudicot, Arabiodpsis thaliana, the genetic basis of lateral organ polarity has been the focus of a number of studies, leading to the identification of key regulators involved in abaxial-adaxial specification of lateral organs, such as, PHABULOSA-like HD-ZIP III gene family and KANADI gene family, for adaxial and abaxial cell fate determination, respectively. Leaf organization in grass species is largely different from that in eudicots, and grass leaves show some unique characters along with abaxial-adaxial axis. For example, mesophyll of grasses shows no distinct differentiation into palisade and spongy parenchyma, and instead, some abaxial- or adaxial-specific tissues differentiate in epidermis. Therefore, it is of interest to study whether the genetic mechanisms for the abaxial-adaxial specification are conserved between grasses and eudicots or not. In this report, we show molecular genetic studies about the functions of PHABULOSA-like and KANADI-like genes in rice by expression analyses and gain-of- or loss-of-function approaches.

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Previously, a high-level expression a D-type cyclin gene was observed in transgenic rice regenerants with multiple shoots caused by overexpression of a T-DNA ipt gene. According to its sequence characteristics, the cyclin gene was classified into type D4;1 (designated as OsCYCD4;1). Though the overexpression result implied that OsCYCD4;1 is regulated by cytokinins, addition of various phytohormones including BA on rice tissues did not increase the transcripts. For more detailed analyses of OsCYCD4;1 expression, transgenic plants carrying OsCYCD4;1 promoter::gus gene were produced. GUS staining of these transgenic plants and in situ hybridization analysis showed that OsCYCD4;1 was not transcribed in meristems, but in procambium at the early stage of vascular development and also differentiated vascular bundles except xylem. Moreover, ectopic expression of OsCYCD4;1 caused disorganized vascular bundles. The results suggested a role of OsCycD4;1 in the development of vascular systems, which is different from those of typical mitotic cyclins.

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The development of the leaf in the monocotyledon progresses almost in one dimension, because cell division is restricted in the base part of leaves. Therefore, it is easy to follow the process of vascular development in the leaf. In order to elucidate the vascular formation system in the monocotyledon, we used rice as a model plant.
We investigated the pattern formation and internal structure of the vascular tissue with the cleared leaf blades and the continuous sections in the basement of the rice plant. From the precise observation, we could divide the vascular development into several stages. Based on this information, we screened mutants defective in vascular formation. As the result, we succeeded in identifying 11 lines of vein pattern mutants and a line of vascular structural mutant. From these results, we will discuss the mechanism of vascular formation in rice leaves.

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We inserted a T-DNA containing a tetramer of the CaMV 35S enhancer coupled with the CaMV 35S minimum promoter into Nipponbare and generated 13,000 activation-tagged rice lines. We selected a Short grain 1 (Sg1) mutant that also showed a semi-dwarf phenotype. Sg1 phenotype was linked to the T-DNA insertion. A full-length cDNA, about 1.4 kb downstream of the inserted T-DNA, was highly expressed in the leaf blades of Sg1 but it was not detected in those of wild-type (WT) plants. In WT plants, this cDNA was expressed mainly in young panicles with developing lemma and palea primordia. The deduced protein shows no homology with other known proteins. These results strongly suggest that transcriptional activation of this novel gene caused the Sg1 phenotype. Transgenic rice lines overexpressing the cDNA are being generated for further characterization.

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The high productivity of sweet potato (Ipomoea batatas) is due to the sink potential of storage root. Although the process of storage root development has been studied anatomically and physiologically, its molecular basis remains largely unknown. To identify the genes involved in the process, we isolated four knotted1-like homeobox (KNOX) sequences from the cDNA of developing storage root, and named Ibkn1 to Ibkn4. Phylogenetic analysis based on the homeodomain sequence showed that the Ibkn1 was homologous to the SHOOT-MERISTEMLESS gene of Arabidopsis. Also, Ibkn2 and Ibkn3 were homologous to the KNAT1 gene, and Ibkn4 was homologous to the KNAT2 gene. Expression analysis showed that Ibkn2, Ibkn3 and Ibkn4 were highly expressed at the stem and the storage root. Ibkn1 was also expressed at the stem, but its expression in the storage root was low. Further expression analyses are ongoing to elucidate the physiological function of these KNOX genes.

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Rice EL5 gene encodes an ubiquitin ligase (E3) containing a trans-membrane motif and a RING-H2 finger domain. Since transgenic rice plants overexpressing mutated EL5 lacking E3 activity showed rootless phenotype, it is suggested that EL5 functions as E3 during crown root and lateral root development after meristem differentiation in rice.
In this study, we conducted expression analyses of the EL5. Northern-blot analyses revealed that EL5 mRNA was abundant in the basal region of shoots and roots. The expression was induced by auxin, cytokinin and jasmonic acid in the basal region, and by cytokinin and jasmonic acid in roots. Transgenic rice plants with the EL5 promoter::GUS gene showed strong GUS activity mainly in vascular bundles of the basal region of shoots. Cytokinin strongly induced the GUS activity in roots. Western-blot analyses indicated that EL5 was localized to membranes after N-terminal processing and regulated by its E3 activity.

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A combined Fourier-Transform Infrared (FTIR) microspectroscopy and principle component analysis (PCA) was used to investigate chemical variations between wild-type and root-hair mutants in Arabidopsis. Using this system, we showed that the gl2 mutation affected cellulose synthesis in roots. There are ten genes encoding cellulose synthase (AtCesA1 to 10) in Arabidopsis, five of them are expressed in the root, and of them, only the AtCesA5 gene was affected by the gl2 mutation.

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A root hair is formed by tip-growth of a root hair cell. It was proposed that phospholipase D (PLD) ζ1 is involved in root-hair cell morphogenesis (Ohashi Y et al. 2003.). This suggested that root-hair cell morphogenesis is controlled by lipid signal transduction. On the other hand, it was revealed that phosphatidyl inositol bisphosphate stimulates many kinds of signaling molecules including Phospholipase D in animal cells. To clarify the mechanism of the root-hair cell morphogenesis, we focused on phosphatidyl inositol phosphate kinase (PIPK). The Arabidopsis genome contains 11 PIPK genes. Northern hybridization analysis revealed that AtPIPK3 was preferentially expressed in roots. T-DNA insertion mutants of AtPIPK3 gene were analyzed, and their root-hair lengths were significantly reduced. In addition, gene expression and the intracellular localization of the AtPIPK3 protein is analyzed. These results suggests that AtPIPK3 gene take part in the root hair tip-growth.

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Many monocots have two whorls of petaloid organs. To explain this floral morphology, modified ABC model was hypothesized that class B genes express in whorl 1 (W1), besides W2 and W3. This model was supported by the expression analysis of class B genes in tulip and agapanthus, but not in asparagus. Thus, applicability of this model in monocots remains unclear.
To inspect this model, we analyzed the tepal morphology and class B genes expression in alstroemeria, a monocot which has distinguishable outer and inner petaloid perianth. Outer and inner tepals differ in shapes but resemble in vascular bundles and epidermal cells. We isolated three class B genes (AlsDEFa, AlsDEFb and AlsGLO). AlsDEFb and AlsGLO were expressed in W1, W2 and W3, whereas AlsDEFa was detected only in W2 and W3. This supports modified ABC model and suggests that AlsDEFa expression may correlate with morphological difference of outer and inner tepals.

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SBP-box genes encode plant-specific transcription factors originally isolated from Antirrhinum as factors binding to promoter of SQUAMOSA, a floral meristem identity gene. In Arabidopsis, 16 SBP-box genes (SPL1-SPL16) were identified, and some of them were shown to have binding ability to the promoter of APETALA1, the Arabidopsis homolog of SQUAMOSA. There are few reports regarding the function of SBP-box genes, probably because of existence of functionally redundant genes. Therefore, we analyzed function of SBP-box genes using CRES-T system in Arabidopsis, in which a chimeric repressor acts dominantly both to endogenous and functionally redundant transcription factors. SPL2, SPL10 and SPL11 sharing high sequence homology were highly expressed in flowers. While loss-of-function mutant of SPL2 showed no visible phenotype, transgenic plants that express the chimeric SPL2 repressor had short petals and pedicels as well as reduced apical dominance. This suggests that SPL2, SPL10 and SPL11 are functionally redundant.

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Cell division and cell expansion have particular importance for developmental process of stem elongation. The receptor-like kinase ERECTA (ER) regulates the inflorescence architecture by affecting cell proliferation of the internodes and pedicels in Arabidopsis. To gain insight into inflorescence development, we have isolated CORYMBOSA1 (CRM1) gene. crm1-1 displays a compact inflorescence due to reduced cell elongation of internodes and pedicels. Double mutants of crm1-1 with er and crm2 show additive phenotype, suggesting that CRM1, ER, and CRM2 promote inflorescence growth via independent pathways. Fine mapping and allelism test confirmed that crm1-1 is allelic to doc1, and is caused by mutation in BIG, which is required for normal auxin transport. In order to investigate the requirement of regions for BIG function in vivo, an allelic series of big mutations, named reduced lateral root formation (rlr), is characterized. Further characterization of the phenotypes of rlr mutants is in progress.

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Arabidopsis FLAKY POLLEN1 (FKP1) is the only gene encoding HMG-CoA synthase in mevalonate pathway, which involves in pollen coat formation and pollen tube elongation. This gene expresses both in tapetum and pollen. To examine which expression is responsible for the pollen coat formation, and for the pollen tube elongation, we joined FKP1 cDNA to tapetum specific (SP11) or pollen specific (LAT52) promoters, and introduced them into the fkp1 mutant. We found the pollen coat formation was recovered only by SP11:FKP1. Examining the pollen tube elongation is in progress.
SHEPHERD (SHD) encodes a Hsp90-type molecular chaperon resident in the ER. The shd pollen grains show defects in exine formation and pollen tube elongation. The pollen tube elongation was recovered by LAT52:SHD, whereas the exine formation was not by both promoters. The exine formation may require the SHD expression in both tissues, or earlier expression than SP11 and LAT52.

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The Arabidopsis acaulis1 (acl1) mutants, originally isolated in a screening for the mutants with reduced stem lengths, have curly leaves, compact rosettes and few flowers. The defect in the stem elongation in acl1 mutants is due to reduced cell size. Vascular cells and guard cells are not fully differentiated in a severe allele. Moreover, callose is accumulated ectopically in the stems of acl1 mutants. The acl1 phenotype is not rescued by phytohormones such as auxin, gibberellin or brassinosteriod. However, when grown at higher temperature, the mature acl1 mutants are indistinguishable from wild-type plants. Also, by adding NH₄NO₃ into the growth medium, the acl1 phenotype was partially rescued. Both acl1-1 (severe allele) and acl1-3 (weak allele) are recessive mutations. Genetic mapping revealed acl1 is located on chromosome 4.

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B3 domain is the plant specific DNA binding domain. More than 40 genes were found to code proteins that contain B3 domain in Arabidopsis. B3 domain family contains ABI3, FUS3, LEC2 and ARFs, which have been shown to play an important role to the plant growth and developments, such as regulation of the expression of seed-storage genes or auxin signaling. However, the functions of most of the B3 genes remain to be clarified.
To analyze the function of B3 family genes, we applied CRES-T system to this family and expressed the chimeric repressor for B3 protein to suppresses the target gene in Arabidopsis. Some of the transgenic plants that expressed the chimeric repressor resulted in the fusion of cotyledon and absence of SAM of seedling. In addition the chimeric repressor affect leaf and flower organ formation, root elongation and embryogenesis. We will discuss the possible function of B3 domain proteins.

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KNOX genes have an important role in maintenance of shoot apical meristem and are divided into two subfamilies, class 1 and 2. The functions of class 1 genes have been characterized by mutant analyses and overexpression experiments, while that of class 2 genes are still unknown, because loss-of-function mutants have not been reported and any phenotype has not been reported in its over-producing transformants.
Physcomitrella patens is a suitable model to analyze genes with unknown function, since gene targeting techniques have been established. We investigate the function of two KNOX class 2 genes, MKN1 and MKN6. Expression patterns of MKN1 mRNA was analyzed by RT-PCR and the transformants in which GUS gene was inserted at the end of MKN1 coding sequence were observed to detect the expression patterns of the fusion protein. MKN1 was expressed in sporophytes, but not in gametophytes. The analyses of MKN6 are ongoing and will be presented.

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Stomata consists of two guard cells. In addition to the physiological roles, a development mechanism of guard cells are interesting.MC79 was isolated as a responsible gene for the Arabidopsis mutant, which showed the abnormal stomata morphology. It showed aberrant location of each guard cells, and "capsule type" stomata. The MC79 encodes a leucine rich repeat receptor like kinase (LRR-RLK). By the observation of the leaf epidermis in the transgenic plant with MC79 pro::MC79-GFP, we confirmed MC79 is expressed in the guared cell in a developmentally specific manner. It was preferentially expressed in meristemoid to guard mother cell. MC79 was also expressed in the epidermal cell at the devision region of the root. We also analyzed MC79-like receptor like kinase (MCL) showed high homology with MC79. In addition, we will report about candidate MC79-interacted clones isolated by Yeast Two-Hybrid screening.

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To investigate how photo-oxidative stress and utilization of nutrients affect leaf senescence, Quercus crispula seedlings were grown under combined conditions of light (100 umol m^-2 s^-1: low, 1000 umol m^-2 s^-1: high), temperature (25C: high, 15C or 10C: low), with or without nutrients supply. Fv/Fm was low in plants grown under high light and low temperature. Under high light and high temperature, Fv/Fm was lower in plants without nutrients than those with nutrients. Without nutrients, xanthophylls increased gradually in plants grown under high light and low temperature. The de-epoxidation rate of xanthophylls was higher in plants grown under high light and low temperature, regardless of nutrients supply. In addition, de-epoxidation rates gradually increased in plants under low light without nutrients, resulting in the similar values to those under high light. It was suggested that the translocation of nutrients increased photo-oxidative stress in senescing leaves in plants without nutrients.

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Hybiscus syriacus L. is known as a plant whose flower life is very short. With the aim of understanding the factors that induce floral senescence of H. syriacus L., various treatments, which affect flower senescence or longevity, were conducted. Sucrose, boric acid and ethanol had no significant effect on Hibiscus flower. The treatments with ethylene, ACC, and ethephon enhanced floral senescence of Hibiscus while AVG showed a strong effect on longevity of this flower. Cycloheximide, MGBG and spermine accelerated flower senescence when treated before full bloom, but such effect was diminished or abolished when treated after full bloom. These chemicals resulted in the ethylene production before blooming. Exogenous application of ethylene before blooming also accelerated floral senescence. Furthermore, endogenous ethylene production before blooming was correlated with flower senescence. These results suggest that ethylene production before flowering has a key role in determination of the timing of Hibiscus flower senescence.

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The progression of leaf senescence is generally defined by chlorophyll degradation and decline of photosynthetic ability. As some morphological change in chloroplast before degradation is recognized, and many senescence–associated genes are reported, the study on chloroplast along the leaf development and senescence would be useful to understand the role of leaves in this period. We monitored the chloroplasts morphologically by microscopic method of Arabidopsis thaliana. Just after the leaves fully expanded, the chloroplast having waved lamella structure appeared among the typical chloroplasts. Every chloroplast in each leaf looked completed such morphological change when the plant had seeds. The ultra-structure of chloroplast with disorientation of the thylakoid membrane and the curling grana structure was observed by electron microscopy. These chloroplasts looked increasing one by one among the cells, but not appearing simultaneously in a cell. The values of chlorophyll a/b ratio were almost the same through the experiment period.

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Chlorophyll degradation is ubiquitous phenomenon found in plants. The removal of the central Mg atom from chlorophyllide a is expected to follow after dephytylation by chlorophyllase. In fact, an activity catalyzing this reaction has been reported in algae and higher plants and considered to be due to an enzyme that had been designated "Mg-dechelatase". In our previous study, however, we reported that only the small molecule substance catalyzes Mg-dechelating reaction from chlorophyllide among some substances that have Mg-dechelating activity with an artificial substrate, chlorophyllin. We named it metal-chelating substance (MCS), and purified and determined its properties. Acetone powder is made from goosefoot (Chenopodium album) that has high activity of Mg-dechelation. Purification of MCS was done using anion exchange and gel-filtration chromatography, and MCS was purified 395-fold with a yield of 37%. We determined molecular mass of MCS using MALDI TOF-MS and tried mass purification and crystallization for structure analyses.

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Only few orthologs of animal apoptosis regulators have been found in plants. Ectopic expression of mammalian inhibitor of apoptosis proteins (IAPs) has recently been shown to affect plant programmed cell death (PCD). We identified two novel IAP-like proteins, AtILP1/2, in Arabidopsis by applying an improved motif searching method. Homologs of AtILPs were found to occur as a novel gene family in other organisms including fungi and animals. Like baculovirus IAP repeats (BIRs) in IAPs, a conserved domain necessary for interaction with caspases, ILPs contain two highly conserved BIR-like domains with a putative C2HC-type zinc finger. Phylogenetic analyses indicated that ILPs are putative paralogs of IAPs. Transient expression of HsILP1 resulted in inhibition of apoptosis in human cells (Higashi et al., 2005), suggesting that AtILP1/2 may also regulate PCD. Possible physiological functions of AtILP1/2 in regulation of PCD triggered by pathogenic elicitors (Kadota et al., 2004) will be discussed.

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To study if vacuolar collapse plays a direct role in the cell death of the seedling of interspecific hybrid, Nicotiana gossei x N. tabacum, which shows hybrid lethality, vacuoles were analyzed with transmission electron microscope. The results showed that several morphological abnormalities and disintegration of vacuolar membrane occurred in the cells of the seedling grown at 26C. However, no such defects were found in the seedlings grown at 37C, a condition suppressing cell death reaction. The activity of vacuolar processing enzyme (VPE), which may be involved in vacuolar collapse of the plants, was higher in the seedling grown at 26C than that at 37C. Application of inhibitor of Caspase-1 (Ac-YVAD-CHO) suppressed the cell death. The present results suggest that through the activity of VPE, the collapse of vacuole plays a direct role in the process of cell death of hybrid seedlings.

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Cell death was compared between two cultured cell lines of interspecific hybrids of Nicotiana. Both lines grow at 37C, but die at 26C. The line TAH5 from N. tabacum x N. africana died faster than GTH4 from N. gossei x N. tabacum. The ROI generated primarily in the cells was H₂O₂ for GTH4 and O₂^- for TAH5. The treatment of DPI and catalase suppressed death of TAH5 and GTH4, respectively. Cell death of GTH4 was promoted by inhibitors of protein phosphatase, and suppressed by inhibitors of protein kinase. However, these treatments did not affect the viability of TAH5. On the other hand, the treatment of inter- and intra-cellular suppressors of Ca²⁺ signaling attenuated the cell death of TAH5, but not GTH4. The results suggest that the execution of cell death in hybrid lethality is under the control of different cellular signaling pathways, depending on the genotype of each F₁ hybrid.

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Cultured cell (GTH4) of Nicotiana gossei x N. tabacum dies at 26C, but not at 37C. After the shift from 37C to 26C, burst of H₂O₂ and activation of SIPK were observed in the cells. However, these were suppressed by U0126, an inhibitor of MEK, and cell death was delayed as well. This suggests that MAPK signaling is involved in the hybrid lethality. To further analyze a role of MAPK signaling, the F₁ hybrids between N. gossei and the transgenic tobacco harboring NtMEK2^DD or NtMEK2^KR, the constitutively active or -inactive MEK inducible with dexamethasone (DEX). The cell lines, GTH^DD and GTH^KR, were generated from the hybrid seedlings. Both lines showed high viability of cell at 37C, but died at 26C under the control condition. However, cell death of GTH^KR at 26C was suppressed by DEX treatment. This suggests that overproduction of NtMEK2^KR disturbed MAPK signaling and, eventually, suppressed cell death.

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Cannabinoids are novel secondary metabolites identified only in Cannabis sativa. Among these, THC and THCA are well known to show novel pharmacological properties including psychoactive and analgesic effects, and therefore have attracted a large attention. Here we show that the cannabinoids THCA and CBCA regulate cell death in C. sativa.
THCA and CBCA are stored in glands on the lower surface of Cananbis leaves. In response to ageing, these cannabinoids leaked from the glands into leaf tissues. Because THCA and CBCA possess the cell death activity for Cannabis cells, the leaf tissues exposed to these cannabinoids caused cell death. When we investigated the mechanism of cell death catalyzed by THCA and CBCA, we found that both cannabinoids induced apoptosis-like cell death. This apoptosis was inhibited by neither ascorbic acid nor caspase inhibitor, indicating that THCA and CBCA induce cell death, independently of hydrogen peroxide and caspase-like proteases.

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Brassica napus leaf protoplasts become swollen and finally degrade, during culture. Both morphological and biochemical approaches revealed that the process through which the death occurred were subjected to apoptotic-like manner. The present results showed that lipid peroxides released into the enzyme solution during the isolation of protoplast, but hydrogen peroxide did not. Greater levels of hydrogen peroxides were accumulated in chloroplasts, while nitrogen oxide (NO) generated in cytoplasm. Mitochondrion did not accumulate hydrogen peroxides. Activation of endonucleases was paralleled with decrease in mitochondria membrane potential. Endnucleases extracted form 2 days after culture were activated by Ca, Mg, Mn, but inhibited by Zn. A endnuclease that were involved in the induction of apoptotic cell death of Brassica napus leaf protoplasts was identified by the active gel staining.

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In yeast and animals, nucleolin functions as a global regulator of ribosome synthesis and their expression is tightly linked with cell proliferation. Between two Arabidopsis genes for nucleolin, expression of AtNucl.1 is inducible by sugars together with many genes for ribosomal proteins (RPs) and snoRNPs. Disruption of AtNucl.1 by T-DNA insertion caused not only reduced pre-rRNA processing but also reduced sugar-induced expression of RP genes. The AtNucl.1-disruptants grew poorly, yet lived longer, than the wild type and showed various phenotypes such as pointed leaves and short valve. Unlike AtNucl.1, expression of AtNucl.2 was hardly detected except in flower buds and AtNucl.2-disruptants grew normally. However, a genetic cross between AtNucl.1- and AtNucl.2-disruptants did not produce seeds homozygous for both mutations suggesting that double disruptants are lethal. Although nucleolin-null yeast mutants can grow even with severely reduced growth rate and increased cold-sensitivity, nucleolin seems to be essential for growth of Arabidopsis.

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We have developed a simple mathematical model that incorporates basic relationships between cell proliferation and volume growth in plant organs, and applied this model to kinematic analysis of root growth in Arabidopsis thaliana, which estimated relative efficiencies of three aspects of root growth; cell proliferation, volume growth, and organ maintenance. Here we report the model-assisted kinematic analysis of temperature effects on root growth in Arabidopsis. Seedlings cultured at 28C, 22C, and 16C were quantitatively compared for root growth patterns, and a remarkable difference was found between 16C and 22C, or 16C and 28C. At 16C, the primary roots had much narrower zones of cell proliferation and volume growth, resulting in lower rates of cell production and volume growth. Analysis with the mathematical model suggested that inefficient cell proliferation and volume growth cause the repression of root growth under the low temperature condition.

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Brassinosteroids (BRs) are plant growth promoting hormones. XTHs (Xyloglucan endotransglucosylases/hydrolases) are BR responsive genes. They may regulate plant growth via cell-wall loosening and reconstruction but the correlations with BR response are not understood.
In this study, we comprehensively screened the BR responsive genes in Arabidopsis thaliana by DNA microarray analysis and MPSS. We then selected the BR responsive genes whose expression correlates with XTHs from the numerous expression data of public database. Here we identify BRR1 as a novel BR responsive ring-finger protein. In two BRR1 knockdown (brr1) lines, production of siRNA from triggered dsRNA and suppression of endogenous BRR1 mRNA were observed. These lines displayed dwarf phenotype. These results show that BRR1 regulates plant growth. We performed DNA microarray analysis between wild type, brr1 seedlings and BL treated brr1 seedlings. We characterized the differentially expressed or BR responsive genes in brr1 by metabolic pathway analysis.

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Since the plant-length affects both the resistance to lodging and the biomass productivity, the plant-length is one of the most important traits in agriculture. Although a considerable number of studies have been conducted on the genetically mechanisms of plant-length regulation, the comprehensive mechanisms of plant-length regulation are not well understand. This is mainly because most of studies have focused on a singular mechanism, whereas the plural mechanisms may be involved in plant-length regulation. In this study, we tried to detect all chromosome regions affecting traits (CRATs) through the vegetative stages using chromosome segment substitution lines (CSSLs).
We detected CRATs affecting plant-length in chromosome No. 1,2,6,7,8,9 and 12. In addition, these CRATs were different by growth stages. As a result of cluster analysis, the CSSLs were classified into some groups by its patterns of the elongation rate. Thus, many chromosome regions involve in plant-length regulation as time specifically.

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It has shown that MDR/PGP-like ABC transporters are involved in transport of the plant hormone auxin. Recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin^{1, 2}. Here, we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development³.
1, Geisler M et al. (2003) Mol. Biol. Cell 14: 4238-4249
2, Geisler M et al. (2005) P. Journal 44: 179-194
3, Santelia D et al. (2005) FEBS Lett. 579: 5399-5406

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Cytokinins (CKs) are mainly transported through vasculatures in the form of nucleosides. Growth analysis of budding yeast (Saccharomyces cerevisiae) cells expressing OsENT2, an equilibrative nucleoside transporter of rice, showed that OsENT2 mediated uptake of isopentenyladenine riboside (iPR, K_m= 32 uM) and trans-zeatin riboside (K_m= 660 uM), suggesting that OsENT2 participates in iPR transport in planta (Plant Physiol., 2005, 138, 196-206). The OsENT2 promoter drove the expression of the ss-glucuronidase reporter gene in the scutellum during germination and in vascular tissues in germinated plants, suggesting a participation of OsENT2 in the retrieval of endosperm-derived nucleosides by the germinating embryo and in the long-distance transport of nucleosides in growing plants, respectively. Further functional analysis of OsENT2 is currently being undertaken by using Tos17 tagged lines and RNAi knock down mutants.

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