Phytochrome (phy) has been shown to be the major photoreceptor involved in photomorphogenetic and physiological responses in plants, and there are three rice genes for phy (PHYA, PHYB and PHYC). Our previous study showed that one of the genes encoding ethylene biosynthetic enzymes, Aco1, was highly expressed in phyAphyBphyC mutants compared to the wild-type (WT) (Kiyota et al., PCP 46: s168, 2005). In this study, we report expression analysis of the Aco1 promoter:GUS constructs in rice. The GUS genes driven by a series of 5'-truncated Aco1 promoters were introduced into both WT and phyAphyBphyC mutant plants to compare the Aco1 promoter activities between them. In phyAphyBphyC mutants, the GUS activity was higher than that in WT plants, although the localization of GUS activity was similar. On the other hand, a GUS activity site specific to WT plants was found in elongated internodes at heading.

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In the previous study, we found that the protein level of phyC in etiolated seedlings is extremely reduced in phyB mutants. In this study, we want to understand the molecular mechanism which controls the phyC level in relation to the phyB. Rice phyAphyB double mutants have reduced level of phyC protein and the etiolated seedlings do not respond to red or far-red light. Overexpression of PHYC gene did not make any differences in the phyAphyB seedlings for their light responses. However, when mutated PHYB gene which lacks a chromophore binding site was expressed, phyC protein amount was recovered to the wild-type level and the transgenic phyAphyB seedlings exhibited distinct responses to red and far-red light, suggesting that the restored phyC is biologically active. These results suggest that phyB protein is indispensable for the stability and therefore, function of phyC in rice photomorphogenesis, probably via the formation of phyB/phyC heterodimer.

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The phototropic response of Arabidopsis thaliana seedlings is initiated by the blue-light photoreceptors, phototropins (phot1 and phot2), and by their signal transducers, NPH3 and RPT2. NPH3 and RPT2 interact with phot1 respectively. RPT2 mRNA levels are induced by irradiation with either blue light or red light in a fluence rate-dependent manner, RPT2 play a role in the phot1 signaling pathway. However, the regulations of RPT2 expression remain unclear. Here, we report that expression of RPT2 mRNA was regulated by phyA, phyB cry1, and cry2 redundantly, and that phyA, cry1, and cry2 play important roles in the phototropic response. We also demonstrated possibility that phot1 induces the expression of RPT2 in seedlings under blue light conditions through translational regulation. These results suggested that expression level of RPT2 was regulated through multiple stages by photoropin, phytochrome and cryptochrome, and that every photoreceptors signal converges on the RPT2.

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Phototropins (phot1 and phot2) consist of two LOV domains (LOV1 and LOV2) in their N-terminus and a Ser/Thr kinase domain in their C-terminus. Although the photochemical properties of LOV domains have been extensively examined, their structural and functional roles remain to be determined. Here, we investigated the roles of phot2 N-terminus (P2N) in planta and in vitro. Size exclusion chromatography suggested that phot2 might exist in a multimeric form around 500 kDa in vivo, for which P2N but not P2C (phot2 C-terminus) is responsible. The multimeric formation was further confirmed by size exclusion chromatography using several recombinant proteins. The calculated molecular weights implied that P2N(1-533) and P2N(116-276) were probably tetramers but P2N(315-533) was a dimer, respectively. Physiological analysis of transgenic plants expressing phot2-deletion mutants insisted that the P2N(1-314) domain is dispensable to mediate the fundamental physiological responses. The functional relevance of the N-terminal domain of phot2 will be discussed.

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Blue-light photoreceptor phototropins (phot1 and phot2) function in phototropism, chloroplast relocation, stomatal opening and leaf flattening. In Arabidopsis, phot1 and phot2 have overlapping and distinct functions. Namely, phot1 exhibits higher sensitivity to blue light than phot2. While both phot1 and phot2 mediate chloroplast photoaccumulation response, only phot2 mediates chloroplast photoavoidance response.
Photoropin consists of two functional domains, the N-terminal photosensory domain and the C-terminal kinase domain. To identify the domain that was responsible for the functional specificity of phot1 and phot2, we swapped the N and C-terminal domains and fused them to GFP (namely P1n/2cG and P2n/1cG). The transgenic plants expressing P1n/2cG, P2n/1cG, PHOT1-GFP and PHOT2-GFP under the PHOT2 promoter were produced and analyzed. For phototropism, the photosensitivity was determined almost exclusively by the N-terminal domain. For chloroplast relocation, both P1n/2cG and P2n/1cG mediated photoaccumulation and photoavoidance responses. Subcellular localization of those chimeric phototropins will be also discussed.

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Blue light receptor phototropin (phot) is involved in various light responses, such as phototropism, chloroplast movement, and stomatal opening, in plants. Phot is chromoprotein consisting of the N-terminal LOV domain and the C-terminal kinase domain.
We previously identified a homologue of ADP-ribosylation factor 1 (ARF1) as a novel interaction partner of phototropin. A recent study further showed that phot specifically interacted with the GTP-bound form of ARF1, through the Ser/Thr kinase domain of phot.
To understand the role of ARF1 in the phot response, we identified phot2 mutant(s) that abolished the interaction with ARF1(GTP). Furthermore, we transformed such mutant phot2-GFP driven by the phot2 promoter in the Arabidopsis phot1phot2 mutant and analyzed the physiological response of the plant and the intracellular localization of mutant phot2:GFP.
In this session, we show the results and discuss the molecular mechanism of blue light signal transduction.

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In Arabidopsis thaliana, two phototropins, PHOT1 and PHOT2, function as blue light photoreceptors. Previously, we have proposed that phototropins might regulate the polar cell elongation in palisade tissue in response to blue light. In the phot1phot2 null mutant, palisade cells exhibit isotropic expansion rather than polar elongation in the leaf-thickness direction. Here, we investigated the effects of phot1 and phot2 on the polar cell elongation in palisade tissue. The phot2 mutant had stronger phenotype for the polar cell elongation than the phot1 mutant. In addition, the defect of phot1phot2 double mutant was completely rescued by 35S-PHOT2-GFP. Hence, phot2 played a major role to regulate the polar-cell elongation in palisade. We also asked where in the leaves phot2 perceives blue light to regulate the polarity of palisade-tissue cells. On the basis of those findings, relationship between the phot2 signals and their physiological consequences will be discussed.

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Phototropin (phot), a blue light sensor in plants, has two photoreceptive domains named LOV1 and LOV2, and Ser/The kinase domain. phot is thought to be a blue-light-regulated protein kinase in which LOV2 acts as a molecular switch of the photoregulation and LOV1 is involved in the regulation of the photosensitivity. In addition, LOV1 is reported to act as a dimer forming site. Then, we studied the molecular basis of the LOV1 dimerization in Arabidopsis phot1 and 2, and obtained the two results. 1) Dimeric crystal structures of both the LOV1 have been solved. phot1-LOV1 forms a dimer by interfacing each β-scaffold in an antiparallel manner and forming a S-S bond. In contrast, phot2-LOV1 dimer cannot form the S-S bond and dimerizes mainly through hydrogen-bonding. 2) Reduction of S-S bond monomerized phot1-LOV1 dimer and two amino acid substitutions involved in the dimer formation resulted in monmerization of phot2-LOV1.

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Phototropin (phot) is a blue light photoreceptor in plants that mediates phototropism, chloroplast relocation, stomata opening and leaf expansion. A phot molecule has two photoreceptive domains, LOV1 and LOV2, in the N-terminal half and the C-terminal half forms Ser/The kinase. Phot kinase is activated by blue light, in which LOV2 act as a light-regulated inhibitory domain. However, little is know regarding the molecular mechanism of the photoregulation.
In order to understand the molecular basis of the photoregulation, binding of LOV domains to kinase domain was analyzed by a docking simulation technique. LOV2 was revealed to bind to the active site of kinase domain to form a stable complex, however, LOV1 was not. The results well agree with the reported role of LOV2 in the photoregulation. In addition, the effects of the introduction of amino acid mutations into the estimated interaction site of LOV2 domain will be reported.

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Phototropins (Phot) are one of the important blue light receptors in plants and are involved in phototropism, stomata opening and chloroplast relocation. The N-terminal half has two light-receptive domains (LOV1, LOV2) each of which binds an FMN and the C-terminal half forms a Ser/Thr kinase (KD) connected to the LOV2 with a linker. The kinase activity is inhibited by LOV2 in the dark. Blue light cancels the inhibition, resulting in both auto- and substrate phosphorylation. But little is known about the light regulation mechanism of the kinase by LOV2.
Bacterially expressed Arabidopsis Phot1 LOV2-KD showed the same spectral changes (dark recovery t_1/2 = 29 s) as that of the LOV2 on blue light. The limited proteolysis indicated that light-induced structural changes occurred in the linker. LOV2-KD also showed kinase activity on the peptide of Phot1-N-terminal region. How the light-induced conformational changes regulate the kinase activity of Phot1 will be discussed.

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Phototropins (phots) are the autophosphorylating protein kinases that mediate phototropism, chloroplast movements, leaf flattening, and stomatal opening. However, the autophosphorylation sites of Arabidopsis phot have not been determined, and the physiological role of autophosphorylation remains unknown. Here, we identified eight phosphorylation sites in the N-terminus, Hinge1 region, kinase domain, and C-terminus in Arabidopsis phot1 by liquid chromatography-tandem mass spectrometry. We substituted the identified Ser and Thr with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses after the transformation of the phot1 phot2 double mutant. We found that phosphorylation of Ser-851 in the kinase activation loop was required for all of the responses, whereas the phosphorylation of the other sites was not. The Ser-851 was rapidly autophosphorylated by blue light in fluence-dependent manners and dephosphorylated gradually upon darkness. These results demonstrate that autophosphorylation of the Ser-851 is a primary step for downstream signaling.

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Arabidopsis phototropins (phot1, phot2) are blue light (BL) receptors that mediate stomatal opening. BL-induced membrane hyperpolarization through the activation of plasma membrane H⁺-ATPase drives K⁺ uptake and elicits stomatal opening. Previous studies suggested the involvement of Ca²⁺ in BL-induced activation of plasma membrane H⁺-ATPase. No evidence is, however, obtained whether phototropins mediate Ca²⁺ increase in guard cells. Here we determined the cytosolic Ca²⁺ concentration in guard cell protoplasts from Arabidopsis transformed with aequorin. We found a transient increase in cytosolic Ca²⁺ in response to BL, which was almost suppressed by DCMU, an inhibitor of photosynthetic electron transport. When BL was superimposed on a background strong red light, which saturated photosynthesis, the BL induced a slight Ca²⁺ increase in wild type but not in phot1 phot2 double mutant. From these results, we concluded that BL induced the Ca²⁺ increase that was dependent on both photosynthesis and phototropins in guard cells.

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AppA is a novel blue-light receptor controlling photosynthesis gene expression in purple bacteria.
The crystal structure of the light-sensing domain called BLUF shares some homology with acyl phosphatase. Other types of blue-light receptors, such as cryptochrome or phototropin, autophosphorylation is associated with their activity. These observations imply that BLUF also transmits a light signal through phosphorylation. Here we found that AppA BLUF domain has a light-dependent autophosphorylation activity. By the site-directed mutagenesis study, the phosphorylated residue was determined to be T30, and R32 was shown to assist the stability of the autophosphorylation reaction. A Q63L mutant protein lacking photo-cycle reaction lost the light dependence of the autophosphorylation reaction, indicating that the autophosphorylation activity is controlled by the light-dependent conformational change around Q63. We also showed that AppA regulates a sensor kinase activity through the phosphoryl-transfer reaction.

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phot1 and phot2 act as blue light receptors for phototropism, chloroplast relocation, stomatal opening and leaf expansion. In the blue light-induced stomatal opening, blue light signal perceived by phot1 and phot2 induces activation of the plasma membrane H⁺-ATPase and stomatal opening. However, signaling pathway between phots and H⁺-ATPase is largely unknown. In this study, we performed the screening of mutants, which show opened stomata phenotype, from EMS-treated phot1 phot2 double mutant in Arabidopsis. One of the isolated mutants, named db10-2, showed always opened stomata phenotype, but this mutant was sensitive to abscisic acid. Interestingly, the H⁺-ATPase in guard cells of db10-2 was always activated, therefore, this mutant may show opened stomata phenotype. Now, we are trying to identify a mutation gene in db10-2 by map-based cloning.

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It has been shown in protonemata of Physcomitrella patens that branch appears at the region irradiated with microbeam blue light (Uenaka et al., 2005). In this study, the effect of fluence rate of microbeam blue light on the branch position was investigated. When microbeam blue light of 10 or 30 W m^-2 was irradiated, branch was induced within the microbeam area (Weak light response, WR). However, branch appeared outside the microbeam area when blue light of 200 or 400 W m^-2 was used (Strong light response, SR). Interestingly, in case of SR, branch formed at the apical side of microbeam, suggesting that cell polarity is also involved in the determination of branch position. When blue light determination of branch position was investigated in PHOTOTROPIN disruptants, reduction in the blue light sensitivity was found. These evidences indicate that branch position in P. patens is regulated through phototropins and cell polarity.

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Under low-light conditions, chloroplasts accumulate in the light to capture weak light efficiently (accumulation response), while under high-light conditions, they escape from the light to avoid their photodamage (avoidance response). We recently identified two homologous genes, KAC1 and KAC2, mediating chloroplast movement in Arabidopsis thaliana. In kac1 mutant, the accumulation response is drastically impaired and also the speed of chloroplast avoidance is slightly slower than in wild type. Although kac2 single mutant did not show any impairment in chloroplast movement, kac1kac2 double mutant lacks both the accumulation and avoidance responses. KAC1 protein and KAC1 mRNA accumulated at much higher level than those of KAC2 in wild type. KAC1 promoter-driven KAC2 expression completely rescued chloroplast photorelocation movement in kac1kac2. These results indicate that the difference between KAC1 and KAC2 is quantitative but not qualitative and that the abundance of KAC proteins is one of the critical determinants for speed of chloroplasts.

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The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid motif that is found in animal, fungi, and plants. A large gene family encoding PPR proteins particularly exists in plants, from a moss to flowering plants. PPR proteins have been shown to play important roles in the wide range of physiological and developmental functions. We previously reported that PPR531-11, one of the moss Physcomitrella patens PPR proteins, was involved in processing of clpP pre-mRNA in chloroplasts. In the present study, we have carried out RNA binding analysis of PPR531-11 using several clpP RNA fragments. The recombinant PPR531-11 expressed in Escherichia coli specifically bound to the upstream region from the cleavage site in the clpP and 5'-rps12 intergenic region. Taken together with the other biochemical data, we propose that the PPR531-11 is a sequence-specific RNA-binding protein and acts as a regulatory factor in plastid RNA processing of clpP pre-mRNA.

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Transcripts of plastids in higher plants are highly modified by C-to-U RNA editing. It exhibits extraordinary phylogenetic dynamics leading to species-specific editing site patterns. Nicotiana tabacum (tobacco) is a natural amphidiploid derived from ancestors of N. sylvestris and N. tomentosiformis. These Nicotiana species offer a significant advantage for evolutional studies of RNA editing in higher plant plastids. Analysis of the first editing site in ndhD mRNAs in these species revealed that editing efficiency in N. tomentosiformis is lower than those in N. tabacum and N. sylvestris, but this level of editing is enough to express the ndhD gene. This result suggests that decrease of editing efficiency in N. tomentosiformis might be due to a defect in function of its CRR4 ortholog. To study this possibility, we are investigating whether CRR4 orthologs have different efficiency in its function among Nicotiana species.

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In rice, the developmental process in leaf formation can be divided into 6 stages (stages P1-P6). We investigated physiological change in the developing leaves at the key stage P4, during which leaf greening and establishment of primary leaf structure occur. Chlorophyll content and Rubisco accumulation were negligible at early P4 and increased rapidly as leaf enters the late P4 phase. Chlorophyll fluorescence ratio (Fv/Fm) also increased markedly and the final value was comparable with that of mature leaves at P5-P6 stages. RNAs mainly transcribed by NEP increased rapidly during early P4 and then declined slowly as PEP-derived RNAs increased. Proportion of carbon in leaf cells was kept constant through the P4 stage, while that of nitrogen decreased with increasing C:N ratio. These observations suggest that in the leaves at P4 developmental stage, chloroplast develops rapidly and effects on the physiological status of leaf cells largely.

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We identified a pale-green mutant, named apg8 (albino or pale-green 8) from screening of RIKEN Arabidopsis Ds tag-lines with knockout genes for nuclear-encoded chloroplast proteins. The gene disrupted by insertion of Ds was found to encode the iron superoxide dismutase, FSD2. FSD from Arabidopsis consists of three members (FSD1, FSD2, FSD3). Phenotype of three alleles of fsd1 showed normal, while two alleles of fsd2 or fsd3 caused pale-green phenotypes, respectively. Moreover, fsd2-1 fsd3-1 showed more severe albino phenotype in the agar plate. The mutant plants were highly sensitive to oxidative stress and had increased levels of reactive oxygen species (ROS) during extended darkness. We also showed that FSD2 and FSD3 proteins form a hetero complex. Taken together, our results provide evidence for essential roles of three FSD members with different functions, and demonstrate that FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development.

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In almost all eukaryotes, mitochondrial (mt) and chloroplast (cp) genes are transmitted to progeny mainly from the maternal parent. The most popular explanation for this phenomenon is simple dilution of paternal mt/cpDNA, because the paternal gametes (sperm) are much smaller than maternal gametes (egg) and contribute a limited amount of mitochondria / plastid to the progeny. Recently, this simple explanation has been challenged in several reports that describe the active digestion of paternal mt/cpDNA in algae, higher plants, slime mold and Medaka fish. However, little is known about how paternal mt/cpDNAs are preferentially degraded whereas maternal DNAs are protected. In order to answer these questions, we employed Chlamydomonas reinhardtii as a model system and focused on the function of zygote specific gene, ezy1. In this session, we will present our latest data obtained by RNAi analysis.

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Plastids send signals that regulate nuclear gene expression, thereby accomplished the assembly of photosynthetic apparatus. At present the plastid genome encodes fewer than 100 ORFs, and most of genes that were encoded by the bacterial ancestor have been moved to the host nuclear genome. This separation of genetic information necessitates the communication between plastids and the nucleus by plastid signals. However the molecular mechanisms underlying this inter-organelle signaling remains to be characterized.
To elucidate the signaling pathways from plastids to the nucleus, there is a significant evidence that the inhibition of protein import into plastids disrupts the expression of nuclear genes encoding plastid proteins. In ppi2 mutant lacking Toc159 protein import receptor, the mRNA levels of photosynthetic proteins are dramatically reduced. In this study, we performed global gene expression analysis of ppi2 mutant using SuperSAGE. We show that specific pathways regulate certain classes of genes were affected in ppi2 mutant.

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In higher plants, cross-talk between the nucleus and plastids is one of the most important intracellular communications. Plastid-to-nucleus retrograde signaling coordinates the nuclear gene expression in response to functional state of chloroplasts. To date, several retrograde signals have been reported, but little is known about their signaling pathways. To investigate the signaling mechanism from plastids to the nucleus, we used the Arabidopsis mutant, ppi2, which lacks the protein import receptor Toc159. We examined the effect of norflurazon on the expression of nucleus-encoded chloroplast proteins involved in retrograde signaling using wild type and ppi2 mutant. The results indicate that the expression of some genes is affected by norflurazon treatment. We also examined genetic interactions between ppi2 and mutations involved in plastid signaling. On the basis of these results, we will discuss the signaling pathway from plastids to the nucleus.

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In higher plants, nuclear-encoded genes related to the plastid-structure/function (e.g. Lhcb, RbcS) were co-coordinately regulated with the functional status of plastids. "Plastid signal" has been hypothesized for this regulatory mechanism. The analysis of gun mutants that have defects in this signaling has revealed the involvement of the tetrapyrrole intermediates (MgProtoIX) as a signaling factor. As oppose to the previous report, MgProtoIX level is 1/100 less accumulated than the control. However, we found that the synthesis of Proto-MgProtoXI-MgProtoIXMe are all elevated in the photobleached plants. Therefore, these intermediates are actively synthesized in the photobleached plants, but they are metablised and/or degraded immediately. We will report the comparison of tetrapyrrole synthesis capacity of gun mutants and discuss about the dynamics of the tetrapyrrole biosynthesis and the plastid signaling. We will also present the analysis of the new gun mutants.

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A white sector of variegated leaves is composed of living cells with aberrant plastids. While recent studies revealed genes responsible for leaf variegation, the precise mechanism forming such sectors remains unknown. Previously, we demonstrated that white sectors of var2 contain undeveloped plastids and that these plastids contain distinct nucleoids, a structure of DNA-protein complexes. Nucleoids, detectable by staining the Technovit thin sections of leaf tissues with DAPI, change their morphologies according to chloroplast differentiation. In this study, we attempt to understand structural changes of plastid nucleoids in developing leaves of several species (Arabidopsis and barley), compare them to those in variegated leaves, and classify plastids of variegated plants based on the status of nucleoid morphologies. Our initial studies showed that plastid nucleoids change their size, number and position during greening. Observation of plastids in various variegated leaves is currently underway.

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To examine the replication and inheritance of plastid DNA, we have established efficient plastid transformation systems for the liverwort Marchantia polymorpha. Progenies of the cross between wild type and spectinomycin-resistant plastid transformant showed antibiotic resistance depending on the maternal genotype of plastid DNA. This indicates that the plastid DNA in liverwort is maternally inherited. To investigate the replication mode of the plastid DNA, two-dimensional agarose gel electrophoresis was performed using vigorously growing liverwort suspension-cultured cells. Replication intermediates that proceeded in the short-single-copy (SSC) region and stopped near the boundary region between the SSC and the inverted repeat regions were detected. This result suggests that the liverwort plastid DNA has a region that stops a replication fork until the other fork in the opposite direction reaches the region and also prevents a replication fork from colliding with the transcriptional machinery for the rRNA operon.

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We have obtained two cDNAs encoding bacterial DNA polymerase I homologs from BY-2 cultured tobacco cells, and named corresponding genes NtPolI-like1 and NtPolI-like2. The two genes exhibited 97-98% identity at nucleotide- and amino acid-levels. The NtPolI-like gene products exhibited DNA polymerase activity, were targeted to both plastids and mitochondria, and were localized in the plastid- and mitochondrial-nuclei. The apparent molecular masses, as well as biochemical properties of the gene products well agreed with those of DNA polymerases biochemically detected in the isolated organelle-nuclei. NtPolI-like transcript levels, NtPolI-like protein levels, and DNA polymerase activities in the organelle-nuclei concomitantly increase in the initial phase of cell proliferation, exactly when organelle DNA synthesis are transiently activated. The concomitant increase strongly suggest that NtPolI-like gene products are involved in the transient activation of organelle DNA synthesis in the initial phase of cell proliferation.

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The regulation of mitochondrial biogenesis and function are essential for all aspects of plant growth and development. However the molecular mechanisms involved are still unclear. Here we describe an analysis of the evolutionarily conserved Miro GTPases from Arabidopsis and show that one of the gene, AtMIRO1, is transcribed ubiquitously throughout the plant tissues. AtMIRO1 encodes a protein that localizes to mitochondria via its C-terminal transmembrane domain. An insertional mutation in the AtMIRO1 gene is lethal during embryogenesis at the early embryo stage and substantially impairs pollen germination and tube growth. The mutant pollen exhibit abnormally enlarged morphology of mitochondria. Our findings suggest that mitochondrial morphology is controlled by AtMIRO1 and is vital during embryogenesis and pollen tube growth.

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Mitochondrial-nucleus is defined as DNA-protein complex of mitochondria, and regarded as the site in which various functions of mitochondrial genomes, such as DNA replication, transcription, are conducted. To understand how these various functions are performed within highly organized structure, identification and characterization of the proteins responsible for the organization of mitochondrial-nuclei are necessary. So, we examined the structural organization of plant mitochondrial-nuclei and the proteins involved in the organization, by using BY-2 cultured tobacco cells. Experiments with isolated mitochondrial-nuclei treated with various agents suggested that the structure of mitochondrial-nuclei is retained by electrostatic interaction between DNA and proteins, and mitochondrial DNA form nucleosome-like structures within mitochondrial-nuclei. Treatment with increasing concentration of NaCl caused gradual dispersion of mitochondrial-nuclei and gradual dissociation of proteins from mitochondrial DNA. Then, we explored DNA-binding proteins in those solubilized by NaCl treatment. At present, in order to identify these proteins, they are analyzed by mass spectrometry.

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In plant vegetative cell, mitochondria are usually small grain-shaped. On the other hand, it was reported that unusually shaped, giant mitochondria (large cup-shaped or long, stretched rod-shaped) appeared in the egg cells of geranium and maize. However, studies on the egg cell mitochondria were reported only in geranium and maize. In this study, to characterize the mitochondria of rice egg cell, we isolated unfertilized egg cells of rice by means of non-enzymatic, manual dissection, and observed the egg cell mitochondria and mitochondrial DNA (mtDNA) simultaneously. These observations showed that the egg cell mitochondria of rice were small grain-shaped, unlike geranium and maize. Quantitative-PCR reveals that an egg cell of rice contained at least 15-fold mtDNA compared with a rice leaf protoplast.

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Mitochondrial fission is achieved by self-assembling large GTPases, dynamin-related proteins (DRPs), that are recruited from the cytosol to mitochondrial fission sites. Mitochondrial fission in Arabidopsis is also mediated by DRP orthologues DRP3A and DRP3B, but little is known about whether other genes are involved in mitochondrial fission. To identify such genes, we screened for arabidopsis mutants with longer and fewer mitochondria. Analysis of such mutants identified a novel gene (ELM1) as the responsible gene. ELM1:GFP seems to surround mitochondria. ELM1 showed interactions with both DPR3A and DRP3B. DRP3A:GFP was observed in the cytosol in the elm1 mutant, not at the ends and constricted sites of mitochondria as observed in the wild type. These results collectively suggest that mitochondrial fission in Arabidopsis is mediated by the plant-specific factor ELM1, which is required for the re-localization of DRP3A from the cytosol to mitochondrial fission-sites.

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Two paralogous Arabidopsis dynamin-rerated proteins, DRP3A and DRP3B were reported to be involved in mitochondrial fission. DRP3A was also shown to be involved in peroxisomal fission. To elucidate the molecular and genetic relationship between DRP3A and DRP3B, we analyzed the molecular interaction between DRP3A and DRP3B, the detailed intracellular localization of DRP3A and DRP3B, and the morphology of mitochondria and peroxisomes in their T-DNA insertion mutants. Yeast two-hybrid assay suggested that DRP3A and DRP3B interacted with each other. The comparison of intracellular localization of the fluorescent fusions of DRP3A and DRP3B expressed by their own promoters suggested that these two proteins were co-localized. Moreover, the analysis of the mitochondrial and peroxisomal morphologies in drp3a, drp3b, drp3a/drp3b and their complementation lines indicated that DRP3A and DRP3B have similar functions that are related to mitochondrial fission, but have different functions that are related to peroxisomal fission.

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Determination of flower organ identity is controlled by many MADS-box genes in rice. It is regulated by a system mostly similar to the ABCDE model in dicot, but there are still many unknown points. We have reported OsMADS6 and OsMADS17 classified in class-G MADS-box genes whose functions are unclear.
Here, we analyze a novel osmads6 mutant allele fm50 which has an amino acid substitution in the MADS-domain. fm50 has more severe phenotype than other osmads6 mutant lines previously reported, i.e. ectopic glumes, disturbance of whorl structure, loss of meristem determinacy.
In addition, we made a construct which can knock down both OsMADS6 and OsMADS17, and introduced it into a weaker allele of osmads6. The phenotype of transgenic line is similar to that of fm50. From these investigations, we will discuss the function and relationship of the two rice class-G genes.

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Genetic and molecular studies in dicots have led to proposal of the ABCDE model for control of floral organ identity. Class-D MADS-box genes belong to class-C subfamily, and they are specifically expressed in ovule. Two class-D genes, OsMADS13 and OsMADS21, have been isolated from rice. However, their functions are still unclear.
Our in situ hybridization analysis showed that OsMADS13 expression is first detected in ovule primordium. As ovule develops, OsMADS13 is expressed in outer and inner integuments, nucellus and adaxial side of carpel.
OsMADS13 RNAi plants have thick integuments and no embryo sac. Ovules of osmads13 mutants caused by Tos17 insertion are homeotically transformed into carpelloid structures. These indicate that OsMADS13 is necessary for normal ovule development.
We are also studying the effect of ectopic expression of OsMADS13 in rice. Based on these observations, we will discuss the role of OsMADS13 in pistil development.

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In Arabidopsis, a petal primordium is formed and developed between sepal and stamen primordia. The identity and initiation of primordial development of the petal have been studied genetically. However, the process of petal maturation is not genetically explored yet. To understand the maturation process of petal development, we analyzed an Arabidopsis mutant folded petals2 (fop2) which had wrinkled petals. The wrinkle in the petal of fop2 was observed only after a petal primordium contacted sepal and stamen primordia. When a sepal was removed before this stage, a petal did not wrinkle. There were no obvious difference in size and shape between mature petals in fop2 and wild type, when the wrinkle was smoothed. These suggest extension of the petal is blocked by sepal and stamen primordia in fop2. FOP2 encodes ABC transporter family protein. We would like to discuss how FOP2 affects the petal maturation process.

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The synergid cells of the female gametophyte play a role in many steps of the angiosperm fertilization process. However, the mechanisms by which the synergid cells become specified and develop their unique features during female gametophyte development are not understood. We identified MYB98 in a screen for Arabidopsis thaliana genes expressed in the female gametophyte. In the context of the ovule, MYB98 is expressed exclusively in the synergid cells, and mutations in this gene affect the female gametophyte specifically. myb98 female gametophytes are affected in two unique features of the synergid cell, pollen tube guidance and the filiform apparatus, but are otherwise normal. MYB98 also is expressed in trichomes and endosperm. Homozygous myb98 mutants exhibit no sporophytic defects, including trichome and endosperm defects. Together, these data suggest that MYB98 controls the development of specific features within the synergid cell during female gametophyte development.

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In Arabidopsis thaliana, a pair of homeobox genes, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR2 (PDF2), play a role in regulating the expression of L1 layer-specific genes. atml1-1 pdf2-1 double mutants show striking defects in the differentiation of shoot epidermal cells. PDF2 and ATML1 belong to the HD-ZIP IV gene family, which consists of 16 genes in the Arabidopsis genome. Expression data on promoter-GUS gene fusions suggested that most of the HD-ZIP IV members are mainly expressed in epidermal tissues. Their transcript levels were reduced in atml1-1 pdf2-1 double mutants. Analyses of T-DNA insertion mutants of each HD-ZIP IV gene revealed that all mutants except known gl2, anl2, and hdg11, exhibit no abnormal phenotypes.
In this study, we constructed double mutants of pdf2-1, atml1-1, or atml1-2, and all other members of this family and examined their phenotypes. We observed reduced fertility in some mutant combinations.

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Nicotinamide adenine dinucleotide (NAD) coenzyme is abundant in pollen grain compared to the other inflorescent tissues of Arabidopsis. Pollen lacking a key NAD biosynthetic gene, nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT), was deficient in tube growth and dormancy. Tracer experiment revealed that the nmnat pollen incompletely eliminated the HPTS-containing moisture, suggesting that nmnat pollen was un-dormant. Under high humidity environment, nmnat/+ plants had long pollen tubes inside the anthers, namely precocious pollen tube growth. Even under dehydrated condition, round pollen with bulges can be distinguished in pollen population of nmnat/+ plants. Un-dormant pollen generally remains to be metabolically active, leading to senescence acceleration. In fact, the longevity of nmnat pollen was quite shorter than that of wild type. Thus, the NAD biosynthesis is essential for acquisition of drought tolerance and transition to pollen dormancy.

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Phosphatidylserine (PS) is a minor phospholipid in plant cells, representing <2% of total lipids. In flowers, however, its content amounts to 10% of total lipids, suggestive of its uncharacterized function. PS is synthesized via a CDP-diacylglycerol pathway in Escherichia coli and yeast, whereas it is synthesized via a base-exchange pathway from phosphatidylcholine or phosphatidylethanolamine in mammals. Arabidopsis thaliana has a putative PS synthase gene for the base-exchange pathway (PSS1; At1g15110), whose gene product remains to be described. To understandPSS1 function in plants, especially in flowers, we herein characterized a T-DNA insertion mutant, designated pss1-1. Homozygous pss1-1 caused recessive embryonic lethality at the four-cell stage. Heterozygotic plants produced abnormal male gametophytes, representing 40% of pss1-1 gametophytes. Transgenic Arabidopsis expressing a PSS1-Enhanced Yellow Fluorescence Protein (EYFP) fusion showed reticular fluorescent images within pollen, suggesting that PSS1 plays an important role in male gametogenesis in A. thaliana.

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To elucidate the mechanisms underlying dioecious flower development, we have identified a SUPERMAN (SUP) homolog, denoted SlSUP, in the dioecious plant Silene latifolia. The introduction of a genomic copy of SlSUP into the Arabidopsis thaliana sup (sup-2) mutant restores an excess-stamen phenotype. SlSUP is a single-copy autosomal gene that is expressed exclusively during the development of the female flower of Silene latifolia. The expression of SlSUP is first detectable in whorls 2 and 3 to persist in the stamen primordia until the ovule has completely matured. When female plants of S. latifolia are infected with the smut fungus Microbotryum violaceum, stamens develop in the female flower. Hence, an infected-female flower seems morphologically to be hermaphroditic. The expression of SlSUP was maintained at stage 5 but markedly reduced until stage 8 in developing stamens of the infected-hermaphrodite-like flower. These expression patterns suggest that SlSUP functions in the suppression of stamen development.

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In plants, the elongation of pedicels and stem internodes produce a highly ordered branching pattern. We have reported corymb-like inflorescence mutations, corymbosa1 (crm1)/big. The CRM1/BIG gene regulates the cell elongation of pedicels and stem internodes through auxin action. To gain new insight into inflorescence development, we screened enhancer and suppresser mutants of crm1. A leafy (lfy) mutaion, was found to be one of the suppressor mutation. Temporal expression analysis revealed that the expression level of LFY in crm1-1 was increased in late developmental stage. We found that pedicels in 35S::LFY-GR treated with DEX during late developmental stage pointed downwards. These results suggest that the corymb-like phenotype in crm1 is partially caused by the later overexpression of LFY that defines the elongation direction of pedicels in stem development. Further analyses of relationship between LFY and CRM1/BIG will be presented.

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Morphologies of flowers and inflorescences in monocots are distinct from those of flowers and inflorescences in eudicots. Especially in grasses, the inflorescences comprise of unique structural units, the spikelet and the floret. It has been regarded that the rice spikelet was originally composed of three florets. The uppermost floret is bisexual and fertile, whereas two lower florets are strongly reduced and sterile. The two lower florets develop only reduced lemma, known as sterile lemma. We have focused on the developmental mechanism of the sterile lemma to elucidate morphological evolution of rice spikelet. Here, we report phenotypic analysis of a rice mutant, sterile lemma of which are longer than those of wild type, and isolation of the gene responsible for the mutation.

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Copper is an essential trance metal for all living organisms. Cu/Zn SOD (CSD1 and CSD2) are involved in scavenging of reactive oxygen species in cytosol and chloroplasts respectively and their expression is down-regulated under copper deficient conditions. Previously, we demonstrated that one of microRNA, miR398, was expressed under low-copper conditions, and degraded CSD1 and CSD2 mRNA directly. In addition, iron superoxide dismutase (FeSOD) is expressed instead of Cu/ZnSODs and complements the function under copper deficient conditions. Furthermore some of copper transporter genes are up-regulated under copper deficient conditions. Thus higher plants have strict strategy for adaptation to copper starving.
In this work, we identified a transcription factor, SPL7, involved in the activation of the miR398 transcription. Additionally, SPL7 activated the transcription of FeSOD and some of copper transporter genes. These results suggest that SPL7 is a key factor to control the adaptation mechanisms to copper deficiency.

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Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide radicals. Plants possess three types of SODs, (i.e., FeSOD, MnSOD and Cu/ZnSOD). We found that copper repressed FeSOD activity in chloroplasts of a moss Barbula unguiculata. This copper-mediated repression involves regulation at the level of transcription.
We showed that multiple GTACT motifs are negative cis-elements of FeSOD gene in response to copper by analysis of FeSOD promoter using transgenic moss plants. SBP (SQUAMOSA promoter binding protein)-domains bind to GTAC core sequence, and then we indicated that PpSBPs bind specifically to GTACT sequences of FeSOD promoter in vitro. The Moss FeSOD gene was negatively regulated by copper in transgenic tobacco plants. Arabidopsis FeSOD (FSD1) gene promoter also contains multiple GTACT motifs, and analysis using transgenic Arabidopsis plants revealed the copper responsiveness of the Arabidopsis FSD1 promoter. These results suggested that the copper-dependent repression via GTACT motif is conserved from moss to land plants.

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Plants induce iron utilization systems under iron deficiency, but the underlying molecular mechanisms remain largely unknown. We identified the rice transcription factor IDEF1 (IDE-binding factor 1), which specifically binds the iron deficiency-responsive cis-acting element IDE1. IDEF1 belongs to an uncharacterized branch of the plant-specific transcription factor family ABI3/VP1, and exhibits the novel sequence recognition property of efficiently binding to the CATGC sequence within IDE1. IDEF1 transcripts are constitutively present in rice roots and leaves. Transgenic tobacco plants constitutively overexpressing IDEF1 transactivate IDE1-mediated expression only in iron-deficient roots. Transgenic rice plants expressing an introduced IDEF1 exhibit substantial tolerance to iron deficiency in both hydroponic culture and calcareous soil. IDEF1 overexpression leads to the enhanced expression of the iron deficiency-induced transcription factor gene OsIRO2, suggesting the presence of a sequential gene regulatory network. These findings reveal novel cis element/trans factor interactions functionally linked to the iron deficiency response.

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Nicotianamine (NA), a metal chelator ubiquitous in higher plants, serves as an antihypertensive substance in humans. To engineer a novel antihypertensive rice that contains higher amounts of NA, the barley nicotianamine synthase gene, HvNAS1, was introduced into rice via Agrobacterium-mediated transformation. The introduced HvNAS1 was driven by pGluB-1, which induces strong gene expression in the endosperm of rice seeds. The NA content in transgenic rice seeds was increased to up to four times that in non-transgenic rice seeds. The Cre/loxP DNA excision (CLX) system was used to remove the selectable marker gene for antibiotic resistance was used. Furthermore, the transgenic rice was crossed with a cleistogamous mutant to prevent gene transfer via pollen dispersal. These two modifications should largely alleviate public concern regarding the use of this transgenic rice.

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In humans, Iron (Fe) and Zinc (Zn) deficiencies are serious problems around the world. Transgenic approaches will be a breakthrough to produce Fe- and Zn-fortified rice varieties to improve human health. Nicotianamine (NA) chelates Fe²⁺ and Zn²⁺ and plays a critical role in plant body to transport these metals to sink tissues including seeds. We aimed to increase Fe and Zn concentrations in rice seeds by overexpressing barley NA synthase gene, HvNAS1, under the control of rice Actin promoter. NA concentrations were significantly increased in shoots of transgenic rice lines than those of non transgenic rice. Fe and Zn concentrations in polished T₁ seeds of transgenic rice lines also increased more than 3 and 2 times respectively. Fe and Zn concentrations in roots and shoots were not significantly increased. These results suggest that translocation of Fe and Zn into seeds is enhanced by overproduction of NA.

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Aiming at phytoremediation of cadmium (Cd)-contaminated soils, we have been studying on plant responses to Cd at molecular levels. To facilitate the molecular dissection of the plant response, we have isolated several Arabidopsis mutants that showed altered responses to Cd. A mutant designated as T-26 is a candidate for the mutants that do incorporate Cd via roots, but fail to transport the incorporated Cd to aboveground tissues. To know the ability of T-26 plants to transport Cd from roots to aboveground tissues, we fed them with 30 μM CdCl₂ via roots for 24 h and measured the Cd concentration of the aboveground tissues. The result showed that the aboveground tissues accumulated Cd at as low as about one third of the concentration noted for those of WT plants. It appeared therefore that T-26 mutant was defective somewhere in the Cd transporting pathway from roots to aboveground tissues.

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Cd accumulation in a major japonica cultivar Sasanishiki (low Cd in grains) and a indica cultivar Habataki (higher Cd in grains) were characterized to determine the traits involved in Cd accumulation in rice. The Cd treatment was first conducted under the hydroponic condition with relatively lower Cd concentration (20μg/L), which can be observed in soil solutions of paddy soils under water-saving conditions with moderate Cd contamination. The greater Cd accumulation was observed in shoots of Habataki through 2-wk Cd treatment, even after 1h Cd treatment. The analysis of xylem sap and transpiration rate indicated that greater ability and activity of root-to-shoot Cd-transport in Habataki would lead to higher Cd accumulation in their shoots. The results of pot experiments also demonstrated the higher Cd level in xylem sap of Habataki, which resulted in higher Cd content in rice grains of the cultivar.

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3-Phosphoglycerate, an intermediate of Calvin-Benson cycle, glycolysis, and pentose phosphate pathway, is converted to serine by 3-phosphoglycerate dehydrogenase and two consecutive reactions. Betaine is an important osmoprotectant, and synthesized by many plants in response to abiotic stresses. Almost all known biosynthetic pathways of betaine are two-step oxidations of choline, recently a novel biosynthetic pathway of betaine from glycine, catalyzed by two N-methyltransferase enzymes, was found in a halotolerant cyanobacterium Aphanothece halophytica. Recent studies suggest that for the accumulation of large amount of betaine, supply of precursors such as ethanolamine, serine, and glycine are important. Here, the potential role of PGDH for betaine synthesis was examined in 1) Aphanothece halophytica and 2) in /Arabidopsis plants which were transformed with two N-methyltransferase genes. Their results will be presented.

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