Unifacial leaves develop abaxialized leaf blades and they have been evolved in a number of divergent species in monocots. The unifacial leaves provide very unique opportunities for the developmental studies of the leaf axes formation in monocots. In addition, the mechanism of the parallel evolution of such drastic changes in leaf polarities is of interest from an evolutionary viewpoint.
We are studying molecular genetic mechanisms of the unifacial leaf development and their evolution, mainly using Juncus prismatocarpus (Juncaceae) as a model. To reveal the genetic frameworks of unifacial leaf development, we established a system of genetics and isolated mutants, which show interesting phenotypes in leaf structures. In this presentation, we will report the phenotypes of these mutants and discuss the genetic mechanism of unifacial leaf development in combination with gene expression studies and comparative molecular genetics in Juncus.

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In Arabidopsis thaliana, mutants defective in ribosomal proteins often develop leaves with pointed shape. We have isolated more than 20 mutants exhibiting such a phenotype. We found that in the asymmetric leaves1 (as1)/as2 backgrounds, which show a weak adaxial defect, some of these mutations strongly enhanced the adaxial defect. Further experiments suggested, however, that not all of pointed-leaves mutants were able to enhance the adaxial defect. To elucidate such differential effects, we cloned the responsible genes from these pointed-leaves mutants. So far, we found that mutants with strong enhancing effects were defective in either a constituent of the 60S subunit or a protein involved in ribosome biogenesis. The cloning of responsible genes from mutants without enhancing effects is in progress. We will discuss possible mechanisms on ribosome-mediated differential gene expression

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During leaf development, growth regulatory factors should localize correctly in adaxial- or abaxial-side of the primodia. However, the mechanisms of the adaxial-abaxial boundary formation are still unknown. To reveal the mechanisms, we focused on the expression domains of PHB, FIL and miR165/166 in Arabidopsis leaf primodia. The analysis of promoter-reporter fusion genes indicated that the expression domains of PHB and FIL were partially overlapped. In contrast, the overexpression of GFP, whose expression was suppressed by miR165/166, showed the adaxial-abaxial boundary. Then, using laser-microdissection technique, total RNA was prepared from the adaxial- or abaxial-side of the primodia, respectively, and the localizations of pre-miR165/166 were checked by semi-qRT-PCR analysis. Among 9 genes, 6 transcripts were found only in abaxial-side. Thus, we concluded that the adaxial-abaxial boundary is determined by suppression of PHB expression via abaxial-specific function of miR165/166. We will discuss about the suppression mechanisms in the meeting.

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Development of shoot apical meristem (SAM) and the adaxial-abaxial (ad-ab) axis of leaf primordial is important for shoot development. It is known that SAM and ad-ab axis mutually control their development. We isolated enf1 mutant, which is defective in both SAM and the ad-ab axis. The expression patterns of the key transcription factors, FIL and WUS were altered in enf1 mutant, suggesting that ENF1 regulate SAM and ad-ab axis via transcriptional regulation of the transcription factors. Transcriptome analysis also revealed that ENF1 regulated several transcription factors. ENF1 gene encoded an enzyme in the primary metabolism, and metabolome analysis showed a drastic change of metabolites in SAM of the mutant. It is suggested that the metabolic condition of cells is responsible to gene expression profile. We will discuss the relationship among metabolite, transcription, and morphogenesis.

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Leaf cells are different between adaxial and abaxial domains of the same leaf.
Such cell differentiations are known to be regulated by several genes expressing specifically in the adaxial or abaxial domains. However, the molecular mechanisms determining these gene expression domains remain to be elucidated. We have analyzed the mechanism regulating FILAMENTOUS FLOWER (FIL), one of abaxial genes, by molecular genetic approach.
We isolated a novel Arabidopsis mutant enlarged fil expression domain2 (enf2) which showed broader FIL expression domain. Recently, we found that in very early stages, FIL expression domain in wild type plant was as broad as in enf2 plant. Thus ENF2 gene may act as regulator to gradually reduce the size of FIL expression domain during leaf development.
Now we are analyzing the transition of FIL expression pattern during leaf development in wild type and enf2 and attempting to reveal the ENF2 function associating with this transition.

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ASYMMETRIC LEAVES2 gene is one of key regulators of morphogenesis of leaves in Arabidopsis thaliana. It encodes a protein with a AS2/LOB domain that consists of the C-motif, the conserved glycine residue and the leucine-zipper-like sequence. The AS2/LOB domain includes a short stretch of basic residues in the C-motif. The AS2 protein localized to a sub-nuclear body that is adjacent to nucleoli and we designated this body as the AS2 body. In order to identify the signal in AS2 required for the localization to the AS2 body, we have made DNA constructs that encoded mutant AS2 proteins with various deletions and amino acid substitutions in the AS2/LOB domain and we have investigated sub-nuclear localization of the mutant proteins. We also have examined whether the localization to the AS2 body might be required for AS2 function in morphogenesis of leaves. We will present latest results of these experiments.

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Leaves are generated as lateral organs from a shoot apical meristem and develop along three axes, proximal-distal, adaxial-abaxial and medial-lateral axes. The ASYMMETRIC LEAVES1 (AS1) and AS2 of Arabidopsis thaliana play important roles to establish these axes to produce flat and symmetric leaves. Microarray analysis using Knowledge-based FuzzyART method, which we developed, revealed that AS1 and AS2 repress not only the class 1 KNOX genes but the abaxial genes ETTIN (ETT) and YABBY5 (YAB5). Levels of expression of BP, ETT and YAB5 in as2 bp knat2 knat6 quadruple mutant and as2 ett arf4 triple mutant were quantified by real-time PCR and revealed that BP and ETT were down-regulated by AS2 in the ett arf4 and bp knat2 knat6 mutants, respectively and that YAB5 were down-regulated in both mutants. Our results suggested that by the repression of the multiple pathways AS1 and AS2 regulate flat and symmetric leaves.

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The ASYMMETRIC LEAVES2 (AS2) and ASYMMETRIC LEAVES1 (AS1) genes of Arabidopsis are required for symmetrical and flat lamina expansion. AS2 encodes a plant specific protein that contains AS2/LOB domain, and AS1 encodes a myb (SANT) domain protein. AS2 and AS1 are thought to act as a transcriptional regulator of certain genes including class 1 KNOX genes. We previously showed that AS2 regulates leaf polarity by repressing ETT/ARF3, KAN2, YAB5, which are abaxial determinants. To assess whether AS2 acts directly regulates ETT, KAN2, YAB5, we used glucocorticoid receptor (GR)-mediated inducible system. After the treatment 35S:AS2-GR plants with DEX, expression of ETT was decreased rapidly, but those of KAN2 and YAB5 were not changed within 12 hours. ETT is known to be a target of trans-acting siRNA, tasiR-ARF, which in turn regulated by miR390. We report how AS2 regulates the ETT expression in the process of leaf development.

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In Arabidopsis thaliana, many genes regulate leaf development through several pathways. The loss-of-function mutations in the ASYMMETRIC LEAVES2 (AS2) genes of Arabidopsis thaliana cause pleiotropic phenotypes in leaves, such as downward curling of leaves, reduced complexity of leaf venation pattern, generation of lobes and defect in the leaf adaxial-abaxial polarity. Our expression analysis suggests that AS2 represses directly or indirectly class 1 knox genes and abaxial factor genes, ETTIN, KANADI2, YABBY5. But the mechanisms for regulating the abaxial factor genes are unknown. To identify new components that function together with AS2 in leaf development, we performed a genetic screen for enhancers and suppressors of the as2-1 mutant. #27, one of enhancer mutants formed filament-like leaves that might be associated with defect in the adaxial-abaxial leaf polarity. We will present characteristic and genetic mapping of the #27 mutant.

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To understand the molecular mechanisms forming the seed size of rice, we are carrying out the identification of causal genes in small and round mutants. We report the identification of the causal gene of a new small and round mutant, srs1 in rice. A map based cloning method was used in the study. First, a mutation site of srs1-1 was suggested to be present in the cDNA (AK120310) sequences located on the long arm of chromosome7. Then, we introduced SRS1 gene covered the cDNA into srs1-1 by Agrobacterium tumefaciens-mediated transformation method. Transformants showed wild type phenotypes. SRS1 gene has ten exons and full length cDNA is encoding 1366 amino acid. Conserved protein domains are not found in SRS1 protein. We also isolated other alleles of srs1, such as srs1-2, srs1-3, srs1-4, and srs1-5 on the basis of the information of the cDNA sequences.

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We report the identification of the causal gene of a new small and round mutant, srs3 in rice. By the map-based cloning method, we found a candidate for the causal gene of srs3-1 in the 5th chromosome, in which a putative gene, Os05g06280.2 were encoded. We introduced the SRS3 cDNA into srs3-1 by Agrobacterium-mediated transformation method. The transformants showed wild type phenotypes. SRS3 consists from 12 exons. The SRS3 protein was consisted with 819 amino acids and grouped into the kinesin 13 family. The kinesin-motor domain containing ATPase activity locates in the region from 3rd exon to 10th exon. The srs3-1 caused one base substitution in the 9th exon, which resulting in the 1 amino acid substitution from leucine492 to phenylalanine492. It was suggested that this mutation might lead to the decrease in motor activity and consequently the development of seeds may be impaired in srs3-1.

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English abstract Low-temperature is an important factor determining germination of rice, but the underlying genetic mechanisms of low-temperature germination(LTG) have not been well elucidated. We constructed a genetic map using recombinant inbred lines derived from a cross Kakehashi(Japanese cultivar) x DunghanShali(European cultivar) in order to locate quantitative trait loci(QTLs) conferring LTG. Three LTG QTLs derived from DunghanShali were mapped. Sequence analysis of QTL region in chromosome 3 identified a base substitution between the parents in a gene coding for a transcription factor containing Histone-like motif. Arabidopsis knockout-mutants with defect in the homologue of this transcription factor gene showed a delay in germination both under normal and cold temperatures. Knockout-mutants also showed slow post-germination growth under cold condition. These observations in Arabidopsis mutants parallel the slow germination of the Japanese cultivar rice in low temperature. Therefore, loss-of-function of this particular gene may cause inefficient LTG in the Japanese cultivar rice.

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Germination inhibition by high temperature (thermoinhibition) is crucial for winter annual seeds to germinate and establish seedling in autumn. In this study, we screened chemicals which suppress and alleviate germination of Columbia seeds at permissive (28^oC) and non-permissive (34^oC) high temperature conditions, respectively. We also screened "chemical suppressors" which revert thremoinhibition resistant germination phenotype of trg1 mutant seeds. Interestingly, the hit chemicals which suppress germination of Columbia seeds at 28^oC did not inhibit germination of the seeds at 22^oC. Most of them did not suppress germination of trg1 seeds, and suppressor chemicals for trg1 did not inhibit germination of the wild type seeds. The hit chemicals may be useful to understand thermoinhibition mechanism and to develop germiantion regulators for crops.

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Seed dormancy and germination is antagonistically regulated by two phytohormones, abscisic acid (ABA) and gibberellins (GA). In the previous meeting, we reported that previously identified Arabidopsis mutant, chotto1 (cho1), which is mutated in a gene encoding a putative double APETALA2 domain transcription factor, exhibits decreased levels of seed dormancy and increased germination potential relative to the wild type after short periods of dry seed storage treatment (after-ripening: AR). AR led to decreases in the endogenous ABA levels and increases in the GA₄ levels after imbibition in wild-type seeds dependent on the length. However, the levels of ABA and GA were changed in cho1 regardless of AR. In agreement with this, transcript levels of ABA catabolism gene and GA biosynthetic genes were up-regulated in cho1 imbibed seeds. In the next meeting, we will report the results of analysis of double mutants between cho1 and ABA-hyper accumulating cyp707a2-1 or GA-deficient ga1-3.

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We examined the role of three redundant Arabidopsis ABA-activated SnRK2 protein kinases, SRK2D, SRK2E, and SRK2I for seed development and germination. During seed development and germination, these proteins are expressed in nucleus. A srk2d srk2e srk2i mutant was sensitive to dehydration and showed severe growth defects during silique and seed development. During seed germination, the triple mutant exhibited a hyper ABA-insensitive phenotype, which was more pronounced than in any single or double mutants and any abi mutants. A viviparous phenotype in the triple mutant was also observed. In-gel kinase experiments showed that the srk2d srk2e srk2i mutant had greatly reduced 42-kD kinase activity, resulting in diminished phosphorylation of ABI5. Transcriptome analyses suggest that SRK2D, SRK2E, and SRK2I protein kinases might control seed maturation and germination through the control of gene expression extensively.

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We isolated a novel ABA hypersensitive mutant named ahg12 (ABA hypersensitive germination 12) in Arabidopsis. Using a map-based cloning, we identified a missense mutation in the RPT5a gene encoding a subunit of 26S proteasome. The ahg12 mutant also showed some other pleiotropic phenotypes. The combination of phenotypes observed in ahg12 is distinct from those of other subunit mutants reported so far. Additionally, the phenotypes are distinct from those of RPT5a disruptant, suggesting that the ahg12 mutation affects a part of RPT5a function. Now, we assume that this mutation is involved in the selectivity of protein degradation depending on the subunit. The ahg12 mutation is an unreported dominant mutation and expected to provide information about the unknown function of the subunit. Detailed analysis of this mutation is now ongoing.

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Post-germinative growth of higher plants is critical phase for seedling establishment. Storage reserve in seed is converted to sucrose. Most of energy is used for growth and not used for synthesis of storage reserves during this stage. To identify transcriptional factor that regulate the carbon partition, we screened Arabidopsis mutants that require sucrose for post-germinative growth using a transcriptional factor deficient mutant library produced by CRES-T (Chimeric Repressor Silencing Technology). Analysis of those mutants allowed to identify several transcription factors. We designated these as LOG (low growth). LOG1 is one of the B-box zinc finger genes. Soluble carbohydrate content of log1 mutant was lower that that of WT during post-germinative stage. Moreover, log1 accumulated excess starch. APL3 and 4 expressions in log1 were approximately three fold higher as compared with those in WT. These results suggest that LOG1 regulates carbon partition through down-regulation of APL3 and 4 expressions.

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The Arabidopsis integument is separated into two integuments (the outer and inner integuments). The cells of outermost cell layer of integument accumulate a large amount of mucilage, pectic polysaccharides, during seed development. We identified that two NAC transcription factors, NARS1 and NARS2, regulated seed morphogenesis redundantly (1). Both NARS1 and NARS2 were specifically expressed in outer integument, and the mucilage was not accumulated in a double knock-out mutant nars1 nars2. To clarify the molecular mechanism underlying the mucilage accumulation, we searched factors whose expressions were regulated by NARS1 and NARS2. DNA microarray analysis revealed that more than 300 genes were down-regulated in nars1 nars2. Furthermore, 15 of 300 genes were down-regulated in the ttg1-1 mutant, which had a deficiency of the mucilage accumulation as nars1 nars2 did. This result suggests that these genes are involved in the mucilage accumulation.
(1) Kunieda et al., Plant Cell, 2008. doi: 10.1105/tpc.108.060160.

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In higher plants, developing seeds are dehydrated in the maturation phase, and subsequently rehydrated during germination. Since aquaporins facilitate the osmotic water transport across biomembranes, they might have some physiological roles during embryo development. In order to reveal physiological roles of plant aquaporins during embryogenesis, we analyzed DcTIP1 encoding a tonoplast intrinsic protein (TIP) in carrot (Daucus carota) somatic embryogenesis. The higher expression of DcTIP1 was detected in somatic embryos and dry seeds. By contrast, the lower expression was detected in seedlings, leaves, petioles, and roots. The expression of DcTIP1 in somatic embryos was significantly increased in response to exogenous abscisic acid (ABA) treatment. Moreover, the upstream region of DcTIP1 included the regions similar to C-ABI3 and DcECP40 promoters. Our results indicate that DcTIP1 expression is abundant in embryos and ABA-inducible, and suggest DcTIP1 might be involved in ABA-inducible desiccation tolerance in carrot somatic and zygotic embryos.

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The anaphase spindle plays an essential role in the chromosome separation. In general, its dynamics shows two phases. The first phase is the kinetochore microtubule shortening (anaphase A) and the second one is the spindle pole separation by sliding of the microtubule overlap zone and by pulling over the aster microtubules (anaphase B). However, in plant cells because they have an acentrosomal spindle, no conspicuous spindle elongation has been reported. We have visualized microtubules and cell nuclei in transgenic tobacco BY-2 cells using GFP-tubulin and histone-RFP and quantified the spindle elongation and chromosome movement. Then we found a clear process of anaphase B in tobacco cells after the start of anaphase A. It suggests that the plant spindle has an elongatable structure. Now we investigate the effects of the cell morphology on spindle dynamics in Arabidopsis thaliana root tips.

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Stomata, which consist of pairs of guard cells, open and close in response to environmental changes. Previous studies suggested actin microfilaments (MFs) were involved in the stomatal movements, however, their dynamics and roles are still unknown. In this study, we acquired microscopic images of GFP-ABD2-labelled MFs with diurnal cycle in Arabidopsis guard cells. From the images, we quantitatively estimated the dynamics including their orientation, bundling and density using our original image analysis systems. As a result, it was revealed that transient actin bundling occurred in the process of stomatal opening. We next performed similar analyze using GFP-mTn expression lines. Although GFP-mTn is useful MF-probes, recent studies have shown that they induced excessive bundling. Our results showed that GFP-mTn-labelled MFs were continually bundled in guard cells. Interestingly, stomatal opening is suppressed in GFP-mTn line. These results suggested that transient actin bundling plays important roles in promotion of stomatal opening.

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Microtubules (MTs) are major cytoskeleton in plant cells and organize their structures by interaction with actin microfilaments (MFs). To investigate the role of MFs in microtubular-structure organization, we prepared MT-MF double-visualized tobacco BY-2 cell lines by using the MT plus-end binding protein, EB1 and the fimbrin actin-binding domain as markers for MTs and MFs, respectively. At the cell surface, both MTs and MFs were localized near the cell cortex but these structures were not observed to be interacted. In the preprophase band (PPB), MFs were not localized in the MT PPB structure but rather surrounding the PPB. At the growing edge of the phragmoplast, MTs seemed to elongate by the guidance of the MFs These observations suggested that in cytoplasm MTs elongate and organized their structures by interaction with MFs but in cell cortex MTs organized their structures without the help of MFs.

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Cortical microtubules are essential cytoskeletal elements that decide the direction of cell expansion by the modulation of the orientation of cellulose microfibrils. Although the cortical microtubules disappear in early M-phase and reorganizes in early G1-phase, this reorganizing system is still unclear. So we prepared a transgenic tobacco BY-2 cell line, which expresses GFP-fused EB1 proteins, The EB1 proteins localized the plus end of the elongating microtubules and the microtubule elongation could be traced. In this cell line, we found that the cortical microtubules largely elongated to the cell end but their orientations were not parallel in the early phase of the cortical microtubule reorganization. A comparable result was observed during the cortical microtubule reorganization in mini-protoplasts in which vacuoles were removed from protoplasts, and the cortical microtubules were disappeared. Now we are examining the cortical microtubule reorganization quantitatively by programming a software, which can trace the EB1 protein movement.

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Plant cells initiate nascent microtubules from gamma- tubulin-containing complexes dispersed on existing microtubules. We simultaneously visualized microtubules (by mCherry-TUB6) and microtubule organizing centers (by gamma-tubulin-complex protein 2-GFP) in transgenic Arabidopsis plants. In interphase epidermal cells, we observed three types of microtubule nucleation events; (1) branching nucleation in which nascent microtubules are formed from the side of existing microtubules at an angle of around 40 degrees, (2) intra-bundle nucleation in which a new microtubule is formed parallele to the existing microtubule, resulting in an instantaneous bundle formation, and (3) de novo nucleation in the absence of existing microtubules. GCP2-GFP still remained on the cell cortex when cortical microtubules were depolymerized by microtubule-disrupting drugs, indicating a microtubule independent association of GCPs to the cell cortex. We are currently analyzing the microtubule nucleation events in a katanin mutant which is defectivein microtubule severing. Contributin of microtubule nucleation to microtubule organization will be discussed.

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Tracheary elements deposit secondary cell walls in appropriate patterns during plant development. To analyze regulatory mechanism of the secondary wall patterns, we have developed in vitro metaxylem vessel differentiation system using Arabidopsis suspension cells harboring inducible VND6, a master regulator of metaxylem vessel differentiation. In this study, we focused on roles of microtubules and actin microfilaments in secondary cell wall patterning. During the differentiation, the cortical microtubules were mainly distributed beneath the region of secondary wall synthesis. Detailed observation revealed that the microtubules soon depolymerized after growing into secondary wall-free pits. Depolymerization of actin microfilaments caused abnormal microtubule distribution and secondary wall patterns. Interestingly, the microtubule stabilizing agent taxol suppressed the effect of actin microfilament depolymerization. Actin microfilaments didn't show significant co-localization with secondary walls. These results suggested that indirect regulation of microtubules by actin microfilaments is essential for appropriate secondary wall patterning.

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The amyloplast sedimentation to the direction of gravity is important for gravity perception in the endodermal cell during shoot gravitropism. To elucidate the mechanism of gravity perception, we have analyzed the sgr9 (shoot gravitropism9) mutant that exhibits weak gravitropism. We have reported that sgr9 exhibits abnormal amyloplast movement, and SGR9 is likely to be localized to amyloplasts. These results suggested that SGR9 is involved in the regulation of amyloplast sedimentation. It has been suggested that actin filaments (F-actin) is involved in amyloplast sedimentation. To analyze the relationship between SGR9 and F-actin, we observed F-actin surrounding amyloplasts both in wild-type and in sgr9. Amyloplast surrounded by F-actin was more freaquently observed in sgr9 than in wild-type. In addition, inhibition of actin polymerization restored gravitropism and amyloplast sedimentation in sgr9. These results suggest that SGR9 might be involved in the modulation of amyloplast-actin interaction on amylopalsts.

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Actin microfilaments are essential intracellular components for organelle movement, cell morphogenesis, and cytoplasmic streaming in plant cells. To examine organization and dynamics of actin microfilaments in an emerging model of basal plants, Marchantia polymorpha (liverwort), we visualized actin microfilaments using Lifeact-Venus. Lifeact is a 17-amino-acid actin binding peptide derived from Abp140p in budding yeast and was reported as a powerful tool to observe the dynamic state of actin microfilaments in mammalian cells. This new probe allowed us to observe dynamic myosin-dependent movement of actin bundles in M. polymorpha cells; sliding, bundling, and branching. We will also discuss advantages and disadvantages of this probe in application to plant studies.

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Intracellular chloroplast positioning is important to optimize photosynthesis. The actin cytoskeleton has been suggested to participate in the chloroplast positioning, however, the manner of participation has not yet been elucidated. To verify our hypothesis that chloroplasts are anchored to the cortical cytoplasm by actin cytoskeleton, we prepared plasma-membrane (PM) ghosts, on which the cytoplasmic side of PM was exposed, from spinach mesophyll protoplasts. Our studies suggested that the attachment of chloroplasts to PM ghosts is actin dependent and regulated by Ca²⁺-calmodulin. From these results, we assumed participation of villin, an actin-binding protein exhibiting the Ca²⁺- calmodulin dependent actin-depolymerizing activity. We detected two kinds of villin-like polypeptides in spinach. Both polypeptides were localized in the vicinity of chloroplasts on PM ghosts. In addition, the anti-villin antibodies induced a disorder of actin filaments and detachment of chloroplasts from the PM ghosts.

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We found that cortical array of microtubules in tobacco BY-2 cells attached to a coverslip was disorganized from transverse to random by centrifugation, and the disorganized microtubules were recovered from random to transverse. The disorganization of microtubules was not observed without attachment to the coverslip and was inhibited in the presence of gadolinium, suggesting that mechano-sensitive ion channel is involved in the disorganization of microtubules. Taxol inhibited the disorganization but not recovery. This suggests that the recovery might be induced by a mechanism other than microtubule-dynamics. The system developed in the present study will be useful to study the mechanism of microtubule organization in plant cells. We will also report on the involvement of phosphorylation, dephosphorylation and actin in the regulation of cortical microtubule organization.

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Peroxisomes undergo dramatic changes in size, shape, number and position within the cell, but the division process of peroxisomes has not been characterized. To better understand molecular mechanism of peroxisome division, we screened a number of Arabidopsis mutants with aberrant peroxisome morphology (apm mutants) based on the different pattern of GFP fluorescence from the parent plant, GFP-PTS1. apm mutants exhibited the tissue-specific patterns of GFP as follows, (1) elongated peroxisomes, (2) enlarged peroxisomes, (3) GFP fluorescence in the cytosol as same as in peroxisomes, and (4) different distribution of peroxisomes. We reported that the mutation in APM1 caused the morphological change to elongated peroxisomes with decrease in number, as a result of inhibition of division, and that APM1 encoded DRP3A, one of 16 dynamin-related family. In this presentation, we would like to discuss the phenotypes of another apm mutant and the double mutants with apm1.

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Peroxisomes are single membrane-bound organelles that are ubiquitously found in eukaryotic cells. In higher plants, peroxisomes have pivotal roles in fatty acid degradation, photorespiration and phytohormone biosynthesis. All peroxisomal proteins are encoded in the nuclear genome and are transported to peroxisomes after protein synthesis in the cytosol. The functions of peroxisomes are plastically controlled under environmental changes. However, precise machineries have not been clarified. To identify the factors required for peroxisomal morphology, we have screened the aberrant peroxisome morphology (apm) mutants, in which peroxisomes display abnormal phenotypes. APM1, APM2 and APM4 have been already identified as Dynamin-related-protein 3A, Peroxin13 and Peroxin12, respectively. In this presentation, we will report characterization of another apm mutant, apm9.

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In the photosynthetic tissues, peroxisome contains enzymes that participate in photorespiration and share the metabolic pathway with chloroplast and mitochondrion. Hence, it has been thought that peroxisome is localized close to chloroplast and mitochondrion for efficient metabolic regulation. Previously, we screened a number of the peroxisome unusual positioning mutants (peup) from EMS treated mutant lines. Some mutants show aggregation of peroxisome (peup1), whereas the other shows that most peroxisomes flow in the cytosol (peup4) and defective in interaction with chloroplast. Here, we further studied the interaction of peroxisome with chloroplast in leaf mesophyll cells of mya2-XI-k double mutants, defective in peroxisome motility. However, the interaction took place normally. To characterize peups mutants, we further analyzed plant growth phenotype under several conditions. These results showed proper localization of peroxisome is essential for photosynthesis and plant normal growth.

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Nucleus is a highly heterogeneous structure, which is composed of double layered nuclear membrane, nuclear pore and nuclear matrix. Because nuclear architecture provides a physical framework for the genetic activities, it is important to know the mechanisms in which structure of the nucleus is built. To reveal the molecular pathway of nuclear biogenesis, we are focusing on nuclear pore complex in Arabidopsis. Nuclear pore complex is a large multi protein complex and is composed of more than 30 types of nucleoporins, which have unique structural domains involving in nuclear transport. To clarify the role of each nucleoporin in Arabidopsis, we expressed chimeric genes encoding nucleoporin fused with fluorescent proteins. Confocal microscopy showed that each nucleoporin localised on nuclear envelope as dot-like structures. We also found that overexpression of nucleoporins leads abnormal plant organ development, suggesting that trafficking between nucleus and cytosol is an important event for plant development.

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The sliding theory of cytoplasmic streaming was proposed about 50 years ago. It is suggested that cytoplasmic streaming is caused by the motor protein, myosin, associated with streaming organelles actively sliding along actin filaments, with the hydrolysis energy of ATP. In GFP-h, which is a transgenic Arabidopsis thaliana expressing green fluorescent protein (GFP) with an endoplasmic reticulum (ER)-retention signal, the ER networks have been visualized together with ER bodies. We focused on the dynamic movements of the ER structures. The ER movement was inhibited by the treatment of an actin depolymerizing reagent or an inhibitor of myosin activity. To identify the Arabidopsis myosin(s) involved in the ER dynamics, we analyzed the ER movement in the myosin XI mutants. We will show and discuss about the involvement of myosin XI in the ER dynamics.

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The endoplasmic reticulum (ER) has dynamic structures; i.e., the polygonal network composed of sheets, tubules, and "three-way junctions". To identify the factors responsible for ER morphology, we isolated the mutants that showed defects in ER morphology, and designated them endoplasmic reticulum morphology (ermo). ermo1 and ermo2 developed a number of ER-derived spherical structures, while ermo3 developed a large aggregate within the cells. The spherical structures in ermo2 also formed a large aggregate. In ermo1 and ermo2, the genes involved in COPI and COPII formation, respectively, were mutated. However, we could not find any defects of membrane trafficking in these mutants, suggesting that ERMO1 and ERMO2 have a novel function in maintaining ER morphology. Alternatively, it is possible that ER-Golgi transport plays a crucial role in ER morphology. In addition, ERMO2 and ERMO3 were shown to be required for keeping organelles distributed throughout the cells.

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Chloroplasts originated from photosynthetic cyanobacterium subsequent to endosymbiotic event. During evolution, most of the cyanobacterial genes were transferred to the host nucleus. Therefore, the precise expression of specific sets of nuclear genes and protein import into plastid are essential for chloroplast biogenesis. One possible cellular mechanism that coordinates these two essential processes is retrograde signaling from plastids to the nucleus. However the molecular mechanisms underlying this inter-organelle signaling remains to be characterized. Down-regulation of photosynthesis-associated genes has been observed in ppi2 (plastid protein import2) mutant lacking the plastid protein import apparatus. Genetic studies revealed that the coordination of plastid protein import and the nuclear gene expression is independent of known signaling components. Instead, it may involve the transcription factor AtGLK1. Overexpression of AtGLK1 in the ppi2 mutant partially restores both photosynthesis-associated gene expression and chlorophyll accumulation. We will discuss the role of AtGLK1 in plastid signaling pathway.

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Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). Physarum polycephalum has highly developed mt-nucleoids and we can isolate them keeping the function as in vivo. Isolated mt-nucleoid consists of at least 70 kinds of proteins. In those proteins, we identified 56kDa-protein (p56) as a DNA binding protein. It has homology with an Yeast mt-nucleoid protein, Mgm101, which is essential for mtDNA maintenance.
p56 showed the sequence-nonspecific and direct DNA-binding activity. Moreover, immunofluorescence and immunoblot analysis revealed that the p56 localized throughout the mt-nucleoid. Furthermore, we developed methods for microinjection into the plasmodium. The knockdown analysis of p56 by microinjection resulted in accumulation of DNA mutation due to oxidation induction medicine and in the increased expression level of certain DNA repair protein. Now, we are studying about the detail on the involvement of the p56 in DNA repair function.

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The plant vacuole has many functions and is essential for growth and development. The vacuolar membrane has quite complex architecture and dynamic features. We previously identified "bulb", as a complex and mobile structure on continuous vacuolar membrane (Saito et al. 2002). To reveal its biological function, we have carried out several approaches to obtaining "further structural evidence", "more direct proof for qualitative difference as a sub-region-like character" and "its function". We obtained a transgenic Arabidopsis line, which visualizes bulbs even in imbibed seeds or the floral organ. In this line, bulbs are observed in a variety of tissues than we already reported. We also found that zig/atvti11 and sgr2-1, which are known to have defects in shoot gravitropism and morphology of the vacuolar membrane in the endodermal cell in flowering stem, are virtually devoid of bulbs. Now we are trying to analyze its ultrastructure by high-pressure freezing and electron microscopy.

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From an environmental standpoint, lignocellulosic biomass is a promising feedstock for conversion to fuels and other useful chemicals. However, commercialization of such biorefinery has not been achieved, largely due to high cost of microbial cellulases, used for enzymatic hydrolysis of cellulose to fermentable sugars. Transgenic plants expressing cellulases at high levels are expected as an alternative approach to cost-effective cellulase production. In this work, we introduced the hyperthermophilic cellulase gene from Pyrococcus horikoshii into the tobacco plastid genome. The transplastomic tobacco leaves accumulated the intact hyperthermophilic cellulase up to comparable level to Rubisco large subunit. Enzyme assays indicated that the active enzyme was retained not only in fresh leaves but also in dry ones. When grown on synthetic medium, there was no apparent difference in phenotype between the transformants and wild-type plants.

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Chloroplast-encoded Ycf4 is essential for the accumulation of Photosystem I (PSI) complex and is a part of large complex (Ycf4 complex) in the green algae Chlamydomonas reinhardtii. The Ycf4 complex was purified and the polypeptide composition was analyzed by immunoblottings and mass spectrometry. In addition to Cop and PsaF, we identified PSI subunits, PsaA, PsaB and PsaD in the purified Ycf4 complex. These polypeptides were easily dissociated from the Ycf4 complex as a chlorophyll-protein complex. Pulse-chase labeling experiments with ³⁵S revealed that the polypeptides on the Ycf4 complex are newly synthesized. These results suggest that an assembly intermediate of PSI complex is loosely bound to the Ycf4 complex. Thus it is conducted that the Ycf4 complex plays a role as a scaffold for PSI complex assembly.

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Photosystem I (PSI) complex consisting of 14-15 core subunits is conserved among higher plants and the green alga Chlamydomonas reinhardtii. In contrast, the number of Light-harvesting Complex I (LHCI) subunit is different; higher plants and Chlamydomonas contain 4-6 and 9 distinctive LHCI subunits, respectively. It remain elusive how 9 LHCI complexes are organized in Chlamydomonas. Here, antibodies against PSI and LHCI subunits were generated and used for analyzing LHCI structure by chemical cross-linking. We detected cross-linked products between PsaG-Lhca9, Lhca9-Lhca2, PsaK-Lhca3, PsaF-Lhca8, Lhca8-Lhca4, Lhca5-Lhca6. Based on the cross-linking experiments, we will discuss on oligomeric structure of LHCI in Chlamydomonas.

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State transitions are required for balancing excitation energy between Photosystem I (PSI) and II (PSII) and are regulated by the redox state of cytochrome b₆f complex. In state 2, when cytochrome b₆f complex is reduced, a part of LHCII is believed to migrate from PSII to PSI to form PSI-LHCI/II supercomplex. We have already demonstrated that monomeric LHCIIs (CP26, CP29 and LhcbM5) reversibly associate with PSI-LHCI supercomplex in state 2. Here we characterized the amount and function of monomeric LHCIIs bound to PSI-LHCI supercomplex in state 2. First, PSI-LHCI/II was dissociated into PSI-LHCI and monomeric LHCIIs and the chlorophyll distribution between these complexes was estimated. In addition, Chls/P700 in PSI-LHCI/II was estimated by measuring light-induced photooxidation of P700. It was concluded that the functional antenna size of PSI-LHCI/II increases 1.2 - 1.4 times more than that of PSI-LHCI by the association of monomeric LHCIIs.

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In all the oxygenic photosynthetic organisms,Chlorophyll (Chl) plays a key role in photosynthetic energy conversion of solar energy into chemical energy. Until recently, Chl a has been believed to be essential for the light harvesting and photochemical reactions in oxygenic photosynthesis. In 1996, a new type of oxygenic photosynthetic organism, Acaryochloris marina (A.marina), was discovered. A.marina performs the oxygenic photosynthesis with Chl d that absorbs light at wavelength above 700nm, at longer wavelength than Chl a. An enigma that how to drive oxygenic photosynthesis using the10-percent-lower-energy photons compared with Chl a arose. We here analyzed the orientation of the pigments in the isolated reaction center pigment-protein complexes to understand the mechanism of excitation transfer and photochemistry with Chl d. We studied orientation and photochemistry of pigments in purified Photosystem I complexes of A.marina by using a Linear Dichroism technique.

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Photosystem I(PS I) pigment-protein super complex of diatoms consists of two peripheral antennas, FCP(fucoxanthin-Chlorophyll(Chl)-binding proteins) I-1, FCP I-2, and core complex (PS I-Core). We studied the ultra-fast excitation energy transfer in isolated FCP-PS I super-complex, PS I-Core complex, FCP I-1 and FCP I-2 isolated from C. gracillis by the upconversion and the streak camera methods at 17 K. Results of a global analysis revealed a major fluorescence decay phase with a 5-ps time constant upon the selective excitation of Chl a at 430 nm. However, it was not observed upon the 460-nm excitation that mainly excited Chl c in FCP. Thus, the 5-ps phase was interpreted as the energy trapping in the core. Two components with 0.86-ps and 14-ps time constants were observed upon the excitation at 460 nm suggesting the two energy-transfer pathways from FCP to the core (FCP I-1-> core and FCP I-2-> core).

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The light-harvesting and primary charge-separation processes in photosystem I (PS I) reaction center have not been revealed completely, because of 1) the large spectral overlap among many antenna chlorophyll(Chl)s, 2) very fast energy transfers among Chl, and 3) the overlap of the time range between the light harvesting and primary charge separation. At cryogenic temperature, the above difficulties 1) and 2) are expected to be overcome because the Chl spectra become very sharp and the energy transfer becomes drastically slower. We studied the ultrafast fluorescence dynamics of PS I at 15 K. The results showed that at 15 K the antenna Chl in PS I are divided into 3 groups; Chl that transfer the excitation energy to P700 with a 5-ps time constant, 300-ps time constant, and transfer the excitation energy to the red Chl with a 30-ps time constant. Stoichiometry of these Chl groups will be discussed.

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Binding properties of the extrinsic PsbO, PsbP and PsbQ in oxygen-evolving photosystem II (PSII) prepared from a green alga, Chlamydomonas reinhardtii, are different from those of spinach PSII¹⁾. The green algal PsbP and PsbQ as well as PsbO directly bind to PSII intrinsic proteins independent of the other extrinsic proteins. To search for the nearest neighbor relationship between these extrinsic proteins and PSII intrinsic proteins, we crosslinked Chlamydomonas PSII with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and then PSII core was prepared by Blue-Native PAGE. Crosslinked products containing PsbO, PsbP or PsbQ were detected in the PSII core by Western blotting using antibodies against these extrinsic proteins, suggesting that each of the three extrinsic proteins interacts electrostatically with different PSII core components. Moreover, crosslinked product between PsbP and PsbQ was found, and its crosslinked site was determined.
¹⁾ Plant Cell Physiol. 44 (2003) 76-84

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Ycf12 is a recently identified low-molecular mass trans-membrane protein of photosystem II (PSII), and its location is unknown in the current crystal structure. We purified PSII dimers from a Ycf12-deletion mutant of Thermosynechococcus elongatus, crystallized, and analyzed the structure of the mutant PSII. Our result showed that Ycf12 is located in the periphery of PSII close to PsbK and PsbZ, and corresponded to an unassigned helix X1 in the 3.0 Å structure reported by the Berlin group. Combining our previous results that X2 is PsbY, it is strongly suggested that the remaining unassigned helix X3 in the 3.0 Å structure corresponds to PsbX. We also purified PSII from a PsbZ deletion mutant, and crystallized PSII from the mutant. The crystals obtained had significantly different unit cell constants than those of wild type, indicating the participation of this subunit in joining the two PSII dimers within the wild type crystals

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PsbM and PsbI are two low-molecular mass subunits of PSII, and PsbM is located in the center of PSII dimer, whereas PsbI is located in its periphery. Thease locations were confirmed by crystal structure analysis of their deletion mutants, and suggest that PsbM is required for the stable formation of PSII dimer, whereas PsbI may not be directly related to the dimer formation or stability. Thylakoids isolated from the two deletion mutants, however, showed a similar decrease in PSII dimer, suggesting that both subunits are required for the dimer formation or stability. Further analysis with purified dimers showed that while dimers lacking PsbM were unstable and decomposed into PSII monomer upon appropriate detergent treatment, dimers lacking PsbI were stable against the same treatment, suggesting that PsbI may be required for the assembly of dimer in vivo but may not be needed for the stability of dimer in vitro.

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We have reported that several small subunits are involved in the stabilization of the dimeric form of Photosystem II (PSII) complex. In this study we investigated the stability of the PSII dimer by blue-native PAGE. Thylakoid membranes prepared from Thermosynechococcus elongatus BP-1 were solubilized with various concentrations of n-dodecyl-β-D-maltopyranoside (DM). It was found that the recovery of the PSII monomer was high at low concentrations of DM, but was reduced at high concentrations. Concomitantly, higher recovery of the PSII dimer was obtained at high concentrations of DM. These results were confirmed by two-dimensional PAGE. Apparently, there was no significant difference in protein composition between the monomeric and dimeric PSII. These results suggest that dimerization of the PSII complex can be induced by action of DM. We will discuss the intrinsic form and interconversion of the PSII complexes in vivo.

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Glutathione is a ubiquitous tripeptide that is synthesized via two reactions dependent on photosynthetic ATP production. In this study, we used Arabidopsis mutants having low levels of glutathione to evaluate the relationship of glutathione with a key Calvin-cycle enzyme, Rubisco. Compared to wild-type plants, these mutants showed 80-90% CO₂ assimilation rates per unit leaf area and 80-90% Rubisco activation state, but they showed no significant difference in Rubisco activity per unit leaf area. Taken together, the decrease in glutathione levels reduces the Rubisco activation state but the glutathione deficiency-dependent decrease in the CO₂ fixation is attributed to any other glutathione-regulated factor than Rubisco. Since the Calvin cycle enzyme aldolase is regulated by glutathione, it is considered that low glutathione levels restrict aldolase activity and resultantly decrease the CO₂ assimilation rate. We will discuss relationship of glutathione levels to aldolase as well as Rubisco on the Calvin cycle regulation.

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